USP30 INHIBITORS AND METHODS OF USE

Information

  • Patent Application
  • 20150344888
  • Publication Number
    20150344888
  • Date Filed
    March 16, 2015
    9 years ago
  • Date Published
    December 03, 2015
    8 years ago
Abstract
Inhibitors of USP30 and methods of using inhibitors of USP30 are provided. In some embodiments, methods of treating conditions involving mitochondrial defects are provided.
Description
RELATED APPLICATIONS

1. Field


Inhibitors of USP30 and methods of using inhibitors of USP30 are provided. In some embodiments, methods of treating conditions involving mitochondrial defects are provided.


2. Background


Mitophagy is a specialized autophagy pathway that eliminates mitochondria through degradation by lysosomes. As such, it removes mitochondria during normal cellular turnover of organelles, during maturation of erythrocytes, and following fertilization to eliminate sperm-derived mitochondria. Mitophagy also mediates the clearance of damaged mitochondria, an important aspect of mitochondria quality control. Defective or excess mitochondria, if left uncleared, may become an aberrant source of oxidative stress and compromise healthy mitochondria through mitochondrial fusion. In yeast, selective blockade of mitophagy causes increased production of reactive oxygen species (ROS) by excess mitochondria and loss of mitochondrial DNA (mt-DNA). Impaired mitochondria quality control could also affect key biosynthetic pathways, ATP production, and Ca2+ buffering, and disturb overall cellular homeostasis.


Parkinson's disease (PD), the second most common neurodegenerative disorder after Alzheimer's disease (AD), is characterized most prominently by loss of dopaminergic neurons in the substantia nigra. Although the pathogenic mechanisms of PD are not clear, several lines of evidence suggest that mitochondrial dysfunction is central to PD. MPTP, a mitochondrial toxin, damages dopamine neurons and produces clinical parkinsonism in humans. Epidemiologic evidence links PD with exposure to pesticides such as rotenone (a complex I inhibitor) and paraquat (an oxidative stressor). Consistent with mitochondrial impairment, reduced complex I activity and high levels of mt-DNA mutations have been found in substantia nigra from PD patients. Similarly, functional and morphological changes in mitochondria are present in genetic models of PD. Perhaps most compellingly, early-onset familial PD can be caused by mutations in Parkin ubiquitin-ligase and PINK1 serine/threonine protein kinase, both of which function to maintain healthy mitochondria through regulating mitochondrial dynamics and quality control.


Genetic studies in flies established that PINK1 acts upstream of Parkin to maintain proper mitochondria morphology and function. PINK1 recruits Parkin from the cytoplasm to the surface of damaged mitochondria, leading to Parkin-mediated ubiquitination of mitochondrial outer membrane proteins and removal of damaged mitochondria by mitophagy. PD-associated mutations in either PINK1 or Parkin impair Parkin recruitment, mitochondrial ubiquitination and mitophagy. Parkin regulates multiple aspects of mitochondrial function such as mitochondrial dynamics and trafficking, and may also influence mitochondria biogenesis. The degradation of a broad range of outer mitochondrial membrane proteins on damaged mitochondria appears to be affected by Parkin. Among these mitochondria associated proteins, MIRO, a component of the mitochondria-kinesin motor adaptor complex, may be a shared substrate of both Parkin and PINK-1.


Parkin expression and/or activity can be impaired through genetic mutations in familial PD or by phosphorylation in sporadic PD. In the context of the inherently high mitochondrial oxidative stress in substantia nigra dopamine neurons, loss of Parkin-mediated mitochondrial quality control could explain the greater susceptibility of substantia nigra neurons to neurodegeneration. Promoting clearance of damaged mitochondria and enhancing mitochondrial quality control could be beneficial in PD.


SUMMARY

In some embodiments, methods of increasing mitophagy in a cell are provided. In some embodiments, the method comprises contacting the cell with an inhibitor of USP30.


In some embodiments, methods of increasing mitochondrial ubiquitination in a cell are provided. In some embodiments, methods of increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or fourteen proteins selected from Tom20, MIRO, MUL1, ASNS, FKBP8, TOM70, MAT2B, PRDX3, IDE, VDAC1, VDAC2, VDAC3, IP05, PSD13, UBP13, and PTH2 in a cell are provided. In some embodiments, the method comprises contacting the cell with an inhibitor of USP30.


In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, or three amino acids selected from K56, K61, and K68 of Tom 20. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K153, K187, K330, K427, K512, K535, K567, and K572 of MIRO. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, or three amino acids selected from K273, K299, and K52 of MUL1. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from K147, K168, K176, K221, K244, K275, K478, K504, and K556 of ASNS. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K249, K271, K273, K284, K307, K317, K334, and K340 of FKBP8. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K78, K120, K123, K126, K129, K148, K168, K170, K178, K185, K204, K230, K233, K245, K275, K278, K312, K326, K349, K359, K441, K463, K470, K471, K494, K501, K524, K536, K563, K570, K599, K600, and K604 of TOM70. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, or four amino acids selected from K209, K245, K316, and K326 of MAT2B. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, or five amino acids selected from K83, K91, K166, K241, and K253 of PRDX3. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K558, K657, K854, K884, K929, and K933 of IDE. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids selected from K20, K53, K61, K109, K110, K266, and K274 of VDAC1. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K31, K64, K120, K121, K277, and K285 of VDAC2. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K20, K53, K61, K109, K110, K163, K266, and K274 of VDAC3. In some embodiments, the method comprises increasing ubiquitination of at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K238, K353, K436, K437, K548, K556, K613, K678, K690, K705, K775, and K806 of IP05. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K2, K32, K99, K115, K122, K132, K161, K186, K313, K321, K347, K350, and K361 of PSD13. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K18, K190, K259, K326, K328, K401, K405, K414, K418, K435, K586, K587, and K640 of UBP13. In some embodiments, the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from 47, 76, 81, 95, 106, 119, 134, 171, 177 of PTH2.


In some embodiments, the cell is under oxidative stress. In some embodiments, methods of reducing oxidative stress in a cell are provided. In some embodiments, a method comprises contacting the cell with an inhibitor of USP30.


In some embodiments, the cell comprises a pathogenic mutation in Parkin, a pathogenic mutation in PINK1, or a pathogenic mutation in Parkin and a pathogenic mutation in PINK1. Nonlimiting exemplary pathogenic mutations in Parkin are shown in Table 1. Thus, in some embodiments, the pathogenic mutation in Parkin is selected from the mutations in Table 1. Nonlimiting exemplary pathogenic mutations in PINK1 are shown in Table 2. In some embodiments, the pathogenic mutation in PINK1 selected from the mutations in Table 2.


In various embodiments, the cell is selected from a neuron, a cardiac cell, and a muscle cell. In some such embodiments, the cell is ex vivo or in vitro. Alternatively, in some such embodiments, the cell is comprised in a subject.


In some embodiments, methods of treating conditions involving mitochondrial defects in a subject are provided. In some embodiments, the method comprises administering to the subject an effective amount of an inhibitor of USP30. In some embodiments, the condition involving a mitochondrial defect is selected from a condition involving a mitophagy defect, a condition involving a mutation in mitochondrial DNA, a condition involving mitochondrial oxidative stress, a condition involving a defect in mitochondrial shape or morphology, a condition involving a defect in mitochondrial membrane potential, and a condition involving a lysosomal storage defect.


In some embodiments, the condition involving a mitochondrial defect is selected from a neurodegenerative disease; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome; Leber's hereditary optic neuropathy (LHON); neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS); Danon disease; ischemic heart disease leading to myocardial infarction; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrongenase deficiency (LCHAD); Leigh disease or syndrome; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrongenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial recessive ataxia syndrome; mitochondrial cytopathy; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; and very long-chain acyl-CoA dehydrongenase (VLCAD) deficiency.


In some embodiments, methods of treating neurodegenerative diseases are provided. In some embodiments, the method comprises administering to a subject an effective amount of an inhibitor of USP30.


In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia.


In some embodiments, methods of treating Parkinson's disease are provided. In some embodiments, the method comprises administering to a subject an effective amount of an inhibitor of USP30.


In some embodiments, methods of treating conditions involving cells undergoing oxidative stress are provided. In some embodiments, the method comprises administering to a subject an effective amount of an inhibitor of USP30.


In some embodiments involving treatment of a subject, the subject comprises a pathogenic mutation in Parkin, a pathogenic mutation in PINK1, or a pathogenic mutation in Parkin and a pathogenic mutation in PINK1 in at least a portion of the subject's cells. In some embodiments, the pathogenic mutation in Parkin is selected from the mutations in Table 1. In some embodiments, the pathogenic mutation in PINK1 is selected from the mutations in Table 2.


In some embodiments, the inhibitor of USP30 is administered orally, intramuscularly, intravenously, intraarterially, intraperitoneally, or subcutaneously. In some embodiments, the method comprises administering at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from levodopa, a dopamine agonist, a monoamino oxygenase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, amantadine, riluzole, a cholinesterase inhibitor, memantine, tetrabenazine, an antipsychotic, clonazepam, diazepam, an antidepressant, and an anti-convulsant.


In any of the methods described herein, the inhibitor of USP30 may be an inhibitor of USP30 expression. Nonlimiting exemplary inhibitors of USP30 expression include antisense oligonucleotides and short interfering RNAs (siRNAs). In any of the methods described herein, the inhibitor of USP30 may be an inhibitor of USP30 activity. Nonlimiting exemplary inhibitors of USP30 activity include antibodies, peptides, peptibodies, aptamers, and small molecules.


In some embodiments, a peptide inhibitor of USP30 comprises the amino acid sequence:












X1X2CX3X4X5X6X7X8X9X10X11CX12
(SEQ ID NO: 48)






wherein:


X1 is selected from L, M, A, S, and V;


X2 is selected from Y, D, E, I, L, N, and S;


X3 is selected from F, I, and Y;


X4 is selected from F, I, and Y;


X5 is selected from D and E;


X6 is selected from L, M, V, and P;


X7 is selected from S, N, D, A, and T;


X8 is selected from Y, D, F, N, and W;


X9 is selected from G, D, and E;


X10 is selected from Y and F;


X10 is selected from L, V, M, Q, and W; and


X12 is selected from F, L, C, V, and Y.


In some embodiments, a peptide inhibitor of USP30 peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 22. In some embodiments, the peptide inhibits USP30 with an IC50 of less than 10 μM. In some embodiments, the IC50 of a peptide inhibitor of USP30 for at least one, at least two, or at least three peptidases selected from USP7, USP5, UCHL3, and USP2 is greater than 20 μM, greater than 30 μM, greater than 40 μM, or greater than 50 μM.


In some embodiments, an antisense oligonucleotide comprises a nucleotide sequence that is at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a region of USP30 mRNA and/or a region of USP30 pre-mRNA. In some embodiments, the region of USP30 mRNA or region of USP30 pre-mRNA is at least at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides long. In some embodiments, the antisense oligonucleotide is 10 to 500 nucleotides long, or 10 to 400 nucleotides long, or 10 to 300 nucleotides long, or 10 to 200 nucleotides long, or 10 to 100 nucleotides long, or 15 to 100 nucleotides long, or 10 to 50 nucleotides long, or 15 to 50 nucleotides long. An antisense oligonucleotide may comprise one or more non-nucleotide components.


In some embodiments, an siRNA comprises a nucleotide sequence that is at least at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a region of USP30 mRNA and/or a region of USP30 pre-mRNA. In some embodiments, the region of USP30 mRNA or region of USP30 pre-mRNA is at least at least 10, at least 15, at least 20, or at least 25 nucleotides long. In some embodiments, the siRNA is 10 to 200 nucleotides long, or 10 to 100 nucleotides long, or 15 to 100 nucleotides long, or 10 to 60 nucleotides long, or 15 to 60 nucleotides long, or 10 to 50 nucleotides long, or 15 to 50 nucleotides long, or 10 to 30 nucleotides long, or 15 to 30 nucleotides long. In some embodiments, an siRNA is an shRNA.


An embodiment of the present invention is an inhibitor of USP30 for the treatment of a condition involving a mitochondrial defect in a subject. In a particular embodiment the condition involving a mitochondrial defect is selected from a condition involving a mitophagy defect, a condition involving a mutation in mitochondrial DNA, a condition involving mitochondrial oxidative stress, a condition involving a defect in mitochondrial shape or morphology, a condition involving a defect in mitochondrial membrane potential, and a condition involving a lysosomal storage defect. in another particular embodiment the condition involving a mitochondrial defect is selected from a neurodegenerative disease; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome; Leber's hereditary optic neuropathy (LHON); neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS); Danon disease; ischemic heart disease leading to myocardial infarction; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrongenase deficiency (LCHAD); Leigh disease or syndrome; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrongenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial recessive ataxia syndrome; mitochondrial cytopathy; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; and very long-chain acyl-CoA dehydrongenase (VLCAD) deficiency. In a more particular embodiment the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia.


Another embodiment of the present invention is an inhibitor of USP30 for the treatment of a neurodegenerative disease in a subject comprising administering to the subject. In a particular embodiment, the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia.


Also an embodiment of the present invention is an inhibitor of USP30 for the treatment of Parkinson's disease in a subject.


In another embodiment of the present invention, the inhibitor of USP30 is administered orally, intramuscularly, intravenously, intraarterially, intraperitoneally, or subcutaneously.


In a particular embodiment of the present invention. the inhibitor of USP30 for the use in a treatment as described herein is combined with at least one additional therapeutic agent. in a further particular embodiment, the at least one additional therapeutic agent is selected from levodopa, a dopamine agonist, a monoamino oxygenase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, amantadine, riluzole, a cholinesterase inhibitor, memantine, tetrabenazine, an antipsychotic, clonazepam, diazepam, an antidepressant, and an anti-convulsant.





BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.



FIG. 1A shows immunostaining of HeLa cells cotransfected with GFP-Parkin, and individual FLAG-tagged DUBs. Following 24 hours of expression, cells were treated with CCP (20 μM, 24 h) and immunostained for GFP, FLAG, and endogenous Tom20. Representative images are shown for FLAG-tagged USP30, DUBA2, UCH-L1, USP15 and ATXN3; other DUBs are not shown. Scale bar, 10 μm. FIG. 1B shows immunostaining of SH-SY5Y cells cotransfected with GFP-Parkin and the indicated control (β-Gal) and USP30 constructs. Following 24 hours of expression, cells were treated with CCCP (20 μM, 24 h) and immunostained for myc, FLAG, and endogenous Tom20 and HSP60 (Scale bar, 5 μm). FIG. 1C shows quantification of percent of cells with Tom20 or HSP60 staining from FIG. 1B (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. N=3 experiments. Error bars represent SEM). FIG. 1D shows quantification of total Tom20 and HSP60 fluorescence intensity per cell from FIG. 1B (**p<0.01 by One-way ANOVA-Dunnett's Multiple Comparison test. n=63, 67 and 54 cells for control (β-Gal), USP30-FLAG and USP30-C77S-FLAG groups, respectively. N=3 experiments. Error bars represent SEM). Figure l E shows quantification of percent of cells containing Parkin clusters from FIG. 1B (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. N=3 experiments. Error bars represent SEM).



FIG. 2A shows immunostaining of transfected USP30-FLAG (red) and mitochondria-targeted GFP (green) in cultured rat hippocampal neurons. Merge is shown in color; individual channels in gray-scale. Scale bar, 5 μm. FIG. 2B shows immunostaining of SH-Sy5Y cells transfected with control or USP30 siRNA. Following 3 days of knockdown, cells were fixed and immunostained for endogenous USP30 and HSP60. USP30 siRNA primarily decreased mitochondrial USP30 antibody staining (Scale bar, 5 μm). Higher magnification images of the boxed regions are shown on the right panel (Scale bar, 2 μm). FIG. 2C shows immunoblots of cytoplasm- and mitochondria-enriched fractions from rat brain with USP30, HSP60, and GAPDH antibodies. FIG. 2D shows immunostaining of SH-SY5Y cells cotransfected with GFP-Parkin and the indicated control (β-Gal) and USP30 constructs. Following 24 hours of expression, cells were treated with CCCP (20 μM, 4 h) and immunostained for GFP, FLAG, and endogenous Tom20 and polyubiquitin chains (detected with the FK2 antibody) (Scale bar, 5 μm). FIG. 2E is a plot showing the quantification of mitochondria-associated polyubiquitin staining intensity normalized by mitochondrial area from FIG. 2D (integrated fluorescence intensity of mitochondrial FK2 staining/area of Tom20 staining) (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=61, 45 and 59 cells for β-Gal, USP30-FLAG and USP30-C77S-FLAG groups, respectively. Error bars represent SEM). FIG. 2F shows immunoblots of cell lysates from GFP-Parkin expressing stable HEK-293 cells transfected with the indicated control (β-Gal) and USP30 constructs. Following 24 hours of expression, cells were treated with CCCP (5 μM, 2 hours) and lysed. FIG. 2G is a plot showing the quantification of immunoblot signal for GFP-Parkin normalized to actin from FIG. 2F (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. N=6 experiments. Error bars represent SEM).



FIG. 3A shows that mt-Keima differentially highlights cytoplasmic (green) and lysosomal (red) mitochondria. Cultured hippocampal neurons were transfected with mt-Keima and GFP. Following 2 days of expression, cells were imaged with 458 nm (shown in green) or 543 nm (shown in red) light excitation. GFP signal was used to outline the cell (shown in white). Scale bar, 5 μm. FIG. 3B shows mt-Keima imaging in neurons transfected with Parkin shRNA knockdown constructs. Scale bar, 5 μm. FIG. 3C is a plot showing the quantification of mitophagy index from FIG. 3B (**p<0.01 and ***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=52-109 cells per group. N=3-6 experiments. Error bars represent SEM). FIG. 3D shows mt-Keima imaging in neurons transfected with PINK1 shRNA knockdown constructs. Scale bar, 5 μm. FIG. 3E is a plot showing the quantification of mitophagy index from FIG. 3D (**p<0.01 and ***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=52-109 cells per group. N=3-6 experiments. Error bars represent SEM). FIG. 3F shows mt-Keima imaging in neurons transfected with PINK1-GFP and Parkin-shRNA#1 (luciferase shRNA and β-Gal as controls). Scale bar, 5 μm. FIG. 3G is a plot showing the quantification of mitophagy index from FIG. 3F (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=55-77 cells. N=3 experiments. Error bars represent SEM).



FIG. 4A shows mt-Keima imaging in cultured hippocampal neurons before and after NH4Cl treatment (50 mM, 2 minutes). mt-Keima signal collected with 543 nm or 458 nm laser excitation sources are shown in red and green, respectively. Scale bar, 5 μm. FIG. 4B shows imaging of mt-Keima and Lysotracker (shown in gray scale) in hippocampal neurons. Scale bar, 5 μm. FIG. 4C shows post-hoc immunostaining for endogenous LAMP-1 in neurons imaged for mt-Keima signal. Immediately following mt-Keima imaging, cells were fixed and stained with anti-LAMP1 antibody (shown in gray scale). Scale bar, 5 μm. FIG. 4D is a plot showing quantification of mitophagy index following 1, 3 and 6-7 days of mt-Keima expression in cultured hippocampal neurons (*p<0.05 and ***p<0.001 using One-way ANOVA-Bonferroni's Multiple Comparison test. n=56-146 cells. N=6 experiments. Error bars represent SEM). FIG. 4E is an immunoblot of HEK-293 cell lysates transfected with FLAG-Parkin cDNA and Parkin shRNA expression constructs. PSD-95-FLAG was co-transfected as control. FIG. 4F is an immunoblot of HEK-293 cell lysates transfected with PINK1-GFP cDNA and PINK shRNA constructs. PSD-95-FLAG was co-transfected as control. FIG. 4G shows an immunoblot of endogenous Parkin in cultured hippocampal neurons infected with Adeno-associated virus expressing the indicated shRNAs. FIG. 4H shows an immunoblot of endogenous PINK1 in cultured hippocampal neurons infected with Adeno-associated virus expressing the indicated shRNAs. FIG. 4I shows mt-Keima imaging in neurons transfected with GFP-Parkin (or GFP as control). Scale bar, 5 μm. FIG. 4J is a plot showing quantification of mitophagy index from FIG. 4I (p=0.52 by Student's t-test. n=61-67 cells. N=3 experiments. Error bars represent SEM).



FIG. 5A shows mt-Keima imaging in neurons transfected with USP30-FLAG or USP30-C77S-FLAG. Scale bar, 5 μm. FIG. 5B shows immunoblots of HEK-293 cell lysates transfected with the indicated cDNA and shRNA constructs. PSD-95-FLAG was co-transfected as control. FIG. 5C shows an immunoblot of endogenous USP30 in cultured hippocampal neurons infected with Adeno-associated virus particles expressing the USP30 shRNA. FIG. 5D shows mt-Keima imaging in neurons transfected with rat USP30 shRNA and human USP30 cDNA expression constructs (luciferase shRNA and β-Gal as controls). Scale bar, 5 μm. FIG. 5E is a plot showing quantification of mitophagy index from FIG. 5A (***p<0.001 by One-way ANOVA-Bonferroni's Multiple Comparison test. 43-122 cells. N=6 experiments. Error bars represent SEM). FIG. 5F is a plot showing the quantification of mitophagy index from FIG. 5B (**p<0.01 and ***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=96-101 cells. N=4 experiments. Error bars represent SEM).



FIG. 6A shows immunoblots of anti-HA-immunoprecipitates for endogenous MIRO and Tom20 in a parental HEK-293 cell line (that lacks GFP-Parkin) transfected with HA-ubiquitin and the indicated constructs. Following 24 hours of expression, cells were treated with CCCP (5 μM, 2 hours) and ubiquitinated proteins were immunoprecipitated with anti-HA beads. Immunoprecipitates and inputs were blotted with the indicated antibodies. FIG. 6B shows immunoblots of anti-HA-immunoprecipitates for endogenous MIRO and Tom20 with USP30 knockdown. GFP-Parkin expressing stable HEK-293 cells were transfected with HA-ubiquitin and the indicated shRNA and cDNA expression constructs. Following 6 days of expression, cells were processed as in FIG. 6A. FIGS. 6C and E show immunoblots of anti-HA-immunoprecipitates for endogenous Miro and Tom20 from cells transfected with the indicated HA-tagged ubiquitin mutants and treated with CCCP (20 μM, 2 hours). FIGS. 6D and F show quantification of immunoblot signals from (C) and (E). Amount of ubiquitination afforded by the ubiquitin mutants are reported relative to wild-type ubiquitin (**p<0.01 and ***p<0.001 compared to ‘wild-type HA-ubiquitin+CCCP’ group, using one-way ANOVA with Dunnett's Multiple Comparison test. 6 denotes ***p<0.001). FIG. 6G shows immunoblots of GFP-Parkin HEK-293 stable cell lysates that were transfected with the indicated FLAG-tagged USP30 constructs and treated with CCCP (5 μM, 1-6 hours). FIG. 6H is a plot showing quantification of immunoblot signals normalized to actin shown in FIG. 6G (*p<0.05, **p<0.01, ***p<0.001 compared to β-Gal control, using Two-way ANOVA with Bonferroni's Multiple Comparison test. Immunoblot signals for all other proteins (VDAC, Mfn-1, Tom70, Hsp60) did not reach significance. N=3-5 experiments).



FIG. 7A shows immunoblots of anti-HA-immunoprecipitates for endogenous MIRO and Tom20 with USP30 overexpression. HEK-293 cells stably expressing GFP-Parkin were transfected with HA-ubiquitin and the indicated constructs. Following 24 hours of expression, cells were treated with CCCP (5 μM, 2 hours) and ubiquitinated proteins were immunoprecipitated with anti-HA beads. Immunoprecipitates and inputs were blotted with the indicated antibodies. FIG. 7B is a plot showing quantification of the immunoblot signal for co-IP′ed MIRO from FIG. 7A. FIG. 7C is a plot showing quantification of the immunoblot signal for co-IP′ed Tom20 from FIG. 7A. Protein levels co-precipitated with anti-HA beads are normalized to ‘β-Gal+CCCP’ group (*p<0.05, **p<0.01 and ***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test, compared to β-Gal+CCCP. N=3-5 experiments. Error bars represent SEM). FIG. 7D shows immunoblots of anti-HA immunoprecipitates for endogenous MIRO and Tom20 with USP30 knockdown. GFP-Parkin expressing stable HEK-293 cells were transfected with HA-ubiquitin and the indicated shRNA plasmids. Following 6 days of expression, cells were processed as in FIG. 7A. FIG. 7E is a plot showing quantification of the immunoblot signal for co-IP′ed MIRO from FIG. 7D. FIG. 7F is a plot showing quantification of the immunoblot signal for co-IP′ed Tom20 from FIG. 7D. Protein levels co-precipitated with anti-HA beads is normalized to luciferase shRNA+CCCP′ group (*p<0.05, **p<0.01 and ***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test, compared to ‘luciferase shRNA+CCCP’. N=4-6 experiments. Error bars represent SEM).



FIG. 8A shows immunoblots of HA-ubiquitin precipitates from GFP-Parkin HEK-293 cells transfected with the indicated constructs. Following transfection and treatment with CCCP (5 μM, 2 hours), ubiquitinated proteins were immunoprecipitated with anti-HA beads, and precipitates and inputs were immunoblotted with the indicated antibodies. FIG. 8B shows mt-Keima imaging in neurons transfected with Tom20-myc and USP30 constructs (β-Gal as control). Scale bar, 5 μm. FIG. 8C shows mt-Keima imaging in neurons transfected with USP30 shRNA and MIRO cDNA constructs (luciferase RNAi and β-Gal as controls). Scale bar, 5 μm. FIG. 8D is a plot showing the quantification of mitophagy index from FIG. 8B (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. n=67-80 cells for all groups. N=3 experiments. Error bars represent SEM). FIG. 8E is a plot showing quantification of mitophagy index from FIG. 8C (*p<0.05 and ***p<0.001 by One-way ANOVA-Bonferroni's Multiple Comparison test. n=72-75 cells for all groups. N=3 experiments. Error bars represent SEM).



FIG. 9A shows extracted ion chromatograms corresponding to K-GG peptides identified from Tom20 in the USP30 knockdown experiment. Relative abundance of each ubiquitinated peptide is shown on the y-axis relative to the most abundant analysis, which precursor ion m/z indicated above each peak. The sequence of each K-GG peptide is shown below in green. Asterisks denote modified lysine residues. FIG. 9B shows extracted ion chromatograms corresponding to K-GG peptides identified from USP30 in the Parkin overexpression experiment. The data are presented in a similar manner as in (A). FIG. 9C shows immunoblots of anti-HA-immunoprecipitates for endogenous USP30 from cells transfected with wild-type, K161N and G430D GFP-Parkin constructs. After 24 hours of expression, cells were treated with CCCP (20 μM, 2 hours) and ubiquitinated proteins were immunoprecipitated with anti-HA beads. Immunoprecipitates and inputs were blotted with the indicated antibodies. FIG. 9D shows quantification of immunoblot signal for co-IP′ed USP30 from (C). Protein levels co-precipitating with anti-HA beads are normalized to the ‘wild-type GFP-Parkin+CCCP’ group. (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test, compared to ‘wild-type GFP-Parkin+CCCP’. N=4 experiments. Error bars represent S.E.M.) FIG. 9E shows immunoblots of lysates prepared from HEK-293 cells transfected with the indicated GFP and GFP-Parkin constructs and treated with CCCP (20 μM). FIG. 9F shows quantification of immunoblot signal for USP30 normalized to actin from (E). (**p<0.01, ***p<0.001 compared to wild-type GFP-Parkin, using Two-way ANOVA with Bonferroni's Multiple Comparison test. N=4 experiments. Error bars represent S.E.M.)



FIG. 10A shows immunostaining in GFP-Parkin-G430D expressing stable SH-SY5Y cells transfected with the indicated siRNAs and cDNA expression constructs. Following 3 days of expression, cells were treated with CCCP (20 μM, 24 hours), and fixed and stained for GFP, FLAG, and endogenous Tom20. Scale bar, 5 μm. FIG. 10B is a plot showing quantification of Tom20 fluorescence intensity from FIG. 10A (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test, Error bars represent SEM). FIG. 10C is a plot showing quantification of GFP-Parkin-G430D puncta area from FIG. 10A (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test, Error bars represent SEM). FIG. 10D shows mt-Keima imaging in neurons transfected with Parkin shRNA and USP30-C77A-FLAG. Scale bar, 5 μm. FIG. 10E is a plot showing quantification of mitophagy index from FIG. 10D (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. N=71-77 cells. N=3 experiments. Error bars represent SEM).



FIG. 11A shows an immunoblot for endogenous USP30 in SH-SY5Y cells transfected with USP30 siRNA for 3 days. FIGS. 11B and 11C show immunostaining in GFP-Parkin-G430D expressing stable SH-SY5Y cells transfected with the indicated siRNAs. Following 3 days of knockdown, cells were treated with CCCP (20 μM, 24 hours), and fixed and stained for GFP and endogenous Tom20. Scale bar, 5 μm. FIG. 11D is a plot showing quantification of fold change in Tom20 staining intensity from FIGS. 11B and 11C normalized to control siRNA (***p<0.001 by One-way ANOVA-Dunnett's Multiple Comparison test. Error bars represent SEM). FIGS. 11E and 11F show immunostaining in GFP-Parkin-G430D (E) and GFP-Parkin-K161N (F) expressing SH-SY5Y cells transfected with USP30 siRNA. Following 3 days of knockdown, cells were treated with CCCP (20 μM, 24 hours), and fixed and stained for GFP and endogenous Tom20 and HSP60. Scale bar, 5 μm. FIGS. 11G and 11H are plots showing quantification of fold change in Tom20 (G) and HSP60 (H) staining intensity from FIGS. 11E and 11F normalized to control siRNA. (*p<0.05, **p<0.01 and ***<0.001 by Student's t-test. N=2-3 experiments. Error bars represent S.E.M.)



FIG. 12A shows transverse sections of indirect flight muscles (IFMs) from wild-type, parkin mutant (park25) and “parkin mutant; dUSP30 knockdown” (park25; Actin-GAL4>UAS-dUSP30RNAi) flies. Electron-dense mitochondria are marked with arrowheads. Mitochondria with reduced and disorganized cristae (hence pale in appearance) are outlined with dashed lines (top panel—Scale bar, 1 μm). Higher magnification images are shown in the lower panels (Scale bar, 0.2 μm). FIGS. 12B and C show quantification of mitochondrial integrity from (A). Percent area of mitochondria containing disorganized cristae over total mitochondrial area (B), and percent of muscle area containing disorganized mitochondria (C) are blindly quantified. (*p<0.05, **p<0.01 and ***p<0.001, compared to wild-type by Two-way ANOVA-Bonferroni's Multiple Comparison test. ***p<0.001 for park25 versus park25; Actin-GAL4>UAS-dUSP30RNAi. 34-55 imaging fields per fly, N=3-4 flies. Error bars represent S.E.M.) FIGS. 12D and E show effect of dUSP30 knockdown and paraquat on climbing assay in Drosophila. Percent of flies climbing >15 cm in 30 seconds, treated with vehicle (5% sucrose) or paraquat (10 mM, 48 hours), for the indicated genotypes. (**p<0.01 and ***p<0.001 by One-Way ANOVA with Bonferroni's multiple comparisons test. N=4-10 experiments. Error bars represent S.E.M.) FIG. 12F shows dopamine neurotransmitter levels per Drosophila head for the indicated genotypes, as determined by ELISA. (*p<0.05 and ***p<0.001 by One-way ANOVA-Bonferroni's Multiple Comparison test. n=28 heads per genotype. N=4 experiments. Error bars represent S.E.M.). FIGS. 12G and H show effect of dUSP30 knockdown and paraquat on survival in Drosophila. Percent of flies still alive, treated with vehicle or paraquat (10 mM, up to 96 hours), for the indicated genotypes. (**p<0.01 and ***p<0.001 using Two-Way ANOVA with Bonferroni's multiple comparisons test. N=3 (G) and 4 (H) experiments. Error bars represent S.E.M.)



FIG. 13A and FIG. 13B shows asymmetric “volcano plot” demonstrating the subset of 41 proteins whose ubiquitination significantly increased (p<0.05) for the “Combo” treatment versus CCCP-treatment alone in both USP30 knockdown (left side) and GFP-Parkin overexpression (right side) experiments. “Combo” refers to cells treated with CCCP and expressing USP30-shRNA, or treated with CCCP and expressing GFP-Parkin, in the two experiments, respectively. For this subset of proteins, fold-increase in ubiquitination (x-axis) and the p-value (y-axis) are reported. Mitochondrial proteins (identified based on the Human MitoCarta database) are shown in red.



FIG. 14 shows inhibition of various peptidases, including USP30, by inhibitory peptides USP303 (“pep3”; SEQ ID NO: 1) and USP308 (“pep8”; SEQ ID NO: 2), as described in Example 10.



FIG. 15A and FIG. 15B shows a graph of residue probability by peptide position for USP303 and certain affinity-matured peptides, along with the signal to noise ratio (“S/N”), ELISA signal (“signal”), number of clones for each sequence (“n”), total number of clones (“total”), and the number of unique sequences (“Uniq”), as described in Example 10.



FIG. 16 shows a graph of signal to noise ratio for USP303 and three affinity matured peptides, as described in Example 10. For each peptide, the targets tested were, from left to right, USP2, USP7, USP14, USP30, UCHL1, UCHL3, and UCHL5. The sequences for each peptide are shown below.



FIG. 17A shows ratiometric mito-roGFP imaging in hippocampal neurons transfected with USP30 shRNA. The “relative oxidation index” was shown in a ‘color scale’ from 0 (mito-roGFP ratio after DTT treatment, 1 mM, shown in black) to 1 (mito-roGFP ratio after aldrithiol treatment, 100 μM, shown in red). FIG. 17B is a plot showing quantification of relative oxidation from FIG. 17A (***p<0.001 by Student's t-test. n=24 cells for luciferase shRNA and 36 cells for USP30 shRNA. N=3 experiments. Error bars represent SEM). FIG. 17C shows quantitative RT-PCR of dUSP30 mRNA. qRT-PCR in Actin-GALA UAS-dUSP30RNAi and Actin-GAL4>UAS-dUSP30RNAi flies, expressed relative to Actin-GAL4. dUSP30 mRNA levels were normalized to Drosophila RpII140 mRNA levels in each group. N=7 experiments. ***p<0.001 by One-Way ANOVA with Bonferroni's multiple comparisons test. FIG. 17D shows climbing assay in control flies (Actin-GAL4). Flies were treated with vehicle control (5% sucrose) or paraquat (10 mM, 48 hours). L-DOPA (1 mM, 48 hours) was administered simultaneously with paraquat, as indicated. (***p<0.001 by One-Way ANOVA-Dunnett's Multiple Comparison test. N=6 experiments. Error bars represent S.E.M.). FIG. 17E shows serotonin levels per fly head, as assessed by ELISA. Flies were treated with paraquat (10 mM, 48 hours) or vehicle control (5% sucrose). (p-values calculated by One-Way ANOVA-Bonferroni's Multiple Comparison test. n=8 heads, N=2 experiments. Error bars represent S.E.M.). FIGS. 17F and G show quantitative RT-PCR measurement of (F) dUSP47 and (G) dYOD1 m RNA levels in flies of the indicated genotypes, expressed as relative to Actin-GAL4 genotype. TaqMan assays Dm01795269_g1 (Drosophila CG5486 (USP47)) and Dm01840115_s1 (Drosophila CG4603 (YOD1)) were used. Dm02134593_g1 (RpII140) was used for normalization. (p**<0.01 and p***<0.001 using One-Way ANOVA-Dunnett's Multiple Comparison test. N=3 replicates. Error bars represent S.E.M.) FIGS. 17H and I show survival curves of flies of the indicated genotype, treated with vehicle or paraquat (10 mM). Graph shows percent flies alive at indicated times after feeding with paraquat. (*p<0.05, p**<0.01, and p***<0.001 using Two-Way ANOVA with Bonferroni's Multiple Comparisons test. N=5 (H) and 4 (1) experiments. Error bars represent S.E.M.)





DETAILED DESCRIPTION

The present inventors have identified USP30, a mitochondria-localized deubiquitinase (DUB) as an atagonist of Parkin-mediated mitophagy. USP30, through its deubiquitinase activity, counteracts ubiquitination and degradation of damaged mitochondria, and inhibition of USP30 rescues mitophagy defects caused by mutant Parkin. Further, USP30 inhibition of USP30 decreases oxidative stress and provides protection against the mitochondrial toxin, rotenone. Since damaged mitochondria are more likely to accumulate Parkin, USP30 inhibition should preferentially clear unhealthy mitochondria. In addition to neurons (such as substantia nigra neurons, which are especially vulnerable to mitochondria dysfunction in Parkinson's disease), long-lived metabolically active cells such as cardiomyocytes also rely on an efficient mitochondria quality control system. In this context, Parkin has been shown to protect cardiomyocytes against ischemia/reperfusion injury through activating mitophagy and clearing damaged mitochondria in response to ischemic stress. Thus, inhibitors of USP30 are provided for us in treating a conditions involving mitochondrial defects, including neurological conditions, cardiac conditions, and systemic conditions.


I. DEFINITIONS

An “inhibitor” refers to an agent capable of blocking, neutralizing, inhibiting, abrogating, reducing and/or interfering with one or more of the activities of a target and/or reducing the expression of the target protein (or the expression of nucleic acids encoding the target protein) Inhibitors include, but are not limited to, antibodies, polypeptides, peptides, nucleic acid molecules, short interfering RNAs (siRNAs) and other inhibitory RNAs, small molecules (e.g., small inorganic molecules), polysaccharides, polynucleotides, antisense oligonucleotides, aptamers, and peptibodies. An inhibitor may decrease the activity and/or expression of a target protein by at least 10% (e.g., by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% decrease) as compared to the expression and/or activity of the target protein that is untreated with the inhibitor.


An “inhibitor of USP30” refers to an agent capable of blocking, neutralizing, inhibiting, abrogating, reducing and/or interfering with one or more of the activities of USP30 and/or reducing the expression of USP30 (or the expression of nucleic acids encoding USP30). In some embodiments, an inhibitor of USP30 reduces the deubiquitinase activity of USP30. In some embodiments, an inhibitor of USP30 reduces deubiquitinase activity by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 100%. Deubiquitinase activity may be reduced by an inhibitor by any mechanism, including, but not limited to, interfering with the active site of USP30, interfering with target recognition, altering the conformation of USP30, interfering with proper subcellular localization of USP30, etc. In some embodiments, an inhibitor of USP30 inhibits USP30 expression, which may be expression as the mRNA (e.g., it inhibits transcription of the USP30 gene to produce USP30 mRNA) and/or protein level (e.g., it inhibits translation of the USP30 mRNA to produce USP30 protein). In some embodiments, an inhibitor of USP30 expression reduces the level of USP30 protein by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or 100%.


The terms “mitophagy” and “mitochondrial degradation” are used interchangeably to refer to the regulated degradation of mitochondria through the lysosomal machinery of a cell.


A “condition involving a mitochondrial defect” refers to a condition involving a defect or defects in mitochondrial function, mitochondrial shape/morphology, mitochondrial membrane potential, and/or mitophagy in a cell population. Conditions involving a mitochondrial defect include, but are not limited to, conditions involving a defect in mitophagy, such that mitophagy occurs in the cell population at a slower rate or to a lesser extent than in a normal cell population. In some embodiments, the defect in mitophagy is accompanied by other mitochondrial defects such that the decreased mitophagy results in the increased presence of defective mitochondria. Conditions involving a mitochondrial defect also include, but are not limited to, conditions involving mutations in mitochondrial DNA that result in altered mitochondrial function. Conditions involving a mitochondrial defect also include conditions involving mitochondrial oxidative stress, in which increased levels of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) in a cell are associated with protein aggregation and/or mitochondrial dysfunction. Mitochondrial oxidative stress may result in mitochondrial dysfunction, or mitochondrial dysfunction may result in oxidative stress. Conditions involving a mitochondrial defect also include, but are not limited to, conditions involving defects in mitochondrial shape/morphology and conditions involving defects in mitochondrial membrane potential. Exemplary conditions involving mitochondrial defects include, but are not limited to, neurodegenerative diseases (such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia); mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome; Leber's hereditary optic neuropathy (LHON); neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS); Danon disease; myoclonic epilepsy with ragged red fibers (MERFF) syndrome; ischemic heart disease leading to myocardial infarction; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrongenase deficiency (LCHAD); Leigh disease or syndrome; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrongenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial recessive ataxia syndrome; mitochondrial cytopathy; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; and very long-chain acyl-CoA dehydrongenase (VLCAD) deficiency.


A “pathogenic mutation” in Parkin or PINK1 refers to a mutation or mutations in the respective protein or gene that results in reduced activity in a cell, and may involve loss of function and/or gain of function (such as dominant negative mutations, for example, Parkin Q311stop). Such reduced activity in a cell may include, but is not limited to, reduced enzymatic activity (such as reduced ubiquitination or kinase activity), reduced activity due to the presence of a dominant negative mutant protein, reduced binding to another cellular factor, reduced activity due to subcellular localization changes, and/or reduced activity due to reduced levels of protein in the cell or in a cellular compartment. In some embodiments, a pathogenic mutation in Parkin and/or PINK1 results in reduced ubiquitination of mitochondria, which may result in reduced mitophagy. Pathogenic mutations may also occur outside of the coding region of the protein, e.g., in an intron (affecting, for example, splicing), the promoter, the 5′ untranslated region, the 3′ untranslated region, etc. Further, Parkin mutations may involve substitutions, deletions, insertions, duplications, etc., or any combination of those. Nonlimiting exemplary pathogenic mutations in Parkin are shown in Table 1. Nonlimiting exemplary pathogenic mutations in PINK1 protein are shown in Table 2. Databases of Parkinson's disease mutations are publicly available, such as Parkinson Disease Mutation Database, http://www.molgen.ua.ac.be/PDmutDB/.









TABLE 1





Exemplary pathogenic mutations in Parkin (PARK2)


















Ala291fs
ex10del
ex4-7del
Gln311Stop


Ala31Asp
ex10dup
ex4del
Gln34fs


Ala398Thr
ex11del
ex4dup
Gln34fs


Arg234Gln
ex11dup
ex5-12del
Gln40Stop


Arg334Cys
ex12dup
ex5-6del
Glu395Stop


Arg33Gln
ex1-4del
ex5-7del
Glu409Stop


Arg33Stop
ex1del
ex5-8dup
Glu444Gln


Arg348fs
ex1dup
ex5-9dup
Glu79Stop


Arg366Trp
ex2-3del
ex5del
Gly179fs


Arg392fs
ex2-3dup
ex5dup
Gly328Glu


Arg42His
ex2-4del
ex6-7del
Gly359Asp


Arg42Pro
ex2-4dup
ex6-8dup
Gly429Glu


Asn428fs
ex2-4trip
ex6del
Gly430Asp


Asn52fs
ex2-5del
ex6dup
IVS1 + 1G > A


Asp280Asn
ex2del
ex7-8del
IVS11 − 3C > G


Asp460fs
ex2dup
ex7-9del
Leu283Pro


Asp53Stop
ex2trip
ex7del
Lys161Asn


c. − 39G > T
ex3-4del
ex7dup
Lys211Asn


Cys212Gly
ex3-4dup
ex8-10del
Lys349fs


Cys212Tyr
ex3-5del
ex8-11del
Met192Leu


Cys238fs
ex3-6del
ex8-9del
Met192Val


Cys268Stop
ex3-7del
ex8del
Met1Leu


Cys289Gly
ex3-9del
ex8dup
partial ex4del


Cys323fs
ex3del
ex9del
Pro113fs/ex3 Δ40 bp


Cys431Phe
ex3dup
ex9dup
Pro133del


ex10-12del
ex4-5del
Gln171Stop
Thr240Arg


ex10-12dup
ex4-6del
Gln311His
Thr240Met


Val56Glu
Val258Met
Trp453Stop
Thr351Pro


prom + ex1del
Val324fs
Trp74fs
Thr415Asn





del = deletion; dup = duplication; fs = frameshift; ex = exon; IVS = intervening sequence; prom = promoter













TABLE 2





Exemplary pathogenic mutations in PINK1




















Tyr258Stop
IVS7 + 1G > A
ex6-8del
Asp297fs



Trp437Stop
Gly440Glu
ex4-8del
Arg492Stop



Thr313Met
Glu239Stop
ex3-8del
Arg464His



Stop582Leu
Gln456Stop
delPINK1
Arg246Stop



Pro196fs
Gln129Stop
Cys92Phe
Ala168Pro



Lys520fs
Gln129fs
Cys549fs
23 bp del ex7



Lys24fs
ex7del
Asp525fs







del = deletion; fs = frameshift; ex = exon






The term “oxidative stress” refers to an increase in reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) in a cell. In some embodiments, oxidative stress leads to protein aggregation and/or mitochondrial dysfunction. In some embodiments, mitochondrial dysfunction leads to oxidative stress.


The term “USP30,” as used herein, refers to any native USP30 (“ubiquitin specific peptidase 30” or “ubiquitin specific protease 30”) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed USP30 as well as any form of USP30 that results from processing in the cell. The term also encompasses naturally occurring variants of USP30, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human USP30 is shown in SEQ ID NO: 26 (Table 4).


The term “Parkin” as used herein, refers to any native Parkin from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed Parkin as well as any form of Parkin that results from processing in the cell. The term also encompasses naturally occurring variants of Parkin, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Parkin is shown in SEQ ID NO: 29 (Table 4).


The term “PINK1” as used herein, refers to any native PINK1 (PTEN-induced putative kinase protein 1) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PINK1 as well as any form of PINK1 that results from processing in the cell. The term also encompasses naturally occurring variants of PINK1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human PINK1 is shown in SEQ ID NO: 30 (Table 4).


The term “Tom20” as used herein, refers to any native Tom20 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed Tom20 as well as any form of Tom20 that results from processing in the cell. The term also encompasses naturally occurring variants of Tom20, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human Tom20 is shown in SEQ ID NO: 27 (Table 4).


The terms “MIRO1” and “MIRO” as used herein, refer to any native MIRO1 (mitochondrial Rho GTPase 1) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed MIRO1 as well as any form of MIRO1 that results from processing in the cell. The term also encompasses naturally occurring variants of MIRO1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human MIRO1 is shown in SEQ ID NO: 28 (Table 4).


The term “MUL1” as used herein, refers to any native MUL1 (mitochondrial ubiquitin ligase activator of NFκB) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed MUL1 as well as any form of MUL1 that results from processing in the cell. The term also encompasses naturally occurring variants of MUL1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human MUL1 is shown in SEQ ID NO: 32 (Table 4).


The term “ASNS” as used herein, refers to any native ASNS (asparagine synthetase [glutamine hydrolyzing]) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ASNS as well as any form of ASNS that results from processing in the cell. The term also encompasses naturally occurring variants of ASNS, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human ASNS is shown in SEQ ID NO: 33 (Table 4).


The term “FKBP8” as used herein, refers to any native FKBP8 (FK506 binding protein 8) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ASNS as well as any form of FKBP8 that results from processing in the cell. The term also encompasses naturally occurring variants of FKBP8, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human FKBP8 is shown in SEQ ID NO: 34 (Table 4).


The term “TOM70” as used herein, refers to any native TOM70 (translocase of outer membrane 70 kDa subunit) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TOM70 as well as any form of TOM70 that results from processing in the cell. The term also encompasses naturally occurring variants of TOM70, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human TOM70 is shown in SEQ ID NO: 35 (Table 4).


The term “MAT2B” as used herein, refers to any native MAT2B (methionine adenosyltransferase 2 subunit beta) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed MAT2B as well as any form of MAT2B that results from processing in the cell. The term also encompasses naturally occurring variants of MAT2B, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human MAT2B is shown in SEQ ID NO: 36 (Table 4).


The term “PRDX3” as used herein, refers to any native PRDX3 (peroxiredoxin III) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PRDX3 as well as any form of PRDX3 that results from processing in the cell. The term also encompasses naturally occurring variants of PRDX3, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human PRDX3 is shown in SEQ ID NO: 37 (Table 4).


The term “IDE” as used herein, refers to any native IDE (insulin degrading enzyme) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed IDE as well as any form of IDE that results from processing in the cell. The term also encompasses naturally occurring variants of IDE, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IDE is shown in SEQ ID NO: 38 (Table 4).


The term “VDAC1” as used herein, refers to any native VDAC1 (voltage-dependent anion selective channel protein 1) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed VDAC1 as well as any form of VDAC1 that results from processing in the cell. The term also encompasses naturally occurring variants of VDAC1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human VDAC1 is shown in SEQ ID NO: 39 (Table 4).


The term “VDAC2” as used herein, refers to any native VDAC2 (voltage-dependent anion selective channel protein 2) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed VDAC2 as well as any form of VDAC2 that results from processing in the cell. The term also encompasses naturally occurring variants of VDAC2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human VDAC2 is shown in SEQ ID NO: 44 (Table 4).


The term “VDAC3” as used herein, refers to any native VDAC3 (voltage-dependent anion selective channel protein 3) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed VDAC3 as well as any form of VDAC3 that results from processing in the cell. The term also encompasses naturally occurring variants of VDAC3, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human VDAC3 is shown in SEQ ID NO: 45 (Table 4).


The term “IP05” as used herein, refers to any native IP05 (importin 5) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed IP05 as well as any form of IP05 that results from processing in the cell. The term also encompasses naturally occurring variants of IP05, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IP05 is shown in SEQ ID NO: 40 (Table 4).


The term “PTH2” as used herein, refers to any native PTH2 (peptidyl-tRNA hydrolase 2, mitochondrial) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PTH2 as well as any form of PTH2 that results from processing in the cell. The term also encompasses naturally occurring variants of PTH2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human PTH2 is shown in SEQ ID NO: 41 (Table 4).


The term “PSD13” as used herein, refers to any native PSD13 (26S proteasome non-ATPase regulatory subunit 13) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PSD13 as well as any form of PSD13 that results from processing in the cell. The term also encompasses naturally occurring variants of PSD13, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human PSD13 is shown in SEQ ID NO: 42 (Table 4).


The term “UBP13” as used herein, refers to any native UBP13 (ubiquitin carboxyl-terminal hydrolase 13) from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed UBP13 as well as any form of UBP13 that results from processing in the cell. The term also encompasses naturally occurring variants of UBP13, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human UBP13 is shown in SEQ ID NO: 43 (Table 4).


The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.


An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.


“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.


In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:





100 times the fraction X/Y


where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.


The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.


A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.


As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.


Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration, in any order.


An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.


The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”


The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.


II. COMPOSITIONS AND METHODS

In various aspects, the invention is based, in part, on inhibitors of USP30 and methods of treating diseases and disorders comprising inhibiting USP30.


A. Exemplary Inhibitors of USP30


The present invention is based in part on the discovery that inhibitors of USP30 activity and/or expression are effective for increasing and/or restoring mitochondrial ubiquitination and mitophagy. In some embodiments, inhibitors of USP30 are effective for treating neurodegenerative diseases, such as Parkinson's disease, as well as conditions that involve mitochondrial defects, such as those involving mitophagy defects, mutations in mitochondrial DNA, mitochondrial oxidative stress, and/or lysosomal storage defects.


Inhibitors of USP30 include inhibitors of USP30 activity and inhibitors of USP30 expression. Nonlimiting exemplary such inhibitors include antisense oligonucleotides, short interfering RNAs (siRNAs), antibodies, peptides, peptibodies, aptamers, and small molecules. In some embodiments, antisense oligonucleotides or short interfering RNAs (siRNAs) may be used to inhibit USP30 expression. In some embodiments, antibodies, peptides, peptibodies, aptamers, and small molecules may be used to inhibit USP30 activity. Some nonlimiting examples of inhibitors of USP30 are described herein. Further inhibitors can be identified using standard methods in the art, including those discussed herein.


Antisense Oligonucleotides


In some embodiments, antisense oligonucleotides that hybridize to USP30 mRNA and/or USP30 pre-mRNA are provided. A nonlimiting exemplary human mRNA sequence encoding USP30 is shown in SEQ ID NO: 30 (Table 4). In some embodiments, an antisense oligonucleotide hybridizes to a region of USP30 mRNA and/or USP30 pre-mRNA and directs its degradation through RNase H, which cleaves double-stranded RNA/DNA hybrids. By mediating cleavage of USP30 mRNA and/or USP30 pre-mRNA, an antisense oligonucleotide may reduce the amount of USP30 protein in a cell (i.e., may inhibit expression of USP30). In some embodiments, an antisense oligonucleotide does not mediate degradation through RNase H, but rather “blocks” translation of the mRNA, e.g., through interference with translational machinery binding or processivity, or “blocks” proper splicing of the pre-mRNA, e.g., through interference with the splicing machinery and/or accessibility of a splice site. In some embodiments, an antisense oligonucleotide may mediate degradation of an mRNA and/or pre-mRNA through a mechanism other than RNase H Any inhibitory mechanism of an antisense oligonucleotide is contemplated herein.


In some embodiments, an antisense oligonucleotide is 10 to 500 nucleotides long, or 10 to 400 nucleotides long, or 10 to 300 nucleotides long, or 10 to 200 nucleotides long, or 10 to 100 nucleotides long, or 15 to 100 nucleotides long, or 10 to 50 nucleotides long, or 15 to 50 nucleotides long. In various embodiments, an antisense oligonucleotide hybridizes to a region of the USP30 mRNA and/or pre-mRNA comprising at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. Further, in various embodiments, an antisense oligonucleotide need not be 100% complementary to a region USP30 mRNA and/or a region of USP30 pre-mRNA, but may have 1 or more mismatches. Thus, in some embodiments, an antisense oligonucleotide is at least 80% complementary, at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to a region of USP30 mRNA and/or a region of USP30 pre-mRNA. In some embodiments, the region of USP30 mRNA or the region of USP pre-mRNA is at least at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides long.


Antisense oligonucleotides may comprise modifications to one or more of the internucleoside linkages, sugar moieties, and/or nucleobases. Further, the sequence of nucleotides may be interrupted by non-nucleotide components, and/or non-nucleotide components may be attached at one or both ends of the oligonucleotide.


Nonlimiting exemplary nucleotide modifications include sugar modifications, in which any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Oligonucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-2′-0-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by modified internucleoside linkages. These modified internucleoside linkages include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all oligonucleotides referred to herein, including antisense oligonucleotides and siRNA.


In some embodiments, one or more internucleoside linkages in an antisense oligonucleotide are phosphorothioates. In some embodiments, one or more sugar moieties in an antisense oligonucleotide comprise 2′ modifications, such as 2′-O-alkyl (such as 2′-OMe) and 2′-fluoro; or are bicyclic sugar moieties (such as LNA). Nonlimiting exemplary nucleobase modifications include 5-methylcytosine. An antisense oligonucleotide may comprise more than one type of modification within a single oligonucleotide. That is, as a nonlimiting example, an antisense oligonucleotide may comprise 2′-O alkyl modifications, bicyclic nucleotides, and phosphorothioate linkages in the same oligonucleotide. In some embodiments, an antisense oligonucleotide is a “gapmer.” Gapmers comprise a central region of deoxyribonucleotides for mediating RNase H cleavage, and 5′ and 3′ “wings” comprising modified sugar moieties that increase the stability of the duplex.


Antisense oligonucleotide design and mechanisms are described, e.g., in van Roon-Mom et al., Methods Mol. Biol., 867: 79-96 (20120); Prakash, Chem. Biodivers., 8: 1616-1641 (2011); Yamamoto et al., Future Med. Chem., 3: 339-365 (2011); Chan et al., Clin. Exper. Pharmacol. Physiol., 33: 533-540 (2006); Kurreck et al., Nucl. Acids Res., 30: 1911-1918 (2002); Kurreck, Eur. J. Biochem., 270: 1628-1644 (2003); Geary, Expert Opin. Drug Metab. Toxicol., 5: 381-391 (2009); “Designing Antisense Oligonucleotides,” available online from Integrated DNA Technologies (2011).


Short Interfering RNAs (siRNAs)


In some embodiments, the expression of USP30 is inhibited with a short interfering RNA (siRNA). As used herein, siRNAs are synonymous with double-stranded RNA (dsRNA) and include double-stranded RNA oligomers with or without hairpin structures at each end (also referred to as small hairpin RNA, or shRNA). Short interfering RNAs are also known as small interfering RNAs, silencing RNAs, short inhibitory RNA, and/or small inhibitory RNAs, and these terms are considered to be equivalent herein.


The term “short-interfering RNA (siRNA)” refers to small double-stranded RNAs that interfere with gene expression. siRNAs are mediators of RNA interference, the process by which double-stranded RNA silences homologous genes. In some embodiments, siRNAs are comprised of two single-stranded RNAs of about 15-25 nucleotides in length that form a duplex, which may include single-stranded overhang(s). In some embodiments, siRNAs are comprised of a single RNA that forms a hairpin structure that includes a double-stranded portion that may be 15-25 nucleotides in length and may include a single-stranded overhang. Such hairpin siRNAs may be referred to as a short hairpin RNA (shRNA). Processing of the double-stranded RNA by an enzymatic complex, for example, polymerases, may result in cleavage of the double-stranded RNA to produce siRNAs. The antisense strand of the siRNA is used by an RNA interference (RNAi) silencing complex to guide mRNA cleavage, thereby promoting mRNA degradation. To silence a specific gene using siRNAs, for example, in a mammalian cell, a base pairing region is selected to avoid chance complementarity to an unrelated mRNA. RNAi silencing complexes have been identified in the art, such as, for example, by Fire et al., Nature 391:806-811, 1998, and McManus et al., Nat. Rev. Genet. 3(10):737-747, 2002.


In some embodiments, small interfering RNAs comprise at least about 10 to about 200 nucleotides, including at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, or greater than 150 nucleotides. In some embodiments, an siRNA is 10 to 200 nucleotides long, or 10 to 100 nucleotides long, or 15 to 100 nucleotides long, or 10 to 60 nucleotides long, or 15 to 60 nucleotides long, or 10 to 50 nucleotides long, or 15 to 50 nucleotides long, or 10 to 30 nucleotides long, or 15 to 30 nucleotides long. In certain embodiments, the siRNA comprises an oligonucleotide from about 21 to about 25 nucleotides in length. In some embodiments, the siRNA molecule is a heteroduplex of RNA and DNA.


As with antisense oligonucleotides, siRNAs can include modifications to the sugar, internucleoside linkages, and/or nucleobases. Nonlimiting exemplary modifications suitable for use in siRNAs are described herein and also, e.g., in Peacock et al., J. Org. Chem., 76: 7295-7300 (2011); Bramsen et al., Methods Mol. Biol., 721: 77-103 (2011); Pasternak et al., Org. Biomol. Chem., 9: 3591-3597 (2011); Gaglione et al., Mini Rev. Med. Chem., 10: 578-595 (2010); Chernolovskaya et al., Curr. Opin. Mol. Ther., 12: 158-167 (2010).


A process for inhibiting expression of USP30 in a cell comprises introduction of an siRNA with partial or fully double-stranded character into the cell. In some embodiments, an siRNA comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a nucleotide sequence found in the USP30 gene coding region or pre-mRNA.


In some embodiments, an siRNA specific to the USP30 gene is synthesized and introduced directly into a subject. In other embodiments, the siRNA can be formulated as part of a targeted delivery system, such as a target specific liposome, which specifically recognizes and delivers the siRNA to an appropriate tissue or cell type. Upon administration of the targeted siRNA to a subject, the siRNA is delivered to the appropriate cell type, thereby increasing the concentration siRNA within the cell type. Depending on the dose of siRNA delivered, this process can provide partial or complete loss of USP30 protein expression.


In other embodiments, an appropriate cell or tissue is provided with an expression construct that comprises a nucleic acid encoding one or both strands of an siRNA that is specific to the USP30 gene. In these embodiments, the nucleic acid that encodes one or both strands of the siRNA can be placed under the control of either a constitutive or a regulatable promoter. In some embodiments, the nucleic acid encodes an siRNA that forms a hairpin structure, e.g., a shRNA.


Various carriers and drug-delivery systems for siRNAs are described, e.g., in Seth et al., Ther. Deliv., 3: 245-261 (2012); Kanasty et al., Mol. Ther., 20: 513-524 (2012); Methods Enzymol., 502: 91-122 (2012); Vader et al., Curr. Top. Med. Chem., 12: 108-119 (2012); Naeye et al., Curr. Top. Med. Chem., 12: 89-96 (2012); Foged, Curr. Top. Med. Chem., 12: 97-107 (2012); Chaturvedi et al., Expert Opin. Drug Deliv., 8: 1455-1468 (2011); Gao et al., Int. J. Nanomed., 6: 1017-1025 (2011); Shegokar et al., Pharmazie., 66: 313-318 (2011); Kumari et al., Expert Opin. Drug Deliv., 11: 1327-1339 (2011).


Antibodies


In some embodiments, an inhibitor of USP30 is an antibody. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. The term “antibody” as used herein refers to a molecule comprising at least complementarity-determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and at least CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to antigen. The term antibody includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′)2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.


In some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region, one or both of which may or may not comprise a respective constant region. A heavy chain variable region comprises heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a heavy chain variable region also comprises at least a portion of an FR1, which is N-terminal to CDR1, and/or at least a portion of an FR4, which is C-terminal to CDR3. Similarly, a light chain variable region comprises light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, a light chain variable region also comprises an FR1 and/or an FR4.


Nonlimiting exemplary heavy chain constant regions include γ, δ, and α. Nonlimiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an a constant region is an IgA antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region) antibodies. Nonlimiting exemplary light chain constant regions include λ and κ.


In some embodiments, an antibody is a chimeric antibody, which comprises at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, chicken, etc.). The human constant region of a chimeric antibody need not be of the same isotype as the non-human constant region, if any, it replaces. Chimeric antibodies are discussed, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984).


In some embodiments, an antibody is a humanized antibody, in which at least one amino acid in a framework region of a non-human variable region (such as mouse, rat, cynomolgus monkey, chicken, etc.) has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region or fragment thereof. In some embodiments, a humanized antibody is an Fab, an scFv, a (Fab′)2, etc. Exemplary humanized antibodies include CDR-grafted antibodies, in which the complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species. Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic. An antibody may be humanized by any method. Nonlimiting exemplary methods of humanization include methods described, e.g., in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-27 (1988); Verhoeyen et al., Science 239: 1534-36 (1988); and U.S. Publication No. US 2009/0136500.


In some embodiments, an antibody is a human antibody, such as an antibody produced in a non-human animal that comprises human immunoglobulin genes, such as XenoMouse®, and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences. Transgenic mice that comprise human immunoglobulin loci and their use in making human antibodies are described, e.g., in Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S. Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806. Methods of making human antibodies using phage display libraries are described, e.g., in Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); and PCT Publication No. WO 99/10494.


The choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo. Such effector function, in some embodiments, includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), and can result in killing of the cell to which the antibody is bound. Typically, antibodies comprising human IgG1 or IgG3 heavy chains have effector function. In some embodiments, effector function is not desirable. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region may be selected or engineered.


Peptides


In some embodiments, an inhibitor of USP30 is a peptide. A peptide is a sequence of amino acids of made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. The amino acid subunits of the peptide may be naturally-occurring amino acids or may be non-naturally occurring amino acids. Many non-naturally occurring amino acids are known in the art and are available commercially. Further, the peptide bonds joining the amino acid subunits may be modified. See, e.g., Sigma-Aldrich; Gentilucci et al., Curr. Pharm. Des. 16: 3185-3203 (2010); US 2008/0318838. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length. In various embodiments, peptide inhibitors may comprise or consist of between 3 and 50, between 5 and 50, between 10 and 50, between 10 and 40, between 10 and 35, between 10 and 30, or between 10 and 25 amino acids. In various embodiments, peptide inhibitors may comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In various embodiments, peptide inhibitors may consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.


Methods of developing peptides that specifically bind a target molecule are known in the art, including phage display methods. See, e.g., U.S. Pat. No. 5,010,175; WO 1996/023899; WO 1998/015833; Bratkovic, Cell. Mol. Life Sci., 67: 749-767 (2010); Pande et al., Biotech. Adv. 28: 849-858 (2010). In some embodiments, following selection of a peptide, the peptide may be modified, e.g., by incorporating non-natural amino acids and/or peptide bonds. A nonlimiting exemplary method of selecting a peptide inhibitor of USP30 is described herein.


Amino acids that are important for peptide inhibition may be determined, in some embodiments, by alanine scanning mutagenesis. Each residue is replaced in turn with a single amino acid, typically alanine, and the effect on USP30 inhibition is assessed. See, e.g., U.S. Pat. Nos. 5,580,723 and 5,834,250. Truncation analyses may also be used to determine not only the importance of the amino acids at the ends of a peptide, but also the importance of the length of the peptide, on inhibitory activity. In some instances, truncation analysis may reveal a shorter peptide that binds more tightly than the parent peptide. The results of various mutational analyses, such as alanine scanning mutagenesis and truncation analyses, may be used to inform further modifications of an inhibitor peptide.


Nonlimiting exemplary peptide inhibitors are described herein, e.g., in Example 10 and FIG. 15. One skilled in the art will appreciate that, in some embodiments, the peptide sequences described herein may be modified in order to generate further peptide inhibitors with desirable properties, such as improved specificity for USP30, stronger binding to USP30, improved solubility, and/or improved cell membrane permeability. In some embodiments, a peptide inhibitor of USP30 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a sequence selected from SEQ ID NOs: 1 to 22.


In some embodiments, a peptide inhibitor comprises the amino acid sequence:












X1X2CX3X4X5X6X7X8X9X10X11CX12
(SEQ ID NO: 48)







wherein:


X1 is selected from L, M, A, S, and V;


X2 is selected from Y, D, E, I, L, N, and S;


X3 is selected from F, I, and Y;


X4 is selected from F, I, and Y;


X5 is selected from D and E;


X6 is selected from L, M, V, and P;


X7 is selected from S, N, D, A, and T;


X8 is selected from Y, D, F, N, and W;


X9 is selected from G, D, and E;


X10 is selected from Y and F;


X11 is selected from L, V, M, Q, and W; and


X12 is selected from F, L, C, V, and Y;


In some embodiments, the peptide inhibits USP30 with an IC50 of less than 10 μM. In some embodiments, X1 is selected from L and M. In some embodiments, X3 is selected from Y and D. In some embodiments, X3 is F. In some embodiments, X4 is selected from Y and F. In some embodiments, X4 is Y. In some embodiments, X5 is D. In some embodiments, X6 is selected from L and M. In some embodiments, X7 is selected from S, N, and D. In some embodiments, X8 is Y. In some embodiments, X9 is G. In some embodiments, X10 is Y. In some embodiments, X11 is L. In some embodiments, X12 is selected from F and L. In some embodiments, X12 is F.


In some embodiments, a peptide inhibitor comprises the amino acid sequence:











(SEQ ID NO: 49)



XAX1X2CX3X4X5X6X7X8X9X10X11CX12XB







wherein X1 to X12 are as defined above, and XA and XB are each independently any amino acid. In some embodiments, XA is selected from S, A, T, E Q, D, and R. In some embodiments, XB is selected from D, Y, E, H, S, and I.


Peptibodies


In some embodiments, an inhibitor of USP30 is a peptibody. A peptibody is peptide sequence linked to vehicle. In some embodiments, the vehicle portion of the peptibody reduces degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, and/or increases biological activity of the peptide. In some embodiments, the vehicle portion of the peptibody is an antibody Fc domain. Other vehicles include linear polymers (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); branched-chain polymers (see, e.g., U.S. Pat. No. 4,289,872 and U.S. Pat. No. 5,229,490; WO 1993/0021259); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide, or peptide vehicle. The peptide portion of the peptibody typically binds to the target, e.g., USP30. In some embodiments, the peptide portion of the peptibody is a peptide described herein.


In some embodiments, peptibodies retain certain desirable characteristics of antibodies, such as a long lifetime in plasma and increased affinity for binding partners (for example, due to the dimerization of Fc domains). The production of peptibodies is generally described, e.g., in WO 2000/0024782 and U.S. Pat. No. 6,660,843.


Aptamers


In some embodiments, an inhibitor of USP30 is an aptamer. The term “aptamer” as used herein refers to a nucleic acid molecule that specifically binds to a target molecule, such as USP30. Aptamers can be selected to be highly specific, relatively small in size, and/or non-immunogenic. See, e.g., Ni, et al., Curr. Med. Chem. 18: 4206 (2011). In some embodiments, a aptamer is a small RNA, DNA, or mixed RNA/DNA molecule that forms a secondary and/or tertiary structure capable of specifically binding and inhibiting USP30.


In some embodiments, an aptamer includes one or more modified nucleosides (e.g., nucleosides with modified sugars, modified nucleobases, and/or modified internucleoside linkages), for example, that increase stability in vivo, increase target affinity, increase solubility, increase serum half-life, increase resistance to degradation, and/or increase membrane permeability, etc. In some embodiments, aptamers comprise one or more modified or inverted nucleotides at their termini to prevent terminal degradation, e.g., by an exonuclease.


The generation and therapeutic use of aptamers are well established in the art. See, e.g., U.S. Pat. No. 5,475,096. In some embodiments, aptamers are produced by systematic evolution of ligands by exponential enrichment (SELEX), e.g., as described in Ellington et al., Nature 346: 818 (1990); and Tuerk et al., Science 249: 505 (1990). In some embodiments, aptamers are produced by an AptaBid method, e.g., as described in Berezovski et al., J. Am. Chem. Soc. 130: 913 (2008). Slow off-rate aptamers and methods of selecting such aptamers are described, e.g., in Brody et al., Expert Rev. Mol. Diagn., 10: 1013-22 (2010); and U.S. Pat. No. 7,964,356.


Small Molecules


In some embodiments, small molecule inhibitors of USP30 are provided. In some embodiments, a small molecule inhibitor of USP30 binds to USP30 and inhibits USP30 enzymatic activity (e.g., peptidase activity) and/or interferes with USP30 target binding and/or alters USP30 conformation such that the efficiency of enzymatic activity or target binding is reduced.


A “small molecule” is defined herein to have a molecular weight below about 1000 Daltons, for example, below about 900 Daltons, below about 800 Daltons, below about 700 Daltons, below about 600 Daltons, or below about 500 Daltons. Small molecules may be organic or inorganic, and may be isolated from, for example, compound libraries or natural sources, or may be obtained by derivatization of known compounds.


In some embodiments, a small molecule inhibitor of USP30 is identified by screening a library of small molecules. The generation and screening of small molecule libraries is well known in the art. See, e.g., Thompson et al., Chem. Rev. 96: 555-600 (1996); and the National Institutes of Health Molecular Libraries Program. A combinatorial chemical library, for example, may be formed by mixing a set of chemical building blocks in various combinations, and may result in millions of chemical compounds. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks theoretically results in the synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds. See, e.g., Gallop et al. 1994, J. Med. Chem. 37: 1233-1250). Various other types of small molecule libraries may also be designed and used, such as, for example, natural product libraries. Small molecule libraries can be obtained from various commercial vendors. See, e.g., ChemBridge, Enzo Life Sciences, Sigma-Aldrich, AMRI Global, etc.


To identify a small molecule inhibitor of USP30, in some embodiments, a small molecule library may be screened using an assay described herein. In some embodiments, the characteristics of each small molecule that inhibits USP30 are considered in order to identify features common to the small molecule inhibitors, which may be used to inform further modifications of the small molecules.


In some embodiments, one or more small molecule inhibitors of USP30 identified, for example, in an initial library screen, may be used to generate a subsequent library comprising modifications of the initial small molecule inhibitors. Using this method, subsequent iterations of candidate compounds may be developed that possess greater specificity for USP30 (versus other DUBs), and/or greater binding affinity for USP30, and/or other desirable properties, such as low toxicity, greater solubility, greater cell permeability, etc.


Various small molecule inhibitors of deubiquitylating enzymes are known in the art, some of which are shown in Table 3.









TABLE 3







Inhibitors of ubiquitin specific proteases










Name
Structure
Target
Reference





HBX 41, 108


embedded image


USP7
Colland et al., Mol. CancerTherap., 8:2286 (2009)





HBX 90, 397


embedded image


USP8
WO 2007/017758;





IU1


embedded image


USP14
Lee et al., Nature, 467: 179-184 (2010)





PR619


embedded image


Broad specificity DUB inhibitor
Tian et al., Assay DrugDevelop.Technol., 9: 165-173 (2011)





Isatin O-acyl oxime


embedded image


UCH-L1
Liu et al., Chemistry &Biology, 10: 837-846 (2003)





Isatin derivative


embedded image


UCH-L3
Liu et al., Neurobiol.Disease, 41: 318-328 (2010); Koharudin et al., PNAS, 107: 6835- 6840 (2010)





PGA1


embedded image


Ubiquitin isopeptidase
Mullally et al., J. Biol. Chem., 276: 30366-73 (2001)





PGA2


embedded image


Ubiquitin isopeptidase
Mullally et al., J. Biol. Chem., 276: 30366-73 (2001)





Δ12-PGJ2


embedded image


Ubiquitin isopeptidase
Mullally et al., J. Biol. Chem., 276: 30366-73 (2001)





Dibenzylidene- acetone (DBA)


embedded image


Ubiquitin isopeptidase
WO 2004/009023





Curcumin


embedded image


Ubiquitin isopeptidase
WO 2004/009023





Shikoccin (NSC-302979)


embedded image


Ubiquitin isopeptidase
WO 2004/009023









The inhibitors shown in Table 3 and the references cited therein, as well as additional inhibitors known in the art, can form the basis for developing additional deubiquitylation enzyme inhibitors, including specific inhibitors of USP30. See also WO 2007/009715. One skilled in the art can, for example, make modifications to any of the above structures to form a library of putative deubiquitylation enzyme inhibitors and screen for modified compounds with specificity for USP30 using the assays described herein.


B. Assays


Various assays may be used to identify and test inhibitors of USP30. For inhibitors that reduce expression of USP30 protein, any assay that detects protein levels may be suitable for measuring inhibition. As an example, protein levels can be detected by various immunoassays using antibodies that bind USP30, such as ELISA, Western blotting, immunohistochemistry, etc. If an inhibitor affects the subcellular localization of USP30, changes in subcellular localization may be detected, e.g., by immunohistochemistry, or by fractionating cellular components and detecting levels of USP30 in the various fractions using one or more antibodies. For inhibitors that reduce levels of USP30 mRNA, amplification-based assays, such as reverse transcriptase PCR (RT-PCR) may be used to detect changes in mRNA levels.


For inhibitors that affect USP30 enzymatic activity, a nonlimiting exemplary assay is as follows: USP30 is contacted with the inhibitor or candidate inhibitor in the presence of a USP30 substrate. Nonlimiting exemplary USP30 substrates include a Ub-β-galactosidase fusion protein (see, e.g., Quesada et al., Biochem. Biophys. Res. Commun 314:54-62 (2004)), Ub4 chains (e.g., Lys-48- and Lys-63-linked Ub), the linear product of UBIQ gene translation; the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates; ubiquitylated remnants resulting from proteasome-mediated degradation, and other small amide or ester adducts. USP30 activity (e.g., processing of Ub substrates) is measured in the presence of USP30 and the inhibitor or candidate inhibitor. This activity is compared with the processing of Ub substrates in the presence USP30 without the inhibitor or candidate inhibitor. If the inhibitor or candidate inhibitor inhibits the activity of USP30, the amount of UB substrate processing will decrease compared to the amount of UB substrate processing in the presence of USP30 without the inhibitor or candidate inhibitor.


A further nonlimiting exemplary assay to determine inhibition and/or specificity is described in Example 10. Briefly, a range of concentrations of inhibitor are mixed with ubiquitin-AMC and USP30 (the inhibitor may be mixed with the substrate, and then USP30 added to start the reaction). If specificity is to be determined, similar reactions may be set up with one or more additional DUBs or ubiquitin C-terminal hydrolases (UCHs) in place of USP30. Immediately after addition of the enzyme, fluorescence is monitored (with excitation at 340 nm and emission at 465 nm). The initial rate of enzymatic activity may be calculated as described in Example 10.


C. Pharmaceutical Formulations


Pharmaceutical formulations of an inhibitor of USP30 as described herein are prepared by mixing such inhibitor having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.


The formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.


Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).


Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the inhibitor, which matrices are in the form of shaped articles, e.g. films, or microcapsules.


The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.


D. Therapeutic Methods and Compositions


Any of the inhibitors of USP30 provided herein may be used in methods, e.g., therapeutic methods. In some embodiments, a method of increasing mitophagy in a cell is provided, the method comprising contacting the cell with an inhibitor of USP30 under conditions allowing inhibition of USP30 in the cell. In some embodiments, a method of increasing mitochondrial ubiquitination in a cell is provided, the method comprising contacting the cell with an inhibitor of USP30 under conditions allowing inhibition of USP30 in the cell. Increased mitophagy may be determined, e.g., using immunofluorescence as described in Example 6. Increased ubiquitination may be determined, e.g., by immunoaffinity enrichment of ubiquitinated peptides after trypsin digestion, followed by mass spectrometry as described in Example 5. In some embodiments, an increase in mitochondrial ubiquitination may be determined by comparing the ubiquitination of a mitochondrial proteins a cell or population of cells contacted with an inhibitor of USP30 with the ubiquitination of mitochondrial proteins in a matched cell or population of cells not contacted with the inhibitor.


In some embodiments, increased mitophagy means a reduction in the average number of mitochondria per cell of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, in a population of cells contacted with an inhibitor of USP30, as compared to matched population of cells not contacted with the inhibitor. In some embodiments, increased mitochondrial ubiquitination means an increase in overall ubiquitination of mitochondrial proteins in a cell or population of cells contacted with an inhibitor of USP30 of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (i.e., 2-fold), at least 150%, or at least 200% (i.e., 3-fold) as compared to a matched cell or population of cells not contacted with the inhibitor.


In some embodiments, a method of increasing ubiquitination of at least one protein selected from Tom20, MIRO, MUL1, ASNS, FKBP8, TOM70, MAT2B, PRDX3, IDE, VDAC, IP05, PSD13, UBP13, and PTH2 in a cell is provided, the method comprising contacting the cell with an inhibitor of USP30 under conditions allowing inhibition of USP30 in the cell. In some such embodiments, ubiquitination increases at at least one, at least two, or three amino acids selected from K56, K61, and K68 of Tom 20; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K153, K187, K330, K427, K512, K535, K567, and K572 of MIRO. In some such embodiments, ubiquitination increases at at least one, at least two, or three amino acids selected from K56, K61, and K68 of Tom 20; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K153, K187, K330, K427, K512, K535, K567, and K572 of MIRO; and/or ubiquitination increases at at least one, at least two, or three amino acids selected from K273, K299, and K52 of MUL1; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K249, K271, K273, K284, K307, K317, K334, and K340 of FKBP8; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from K147, K168, K176, K221, K244, K275, K478, K504, and K556 of ASNS; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K78, K120, K123, K126, K129, K148, K168, K170, K178, K185, K204, K230, K233, K245, K275, K278, K312, K326, K349, K359, K441, K463, K470, K471, K494, K501, K524, K536, K563, K570, K599, K600, and K604 of TOM70; and/or ubiquitination increases at at least one, at least two, at least three, or four amino acids selected from K209, K245, K316, and K326 of MAT2B; and/or ubiquitination increases at at least one, at least two, at least three, at least four, or five amino acids selected from K83, K91, K166, K241, and K253 of PRDX3; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K558, K657, K854, K884, K929, and K933 of IDE; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids selected from K20, K53, K61, K109, K110, K266, and K274 of VDAC1; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K31, K64, K120, K121, K277, and K285 of VDAC2; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K20, K53, K61, K109, K110, K163, K266, and K274 of VDAC3; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K238, K353, K436, K437, K548, K556, K613, K678, K690, K705, K775, and K806 of IP05; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K2, K32, K99, K115, K122, K132, K161, K186, K313, K321, K347, K350, and K361 of PSD13; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K18, K190, K259, K326, K328, K401, K405, K414, K418, K435, K586, K587, and K640 of UBP13; and/or ubiquitination increases at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from K47, K76, K81, K95, K106, K119, K134, K171, K177 of PTH2. In some embodiments, ubiquitination of one or more additional proteins increases upon contacting a cell with an inhibitor of USP30. Nonlimiting exemplary proteins whose ubiquitination may be increased in the presence of an inhibitor of USP30 are listed in Appendix A, which is incorporated herein by reference. Increased ubiquitination of a target protein can be determined, e.g., by immunoaffinity enrichment of ubiquitinated peptides after trypsin digestion, followed by mass spectrometry, as described in Example 5. In some embodiments, an increase in ubiquitination may be determined by comparing the ubiquitination of a target protein a cell or population of cells contacted with an inhibitor of USP30 with the ubiquitination of the same target protein in a matched cell or population of cells not contacted with the inhibitor.


In some embodiments, increased ubiquitination of a protein means an increase in ubiquitination of the protein in a cell or population of cells contacted with an inhibitor of USP30 of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% (i.e., 2-fold), at least 150%, or at least 200% (i.e., 3-fold) as compared to a matched cell or population of cells not contacted with the inhibitor.


In some embodiments, the cell is under oxidative stress. Further, in some embodiments, a method of reducing oxidative stress in a cell is provided, the method comprising contacting the cell with an inhibitor of USP30 under conditions allowing inhibition of USP30 in the cell.


In any of the foregoing methods, the cell may comprise a pathogenic mutation in Parkin, a pathogenic mutation in PINK1, or a pathogenic mutation in Parkin and a pathogenic mutation in PINK1. Nonlimiting exemplary pathogenic mutations in Parkin and PINK1 are shown, e.g., in Tables 1 and 2 herein.


In some embodiments, the cell is a neuron. In some embodiments, the cell is a substantia nigra neuron. In some embodiments, the cell is a cardiac cell. In some embodiments, the cell is a cardiomyocyte cell. In some embodiments, the cell is a muscle cell.


In some embodiments of any of the foregoing methods, the cell is comprised in a subject. In some embodiments of any of the foregoing methods, the cell may be in vitro or ex vivo.


In another aspect, an inhibitor of USP30 for use as a medicament is provided. In further aspects, an inhibitor of USP30 for use in a method of treatment is provided. In some embodiments, a method of treating a condition involving a mitochondrial defect in a subject is provided, the method comprising administering to the subject an effective amount of an inhibitor of USP30. A condition involving a mitochondrial defect may involve a mitophagy defect, one or more mutations in mitochondrial DNA, mitochondrial oxidative stress, defects in mitochondrial shape/morphology, mitochondrial membrane potential defects, and/or a lysosomal storage defect. Nonlimiting exemplary conditions involving mitochondrial defects include neurodegenerative diseases; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome; Leber's hereditary optic neuropathy (LHON); neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS); Danon disease; ischemic heart disease leading to myocardial infarction; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrongenase deficiency (LCHAD); Leigh disease or syndrome; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrongenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial recessive ataxia syndrome; mitochondrial cytopathy; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; and very long-chain acyl-CoA dehydrongenase (VLCAD) deficiency. Nonlimiting exemplary neurodegenerative diseases that involve mitochondrial defects include Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia. Additional exemplary neurodegenerative diseases that may involve mitochondrial defects include, but are not limited to, intracranial hemorrhage, cerebral hemorrhage, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, progressive muscular atrophy, primary lateral sclerosis (PLS), pseudobulbar palsy, progressive bulbar palsy, spinal muscular atrophy, inherited muscular atrophy, invertebrate disk syndromes, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies, prophyria, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, demyelinating diseases, Guillain-Barre syndrome, multiple sclerosis, Charcot-Marie-Tooth disease, prion disease, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia. In some such embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.


In some embodiments, an inhibitor of USP30 is provided for use in the manufacture or preparation of a medicament. In some such embodiments, the medicament is for treatment of conditions involving a mitochondrial defect, such as, for example, conditions involving a mitophagy defect, conditions involving mutations in mitochondrial DNA, conditions involving mitochondrial oxidative stress, conditions involving defects in mitochondrial shape/morphology, conditions involving defects in mitochondrial membrane potential, and conditions involving lysosomal storage defects. In further embodiments, the medicament is for use in a method of treating a condition involving a mitochondrial defect, the method comprising administering to an individual having the condition involving a mitochondrial defect an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.


An “individual” according to any of the embodiments herein may be a human.


In a further aspect, pharmaceutical formulations comprising any of the inhibitors of USP30 provided herein, e.g., for use in any of the above therapeutic methods are provided. In one embodiment, a pharmaceutical formulation comprises any of the inhibitors of USP30 provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the inhibitors of USP30 provided herein and at least one additional therapeutic agent, e.g., as described below.


Inhibitors of USP30 can be used either alone or in combination with other agents in a therapy. For instance, an inhibitor of USP30 may be co-administered with at least one additional therapeutic agent.


Exemplary therapeutic agents that may be combined with an inhibitor of USP30, e.g., for the treatment of Parkinson's disease, include levodopa, dopamine agonists (such as pramipexole, ropinirole, and apomorphine), monoamine oxygenase (MAO) B inhibitors (such as selegiline and rasagiline), catechol O-methyltransferase (COMT) inhibitors (such as entacapone and tolcapone), anticholinergics (such as benzotropine and trihexylphenidyl), and amantadine. A further exemplary therapeutic agent that may be combined with an inhibitor of USP30, e.g., for the treatment of amyotrophic lateral sclerosis, is riluzole. Exemplary therapeutic agents that may be combined with an inhibitor of USP30, e.g., for the treatment of Alzheimer's disease, include cholinesterase inhibitors (such as donepezil, rivastigmine, galantamine, and tacrine), and memantine. Exemplary therapeutic agents that may be combined with an inhibitor of USP30, e.g., for the treatment of Huntington's disease, include tetrabenazine, antipsychotic drugs (such as haloperidol and clozapine), clonazepam, diazepam, antidepressants (such as escitalopram, fluoxetine, and sertraline), and mood-stabilizing drugs (such as lithium), and anti-convulsants (such as valproic acid, divalproex, and lamotrigine).


Administration “in combination” encompasses combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the inhibitor of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. In some embodiments, administration of the inhibitor of USP30 and administration of an additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of one another Inhibitors of the invention can also be used in combination with other types of therapies.


An inhibitor of the invention (and any additional therapeutic agent) can be administered by any suitable means, including oral, parenteral, intrapulmonary, intranasal, and intralesional administration. Parenteral administration includes, but is not limited to, intramuscular, intravenous, intraarterial, intracerebral, intracerebroventricular, intrathecal, intraocular, intraperitoneal, and subcutaneous administration. An inhibitor of the invention (and any additional therapeutic agent) may also be administered using an implanted delivery device, such as, for example, an intracerebral implant. Nonlimiting exemplary central nervous system delivery methods are reviewed, e.g., in Pathan et al., Recent Patents on Drug Delivery & Formulation, 2009, 3: 71-89. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.


Inhibitors of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The inhibitor need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of inhibitor present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.


For the prevention or treatment of disease, the appropriate dosage of an inhibitor of USP30 (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of inhibitor, the severity and course of the disease, whether the inhibitor is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the inhibitor, and the discretion of the attending physician. The inhibitor is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of inhibitor can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the inhibitor would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the inhibitor). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.


It is understood that any of the above formulations or therapeutic methods may be carried out using more than one inhibitor of USP30.


E. Articles of Manufacture


In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described herein is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the disorder and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an inhibitor of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an inhibitor of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.


III. EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.


Example 1
Materials and Methods

DUB cDNA Overexpression Screen:


To identify regulators of mitophagy, individual cDNAs from a FLAG-tagged DUB library were cotransfected into HeLa cells with GFP-Parkin using Lipofectamine 2000 (Invitrogen) (1:3 DUB-FLAG: GFP-Parkin cDNA ratio). After 24 hours of expression, cells were treated with 10 μM CCCP for 24 hours, and fixed and stained using anti-Tom20 (Santa Cruz Biotechnology), anti-GFP (Ayes Labs) and anti-FLAG (Sigma) primary antibodies. Following staining with secondary antibodies, images of random fields were acquired with a Leica SP5 Laser Scanning Confocal Microscope using a 40×/1.25 oil-objective (0.34 μm/pixel resolution, 1 μm confocal z-step size). Percent of GFP-Parkin and FLAG-DUB cotransfected cells containing Tom20 staining was scored blindly.


Hippocampal Culture, Transfection and Mt-Keima Imaging:

Dissociated hippocampal neuron cultures were prepared as described (Seeburg et al., Neuron 58: 571-583 (2008)) and transfected using Lipofectamine LTX PLUS at DIV 8-10. Constructs were expressed for 1-3 days for overexpression experiments and 3-4 days for knockdown experiments. mt-Keima-transfected neurons were imaged with a Leica TCS SP5 Laser Scanning Confocal microscope with a 40×/1.25 oil objective (0.07 μm/pixel resolution, 1 μm confocal z-step size). Cells were kept in a humidified chamber maintained at 37° C./5% CO2 during imaging. Two images were acquired using a hybrid detector in sequential mode with 458 nm (neutral pH signal) and 543 nm (acidic pH signal) laser excitation, and emission fluorescence collected between 630-710 nm. All image quantification was performed by custom-written macros in ImageJ. For mt-Keima quantification, cell bodies were manually outlined and total area of high ratio (543 nm/458 nm) lysosomal signal was divided by the total area of somatic mitochondrial signal (mitophagy index).


Mass Spectrometry

To determine Parkin substrates, HEK-293 GFP-Parkin inducible cells were treated with doxycycline for 24 hours, and then treated with 5 μM CCCP or DMSO vehicle control for 2 hours. To determine USP30 substrates, HEK-293T cells were transfected with human USP30 shRNA using Lipofectamine 2000 (Invitrogen) for 6 days, then treated as before. In both experiments, cells were lysed (20 mM HEPES pH 8.0, 8M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate), sonicated, and cleared by centrifugation prior to proteolytic digestion and immunoaffinity enrichment of peptides bearing the ubiquitin remnant, and mass spectrometry analysis.


Preparation of Cellular Lysates and Immunoprecipitation

For total lysate experiments, transfected HEK-293 cells were lysed at 24 hours post-transfection in SDS sample buffer (Invitrogen) containing sample reducing agent (Invitrogen). For immunoprecipitation experiments, cells were lysed at 24 hours (overexpression experiments) or 6 days (knockdown experiments) post-transfection in TBS buffer containing 0.5% SDS, and lysates were diluted with a buffer containing 1% Triton-X-100 and protease and phosphatase inhibitors. Ubiquitinated proteins were immunoprecipated from lysates of HA-ubiquitin transfected cells with anti-HA affinity matrix beads (Roche Applied Science). Inputs and precipitates were resolved by SDS-PAGE and analyzed by immunoblotting.


Statistical Analysis

Error bars indicate standard error of the mean (S.E.M.), To compute p values, non-paired Student's t-test, One-way ANOVA with Dunnett's Multiple Comparison test (for comparisons to a single condition) or Bonferroni's Multiple Comparison test (for comparisons between multiple conditions), and Two-way ANOVA were used, as indicated in figure legends. All statistical analysis was performed in GraphPad Prism v.5 software.


DNA Construction

For the DUB overexpression screen, a FLAG-tagged DUB library consisting of 100 cDNAs was used. For transfection, the following constructs were subcloned into β-actin promoter-based pCAGGS plasmid: USP30-FLAG (rat), USP30-FLAG (human), GFP-Parkin (human), FLAG-Parkin (human), PINK1-GFP (human), myc-Parkin (human), RHOT1 (MIRO)-myc-FLAG (human), TOM20-myc (human), HA-ubiquitin, PSD-95-FLAG, and mt-mKeima (Katayama et al., Chemistry & Biology 18: 1042-1094 (2011)). Point mutations were generated using QuikChange II XL (Agilent Technologies) for the following constructs: USP30-C77S-FLAG (rat), USP30-C77A-FLAG (rat), USP30-C77S-FLAG (human), GFP-Parkin K161N (human), and GFP-Parkin G430D (human). Mito-tagGFP2 (Evrogen), Tom20-3KR-myc, and HA-ubiquitin mutants (Blue Heron) were purchased. β-Gal (Seeburg and Sheng, J. Neurosci. 28: 6583-6591 (2008)) and mito-ro-GFP (Dooley et al., J. Biol. Chem. 279: 22284-22293 (2004)) expression plasmids were previously described. Short-hairpin sequences targeting the following regions were cloned into pSuper or pSuper-GFP-neo plasmids: rat PINK1 #1 (TCAGGAGATCCAGGCAATT), rat PINK1 #2 (CCAGTACCTTGAAGAGCAA), rat Parkin #1 (GGAAGTGGTTGCTAAGCGA), rat Parkin #2 (GAGGAAAAGTCACGAAACA), rat USP30 (CCAGAGCCCTGTTCGGTTT), human USP30 (CCAGAGTCCTGTTCGATTT), and firefly luciferase (CGTACGCGGAATACTTCGA).


Antibodies and Reagents:

The following antibodies were used for immunocytochemistry: rabbit anti-TOM20, mouse anti-TOM20, goat anti-HSP60 (Santa Cruz Biotechnology); mouse anti-FLAG, rabbit anti-FLAG, mouse anti-myc (Sigma-Aldrich); and chicken anti-GFP (Ayes Labs).


The following antibodies were used for immunoblotting: rabbit anti-TOM20, goat anti-HSP60 (Santa Cruz Biotechnology); mouse anti-MFN1, HRP-conjugated anti-FLAG, mouse anti-myc, rabbit anti-USP30, rabbit anti-RHOT1 (MIRO), rabbit anti-TIMM8A (Sigma-Aldrich); rabbit anti-GFP, chicken anti-GFP (Invitrogen); HRP-conjugated anti-GAPDH, HRP-conjugated anti-α-actin, HRP-conjugated anti-β-tubulin, rabbit anti-VDAC (Cell Signaling Technology); rabbit anti-TOM70 (Proteintech Group); anti-ubiquitin (FK2) (Enzo Life Sciences); mouse anti-LAMP1 (StressGen); HRP-conjugated anti-HA (Roche); and anti-USP30 rabbit (generated by immunizing rabbits with purified human USP30 amino acids 65-517).


For immunoprecipitation experiments anti-FLAG M2 affinity gel beads (Sigma) and anti-HA affinity matrix beads (Roche Applied Science) were used.


Adeno-associated virus type2 (AAV2) particles expressing Parkin, PINK1 and USP30 shRNAs were produced by Vector Biolabs, Inc. from pAAV-BASIC-CAGeGFP-WPRE vector containing the H1 promoter and shRNA expression cassette of the pSuper vectors.


The following reagents were purchased as indicated: blasticidin S, zeocin, Lipofectamine 2000, Lipofectamine LTX PLUS, LysoTracker Green DND-626 (Invitrogen); PhosSTOP phosphatase inhibitor tablets, cOmplete EDTA-free protease inhibitor tablets, DNase I (Roche Applied Science); carbonyl cyanide 3-chlorophenylhydrazone (CCCP), doxycycline, dimethyl sulfoxide, ammonium chloride, rotenone, DTT, aldrithiol, paraquat dichloride (Sigma-Aldrich); N-Ethylmaleimide (Thermo Scientific); and hygromycin (Clontech Laboratories).


Transfection and Immunocytochemistry:

All heterologous cells were transfected with Lipofectamine 2000 for cDNA expression and Lipofectamine RNAiMAX for siRNA knockdown experiments, according to manufacturer's instructions (Invitrogen). siRNAs were purchased from Dharmacon as siGenome pools (non-Silencing pool #2 was used control siRNA transfection). Hippocampal cultures were prepared as described previously (Seeburg et al., Neuron 58: 571-583 (2008)) and transfected with Lipofectamine LTX PLUS (Invitrogen) with 1.8 μg DNA, 1.8 μl PLUS reagent and 6.3 μl LTX reagent. Following drug treatments, cells were fixed with 4% paraformaldehyde/4% sucrose in phosphate-buffered saline (PBS, pH 7.4) (Electron Microscopy Sciences). Following permeabilization (0.1% Triton-X in PBS), blocking (2% BSA in PBS) and primary antibody incubation, antibodies were visualized using Alexa dye-conjugated secondary antibodies (Invitrogen). All immunocytochemistry images were acquired with a Leica SP5 laser scanning microscope with a 40×/1.25 oil objective (0.34 μm/pixel resolution, 1 μm confocal z-step size).


HEK293 and SH-SY5Y Stable Cell Line Generation

Stably transfected HEK cell lines expressing GFP-Parkin (human) wild-type, K161N, and G430D were generated by co-transfecting FLP-In 293 cells with a pOG44 Flp-recombinase expression vector (Invitrogen) and a pcDNA5-FRT vector (Invitrogen) expressing the corresponding constructs under a CMV promoter. Cell lines were selected and maintained using 50 μg/mL hygromycin selection. Inducible HEK stable cell line expressing GFP-Parkin (human) was generated by co-transfecting FLP-In T-Rex 293 cells with pOG44 and a pcDNA5-FRT-TO vector (Invitrogen) expressing GFP-Parkin (human). The line was selected and maintained using 50 μg/mL hygromycin and 15 μg/mL blasticidin. SH-SY5Y stable cells were generated similarly with a Flp-In inducible parental cell line using pcDNA5-FRT-TO and maintained under 75 μg/ml hygromycin and 3 μg/ml blasticidin.


Isolation and Identification of Ubiquitin Modifications by Mass Spectrometry

To identify Parkin substrates, HEK 293T cells stably expressing inducible GFP-Parkin (human) were induced using doxycycline (1 μg/mL) for 24 hours, then treated with 5 μM CCCP or DMSO vehicle control for 2 hours. To determine USP30 substrates, HEK 293T cells were transfected with human USP30 shRNA using Lipofectamine 2000 (Invitrogen) for 6 days, then treated as above.


Immunoaffinity isolation and mass spectrometry methods were used to enrich and identify K-GG peptides from digested protein lysates as previously described (Xu et al., Nat. Biotech., 28: 868-873 (2010); Kim et al., Mol. Cell, 44: 325-340 (2011)). Cell lysates were prepared in lysis buffer (8M urea 20 mM HEPES pH 8.0 with 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM B-glycerophosphate) by brief sonication on ice. Protein samples (60 mg) were reduced at 60° C. for 20 min in 4.1 mM DTT, cooled 10 min on ice, and alkylated with 9.1 mM iodoacetamide for 15 min at room temperature in the dark. Samples were diluted 4X using 20 mM HEPES pH 8.0 and digested in 10 μg/ml trypsin overnight at room temperature. Following digestion, TFA was added to a final concentration of 1% to acidify the peptides prior to desalting on a Sep-Pak C18 cartridge (Waters). Peptides were eluted from the cartridge in 40% ACN/0.1% TFA, flash frozen and lyophilized for 48 hr. Dry peptides were gently resuspended in 1.4 ml 1×IAP buffer (Cell Signaling Technology) and cleared by centrifugation for 5 min at 1800×g. Precoupled anti-KGG beads (Cell Signaling Technology) were washed in 1×IAP buffer prior to contacting the digested peptides.


Immunoaffinity enrichment was performed for 2 hours at 4° C. Beads were washed 2X with IAP buffer and 4X with water prior to 2X elution of peptides in 0.15% TFA for 10 min each at room temperature. Immunoaffinity enriched peptides were desalted using STAGE-Tips as previously described (Rappsilber et al., Anal. Chem., 75: 663-670 (2003)).


Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on an LTQ-Orbitrap Velos mass spectrometer operating in data dependent top 15 mode. Peptides were injected onto a 0.1×100-mm Waters 1.7-um BEH-130 C18 column using a NanoAcquity UPLC and separated at 1 ul/min using a two stage linear gradient where solvent B ramped first from 2% to 25% over 85 min and then 25% to 40% over 5 min. Peptides eluting from the column were ionized and introduced to the mass spectrometer using an ADVANCE source (Michrom-Bruker). In each duty cycle, one full MS scan collected was at 60,000 resolution in the Orbitrap followed by up to 15 MS/MS scans in the ion trap on monoisotopic, charge state defined precursors (z>1). Ions selected for MS/MS (+20 ppm) were subjected to dynamic exclusion for 30 sec duration.


Mass spectral data were converted to mzxml for loading into a relational database. MS/MS spectra were searched using Mascot against a concatenated target-decoy database of tryptic peptides from human proteins (Uniprot) and common contaminants. Precursor ion mass tolerance was set to +50 ppm. Fixed modification of carbamidomethyl cysteine (+57.0214) and variable modifications of oxidized methionine (+15.9949) and K-GG (+114.0429) considered. Linear discriminant analysis (LDA) was used to filter peptide spectral matches (PSMs) from each run to 5% false discovery rate (FDR) at the peptide level, and subsequently to a 2% protein level FDR as an aggregate of all runs (<0.5% peptide level FDR). Localization scores were generated for each K-GG PSM using a modified version of the AScore algorithm and positions of the modifications localized accordingly as the AScore sequence. (Beausoleil et al., Nat. Biotech. 24(10): 1285-1292 (2006)). Given work showing that trypsin cannot cut adjacent to ubiquitin modified lysines PSMs where the AScore sequence reports a -GG modification on the C-terminal lysine are dubious (Bustos et al., Mol. Cell. Proteomics, published online Jun. 23, 2012, doi: 10.1074/mcp.R112.019117; Seyfried et al., Anal. Chem., 80: 4161-4169 (2008)). Possible exceptions to this would be lysines at the C-termini of proteins (or in vivo truncation products), PSMs stemming from in source fragmentation of a bona fide K-GG peptide. To establish the most reliable dataset for downstream analysis, PSMs where the AScore sequence reports a C-terminal lysine were split into two groups: those with an available internal lysine residue to which the -GG could be alternatively localized, and those which lacked an available lysine. PSMs bearing a C-terminal K-GG but lacking an available lysine were removed from consideration in downstream analyses. For the remaining PSMs, the -GG modification was relocalized to the available lysine closest to the C-terminus.


Confidently identified peptides with ambiguous localization (AScore<13) bearing only a single internal lysine residue were reported with the modification localized to that internal lysine. Peptides where the modification has been assigned to the C-terminal lysine with an AScore>13 were discarded based on evidence suggesting that trypsin cannot cleave at a ubiquitin modified lysine residue.


A modified version of the VistaGrande algorithm, termed XQuant, was employed to interrogate the unlabeled peak areas for individual K-GG peptides, guided by direct PSMs or accurate precursor ion and retention time matching (cross quantitation). For direct PSMs, quantification of the unlabeled peak area was performed as previously described using fixed mass and retention tolerances (Bakalarski et al., J. Proteome Res., 7: 4756-4765 (2008)). To enable cross quantitation within XQuant, retention time correlation across pairwise instrument analyses was determined based on high-scoring peptide sequences identified by between one and four PSMs across all analyses within an experiment. Matched retention time pairings were modeled using a linear least squares regression model to yield the retention time correlation equation. In instrument analyses where a peptide was not identified by a discrete MS/MS, cross quantification was carried out by seeding the XQuant algorithm with the calculated mass of the precursor ion and its predicted retention time derived from the regression model. While the m/z tolerance was fixed, the retention time tolerance was dynamically adjusted for each pairwise instrument run. In cases where peptides were not confidently identified within a given instrument run but were identified in multiple other runs, multiple cross quantification events were performed to ensure data quality. XQuant results were filtered to a heuristic confidence score of 83 or greater, as previously described (Bakalarski et al., J. Proteome Res., 7: 4756-4765 (2008)). Full scan peak area measurements arising from multiple quantification events of the same m/z within a single run were grouped together if their peak boundaries in retention time overlapped. From such a group, the peak with the largest total peak area was chosen as its single representative.


To identify candidate substrates of Parkin and USP30, graphical analysis and mixed-effect modeling were applied to the XQuant data. A mixed-effect model was fit to the AUC data for each protein. “Treatment” (e.g. Control, Parkin overexpression/USP30 knockdown, CCCP, Combo) was a categorical fixed effect and “Peptide” was fit as random effect. False discovery rates (FDR) are calculated based on the P-values of each treatment vs. Control. Fold-changes and P-values of mean AUC from Combo vs. Control and Combo vs. CCCP were utilized in preparing plots. Mixed-effect model was fit in R by ‘nlme’ (Pinheiro et al., nlme: Linear and Nonlinear Mixed Effects Models. R package version 3, 1-101 (2011)).


Preparation of Cell Lysates, and Immunoprecipitation

For total lysate experiments, cells were lysed after 24 hours in SDS sample buffer (Invitrogen) containing sample reducing agent (Invitrogen) and boiled at 95° C. for 10 minutes. Total lysates were resolved by SDS-PAGE and analyzed by immunoblotting. For immunoprecipitation experiments, cells were treated with 5 μM MG132 and the indicated concentrations and durations of CCCP at 24 hours (overexpression experiments) or 6 days (knockdown experiments) in 0.5% SDS in Tris-Buffered Saline (10 mM TRIS, 150 mM NaCl, pH 8.0) and boiled at 70° C. for 10 minutes. Lysates were diluted in immunoprecipitation buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton-X, protease inhibitors (Roche Applied Science), phosphatase inhibitors (Roche Applied Science), DNAse I (Roche Applied Science), 2 mM N-Ethylmaleimide (Thermo Scientific), pH 7.4), cleared by centrifugation at 31,000 g for 10 minutes, and incubated overnight with anti-HA affinity matrix beads (Roche Applied Science). Inputs and anti-HA immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting.


Mitochondria Fractionation

Subcellular fractionation was performed using the FOCUS SubCell Kit (G Biosciences) from ˜P60 adult male rat forebrain.



Drosophila Stocks

The following Drosophila lines were obtained for analysis: y, w; Actin5C-GAL4/CyO, y+(Bloomington Drosophila Stock Center, 4414), UAS-CG3016RNAi (referred to here as UAS-dUSP30RNAi; NIG-Fly Stock Center, 3016R-2). For USP30 knockdown experiments, Actin5C-GAL4 and UAS-dUSP30RNAi were recombined onto the same chromosome using standard genetic techniques.


Flies were raised on Nutri-Fly “German Food” Formulation (Genesee, 66-115), prepared per manufacturer's instructions. All flies were raised at 25° C. and crossed using standard genetic techniques. All experiments were performed using age-matched male flies.


Quantitative RT-PCR

RNA and subsequent cDNA was obtained from single flies following manufacturer's instructions (Qiagen RNeasy Plus kit, Applied Biosystems High Capacity cDNA Reverse Transcription kit). Quantitative RT-PCR was performed using an Applied Biosystems ViiA7 Real-Time PCR system using TaqMan Assays Dm01796115_g1 and Dm01796116_g1 (Drosophila CG3016 (USP30)), Dm01795269_g1 (Drosophila CG5486 (USP47)), and Dm01840115_s1 (Drosophila CG4603 (YOD1)). Dm02134593_g1 (RpII140) was used as a control.


Determination of Ingested Paraquat Concentration

1-day old adult males were fed a solution containing 5% sucrose only (in water) or 5% sucrose+10 mM paraquat (in water) on saturated Whatman paper. After 48 hours of treatment, 15 flies were collected per condition and homogenized in 100 μL water. Standard curve samples were generated by spiking appropriate amounts of paraquat to homogenates from untreated flies. Then the samples were vortex mixed, 200 μl of acetonitrile containing internal standard (Propranolol) was added. The samples were vortexed again and centrifuged at 10,000×g for 10 min. Supernatants were transferred to a new plate that contained 200 μl of water and analyzed by LC-MS/MS to quantify for concentrations of paraquat. The LC-MS/MS consisted of an Agilent 1100 series HPLC system (Santa Clara, Calif.) and an HTS PAL autosampler from CTC Analytics (Carrboro, N.C.) coupled with a 4000 Q TRAP® MS and TurbolonSpray® ion source from Applied Biosystems (Foster City, Calif.). HPLC separation was performed on a Waters Atlantis dC18 column (3 μm 100×2.1 mm) with a Krud Katcher guard column from Phenomenex. Quantitation was carried out using the multiple reaction monitoring (MRM) with transition 185.1→165.1 for paraquat and 260.2→183.1 for propranolol. The lower and upper limit of the assay is 10 μM and 1000 μM, respectively. The quatitation of the assay employed a calibration curve which was constructed through plotting the analyte/IS peak area ration versus the nominal concentration of paraquat with a weighed 1/x2 linear regression.


Transmission Electron Microscopy of Drosophila Indirect Flight Muscles

Adult male thoraxes were isolated from the remainder of the body, then longitudinally hemi-sectioned and immediately fixed and processed as previously described (Greene et al., Proc. Natl. Acad. Sci. USA, 100: 4078-4083 (2003)).


Climbing Assays

Climbing assays were performed using the following Drosophila lines: y, w; Actin5C-GAL4/CyO, y+(Actin only); y, w; UAS-CG3016-RNAi/CyO, y+(RNAi only); y, w; UAS-CG3016RNAi Actin5C-GAL4/CyO, y+(USP30 knockdown).


1-day old adult males were fed a solution containing 5% sucrose only (in water) or 5% sucrose+10 mM paraquat (in water) on saturated Whatman paper. After 48 hours of treatment, flies were anesthetized using carbon dioxide and transferred in groups of ten to vials containing only 1% agarose for a 1-hour recovery period from the effects of carbon dioxide. The flies were then transferred into a new glass tube, gently tapped to the bottom and scored for their ability to climb. The number of flies climbing vertically >15 cm in 30 seconds was recorded.


Survival Assays

Ten adult 1-day old males per vial were fed a solution containing 5% sucrose only (in water) or 5% sucrose+10 mM paraquat (in water) on saturated Whatman paper. The number of live flies was counted at described intervals.


MultiTox Cell Death Assay

SH-SY5Y cells transfected with control or USP30 siRNAs are treated with rotenone in normal growth medium (DMEM/F12 and 1× GlutaMax) containing 1% Fetal Bovine Serum. Following 24 hours of incubation, Multi-Tox Fluor assay (Promega) is used to measure cell viability according to manufacturer's instructions. GF-AFC fluorescence is normalized to bis-AAF-R110 fluorescence for each condition and presented as a fraction of control (control RNAi+DMSO).


Example 2
USP30 Antagonizes Parkin-Mediated Clearance of Damaged Mitochondria

To identify DUBs that regulate mitochondria clearance, a FLAG-tagged human DUB cDNA library (97 DUBs) was screened in an established mitochondrial degradation assay (Narendra et al., J. Cell Biol., 183: 795-803 (2008)). In this assay, mitochondria depolarization induced by protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μM, 24 hours) results in marked loss of mitochondria in cultured cells overexpressing Parkin (as measured by staining for mitochondria outer membrane protein marker Tom20). CCCP treatment led to a robust disappearance of Tom20 staining in the great majority of cells transfected with GFP-Parkin (>80% of Parkin-transfected cells lacked Tom20 staining after CCCP—FIG. 1A). Individual FLAG-tagged DUB cDNAs were cotransfected with GFP-Parkin, and their effects on CCCP-induced mitochondrial (Tom20) clearance were measured. Out of the library of ˜100 different DUBs, 2 DUBs, USP30 and DUBA2, robustly blocked the loss of Tom20 staining in CCCP-treated GFP-Parkin-transfected cells, whereas others had little effect (FIG. 1A—% of cells with Tom20 staining: control (β-Gal): 15.3%, USP30: 97.4%, DUBA2: 94.7%, UCH-L1: 36%, USP15: 23.3%, ATXN3: 8.3%; other negative DUBs not shown). USP30 rather than DUBA2 was selected for further study since USP30 has been reported to be localized in the mitochondrial outer membrane with its enzymatic domain putatively facing the cytoplasm (Nakamura and Hirose, Mol. Biol. Cell, 19: 1903-1914 (2008)); thus it would be in the right subcellular compartment to counteract the action of Parkin on mitochondria. The specific mitochondrial association of USP30 was confirmed by immuno-colocalization of transfected USP30-FLAG and of endogenous USP30 with mitochondrial markers in neurons (FIG. 2A, B), as well as by cofractionation of USP30 with purified mitochondria from rat brain (FIG. 2C).


The ability of USP30 overexpression to prevent CCCP-induced mitophagy was also shown in a different cell line (dopaminergic SH-SY5Y cells) transfected with myc-Parkin (FIG. 1B). To confirm that the effects of USP30 were not specific to Tom20, whether USP30 overexpression also prevented the CCCP-induced loss of the mitochondrial matrix protein HSP60 was tested. Indeed, USP30 overexpression also prevented the CCCP-induced loss of HSP60, implying USP30 blocks en masse degradation of the organelle (FIG. 1B-D). In contrast, expression of an catalytically-inactive USP30 C77S mutant (Nakamura and Hirose, Mol. Biol. Cell, 19: 1903-1914 (2008)) was ineffective at preventing Parkin-mediated mitochondria degradation, supporting the idea that USP30 counteracts mitophagy through deubiquitination of mitochondrial substrates (FIG. 1B-D).


Since USP30 enzymatic activity was necessary for blocking mitophagy, whether USP30 and Parkin have opposing effects on mitochondria ubiquitination was examined. As reported previously, short-term CCCP treatment (20 μM, 4 hours) caused Parkin redistribution to mitochondria (marked by Tom20) and led to accumulation of ubiquitination signal on mitochondria (measured by staining with polyubiquitin antibody FK2, FIG. 2D; (Lee et al., J. Cell Biol., 189: 671-680 (2010)). When USP30 was co-expressed with Parkin, the amount of ubiquitin signal accumulated on mitochondria was reduced by ˜75% —an effect that also required USP30 enzymatic activity (FIGS. 2D, E). These data support the idea that USP30 functions as a DUB that opposes the ubiquitin ligase action of Parkin on mitochondrial proteins, thereby inhibiting mitophagy.


Previous studies indicated that Parkin pathogenic mutants defective in ligase activity cannot support mitochondrial degradation in response to CCCP, leading to clustering of uncleared mitochondria in the perinuclear region in association with translocated Parkin (Geisler et al., Nat. Cell Biol., 12: 119-131 (2010); Lee et al., J. Cell Biol., 189: 671-680 (2010)). Remarkably, in cells co-transfected with USP30 plus Parkin and treated with CCCP, wild-type myc-Parkin behaved similarly to mutant Parkin in that it remained associated with the perinuclear clusters of non-degraded mitochondria (FIG. 1B (white arrow), E). Co-expression of USP30 did not alter Parkin expression level (FIGS. 2F, G). These data indicate that USP30 blocks mitophagy by enzymatic removal of ubiquitin signal on damaged mitochondria, rather than by inhibiting the translocation of Parkin to mitochondria.


Example 3
Pink1, Parkin Required for Mitophagy in Neurons

To measure mitophagy in neurons, mt-Keima, a ratiometric pH-sensitive fluorescent protein that is targeted into the mitochondrial matrix, was monitored. A low ratio mt-Keima-derived fluorescence (543 nm/458 nm) reports neutral environment whereas a high ratio fluorescence reports acidic pH (Katayama et al., Chemistry & Biology 18: 1042-1094 (2011)). Thus mt-Keima enables differential imaging of mitochondria in the cytoplasm and mitochondria in acidic lysosomes. Because mt-Keima is resistant to lysosomal proteases, it allows for measurement of cumulative lysosomal delivery of mitochondria over time.


Following transfection in rat dissociated hippocampal cultures, mt-Keima signal accumulated initially in elongated structures characteristic of mitochondria and with low 543/458 ratio values (shown in green—FIG. 3A). After 2-3 days of expression, multiple round mt-Keima structures with high ratio (acidic) signal also appeared throughout the cell body (shown in red—FIG. 3A). These round mt-Keima-positive structures most likely represent lysosomes since (1) neutralizing cells with NH4Cl completely reversed the high ratio (543/458) pixels to low ratio signal specifically in these round structures without affecting the tubular-reticular mitochondrial signal (FIG. 4A); (2) an independent lysosomal marker dye (lysotracker green DND-26) stained high ratio mt-Keima structures, though there were also many Lysotracker-positive structures that were not associated with mt-Keima (FIG. 4B); (3) in post-hoc immunostaining experiments, high ratio pixels colocalized with endogenous lysosomal protein LAMP-1 (FIG. 4C). Since almost all of the “acidic” mt-Keima signal was found in neuronal cell bodies (cell body contained 95.6±2.2% of the total high ratio (543/458) signal), the ratio of the area of lysosomal (red) signal/mitochondrial (green) signal within the cell body was used as a measure of lysosomal delivery of mitochondria in neurons (“mitophagy index”) (Katayama et al., Chemistry & Biology 18: 1042-1094 (2011)). As quantified by this mitophagy index, the abundance of mt-Keima in lysosomes increased over a time course of days (FIG. 4D), implying active mitophagy in cultured neurons under basal conditions.


In heterologous cells, Parkin overexpression can drive mitochondrial degradation upon mitochondria depolarization; however, it is not yet established whether endogenous Parkin and PINK1 are required for mitophagy in non-neural or neural cells (Youle and Narendra, Nat. Rev. Mol. Cell Biol. 12: 9-14 (2011)). To examine the role of the PINK1/Parkin pathway in neuronal mitophagy, Parkin or PINK1 was knocked down using small hairpin RNAs (shRNAs) expressed from pSuper-based vectors. These Parkin and PINK1 shRNAs efficiently knocked down the cDNA-driven expression of their respective targets in heterologous cells (FIGS. 4E, F), and suppressed the protein levels of endogenous Parkin or PINK1 in neuronal cultures by ˜80% and ˜90%, respectively (FIGS. 4G, H). Compared to control luciferase shRNA, neurons transfected with Parkin shRNAs (two independent sequences) showed ˜50% reduction in the mitophagy index, indicative of decreased mitochondria delivery to lysosomes (FIGS. 3B, C). PINK1 shRNAs were even more effective in reducing the acidic mt-Keima signal (˜80-90% reduction in mitophagy index (FIGS. 3D, E)). Previous genetic studies placed PINK1 upstream of Parkin in maintaining healthy mitochondria (Clark et al., Nature, 441: 1162-1166 (2006); Park et al. Nature, 441: 1157-1161 (2006)). Consistent with the genetic epistasis, our mt-Keima experiments showed that PINK1 overexpression strongly enhanced mitophagy in neurons, an effect that was completely eliminated by Parkin knockdown (FIGS. 3F, G). On the other hand, Parkin overexpression by itself had no apparent effect on basal mitophagy, as measured by the mt-Keima assay (FIGS. 4I, J). Thus, neuronal mitophagy requires both PINK1 and Parkin, with PINK1—apparently limiting—acting upstream of Parkin.


Example 4
USP30 Antagonizes Mitophagy in Neurons

Next, whether USP30 suppresses mitophagy in neurons as in heterologous cells was investigated. Compared with control neurons transfected with β-Gal and mt-Keima, co-expression of wild-type USP30 caused a ˜60% reduction in mitophagy index at 3 days, indicating that USP30 inhibits lysosomal delivery of mitochondria in neurons (FIGS. 5A, E). In contrast, overexpression of enzymatically-inactive USP30 (C77S or C77A) induced a robust increase in mitophagy signal (FIGS. 5A, E). The enhanced delivery of mitochondria to lysosomes likely reflects a dominant-negative action of catalytically-inactive USP30, presumably by interacting with substrates or pro-mitophagy ubiquitin chains, and sequestering them from endogenous USP30 (Berlin et al., J. Biol. Chem., 285: 34909-34921 (2010); Bomberger et al., J. Biol. Chem., 284: 18778-18789 (2009); Ogawa et al., J. Biol. Chem., 286: 41455-41465 (2011)).


To test the function of endogenous USP30, USP30 was knocked down using shRNAs. In heterologous cells, rat USP30 shRNA plasmid specifically eliminated the expression of transfected rat USP30 cDNA (FIG. 5B). The same rat USP30 shRNA led to a ˜85% reduction in endogenous USP30 in neuronal cultures (FIG. 5C). In neurons, USP30 knockdown increased the lysosomal delivery of mt-Keima (˜60% increase in mitophagy index), relative to negative control luciferase shRNA (FIGS. 5D, F). Co-transfection of shRNA-resistant human USP30 cDNA “rescued” this effect, i.e. it restored the brake on mitochondrial degradation, indicating that USP30 shRNA was not exerting a non-specific effect (FIGS. 5B, D, F). In fact, neurons co-transfected with human USP30 cDNA plus rat USP30 shRNA showed lower levels of lysosomal accumulation of mt-Keima than controls, similar to neurons overexpressing wild-type USP30 by itself (FIGS. 5D, F). Moreover, enzymatically-inactive human USP30 (C77S) failed to reverse the enhanced mitophagy induced by USP30 shRNA, and actually enhanced mitophagic activity even more than USP30 shRNA (FIGS. 5D, F), the latter result suggesting that USP30 knockdown is incomplete. These results provide strong evidence that endogenous USP30 restrains mitophagy in neurons through its DUB activity.


Example 5
USP30 Deubiquitinates Multiple Mitochondrial Proteins

Since Parkin and USP30 antagonistically regulate mitochondrial degradation, it was hypothesized that this E3 ligase and DUB act on some common substrates. To identify Parkin and USP30 substrates, global ubiquitination in cells was analyzed by mass spectrometry (MS) following immunoaffinity enrichment of ubiquitinated peptides from trypsin-digested extracts using the ubiquitin branch-specific (K-GG) antibodies. Global ubiquitination was analyzed and quantified by MS in HEK-293 cells in two different sets of conditions: 1) inducible Parkin overexpression, or 2) USP30 knockdown (USP30 knockdown efficiency was 85±5% (see FIG. 7C)). In each set, cells were treated with CCCP (5 μM, 2 hours) or vehicle control (DMSO). In aggregate, MS analysis revealed >15,000 unique ubiquitination sites on ˜3200 proteins of which a subset responded to either CCCP alone (endogenous Parkin and USP30 levels) or Parkin overexpression/USP30 knockdown (see Appendix A for a list of the ˜3200 proteins). MS identified 233 and 335 proteins whose ubiquitination increased by parkin overexpression or USP30 knockdown, respectively (i.e. exhibited significantly more ubiquitination in ‘parkin overexpression+CCCP’ or ‘USP30 knockdown+CCCP’ vs. CCCP-alone. 41 of these proteins were regulated by both Parkin overexpression and USP30 knockdown (FIG. 13). Twelve of these 41 proteins are mitochondrial or associated with mitochondria (Tom20, MIRO1, FKBP8, PTH2, MUL1, MAT2B, TOM70, PRDX3, IDE, and all three VDAC isoforms—based on Human MitoCarta database). Others included nuclear import proteins (e.g. IP05), demethylases (e.g. KDM3B), and components of the ubiquitin/proteasome system (e.g., PSD13, UBP13) (FIG. 13).


We focused additional studies on two mitochondrial proteins—Tom20 and MIRO—that showed large increases in ubiquitination with USP30 knockdown (USP30 shRNA+CCCP/CCCP ubiquitination ratio for Tom20=3.52, p=0.005; for MIRO=2.95, p=0.019; see FIG. 13, left). Tom20 and MIRO also showed large magnitude and highly significant increases in ubiquitination with Parkin overexpression (FIG. 13, right). To confirm that USP30 can deubiquitinate these proteins, cell lines stably overexpressing GFP-Parkin were transfected with HA-ubiquitin and immunoprecipitated (IP) ubiquitinated proteins using anti-HA antibodies. Following mitochondrial depolarization (CCCP, 5 μM, 2 hours), GFP-Parkin stable cells showed robust enhanced ubiquitination of endogenous MIRO, as measured by immunoblotting for MIRO in the anti-HA-immunoprecipitates (FIG. 7A). In control transfections without HA-ubiquitin, anti-HA-beads did not immunoprecipitate MIRO, indicating the specificity of MIRO ubiquitination signal (FIG. 7A, left lanes). Compared to β-Gal control, cotransfection of wildtype USP30, but not DUB-dead USP30-C77S, decreased the amount of ubiquitininated MIRO by ˜85% (FIGS. 7A, B). Similarly, wildtype USP30 overexpression reduced the ubiquitination of Tom20 (FIG. 7A); whereas USP30-C77S actually increased basal Tom20 ubiquitination ˜2-fold (without CCCP), and CCCP-induced ubiquitination ˜8-fold, consistent with a dominant-negative mechanism (FIGS. 7A, C). CCCP did not induce detectable Tom20 or MIRO ubiquitination in the parental HEK-293 cell line (lacking GFP-Parkin) (FIG. 6A). In this cell line, however, overexpression of USP30-C77S was still able to enhance basal Tom20 ubiquitination and USP30 to suppress it (FIG. 6A). Taken together, our data indicate that MIRO and Tom20 are substrates of USP30 and that USP30 can counteract Parkin-mediated ubiquitination of both MIRO and Tom20 following mitochondria damage.


It was found that a subset of the shared substrates were regulated by USP30 even under basal conditions (exemplified by Tom20, discussed above). MUL1, ASNS and FKBP8—but not MIRO—were substrates that behaved similarly to Tom20; i.e. they also exhibited a basal increase in ubiquitination with USP30 knockdown in the absence of CCCP. Thus, USP30 basally deubiquitinates this set of proteins, possibly counterbalancing against a mitochondrial E3 ligase that is active in the absence of CCCP and that acts on Tom20 but not MIRO. On the other hand, proteins such as TOM70, MAT2B and PTH2 behaved similarly to MIRO in that they exhibited enhanced ubiquitination with USP30 knockdown only following CCCP, suggesting that USP30 engages in deubiquitination of these proteins only after Parkin is recruited to mitochondria. Parkin, following recruitment to damaged mitochondria, may target both Tom20 and MIRO types of USP30 substrates, shifting the balance towards their polyubiquitination.


Using the same experimental system (cells overexpressing GFP-Parkin and HA-ubiquitin), the function of endogenous USP30 was tested by shRNA suppression. USP30 knockdown did not affect basal ubiquitination of MIRO (in the absence of CCCP-induced mitochondria damage). After mitochondrial depolarization (CCCP 5 μM, 2 hours), however, and consistent with the MS experiments, USP30 knockdown increased the level of ubiquitinated MIRO ˜2.5-fold, as measured in HA-ubiquitin immunoprecipitates (FIGS. 7D, E). Notably, USP30 knockdown increased both basal and CCCP-induced Tom20 ubiquitination, similar to enzymatically-inactive USP30 (FIGS. 7D, F). The increase in MIRO and Tom20 ubiquitination caused by USP30 shRNA was prevented by expression of the rat USP30 cDNA that is insensitive to human USP30 shRNA, indicating the specificity of the RNAi effect (FIG. 6B). Thus, these biochemical data corroborate the MS findings that endogenous USP30 acts as a brake on ubiquitination of both Tom20 and MIRO.


Parkin has previously been shown to assemble K27-, K48- and K63-type polyubiquitin chains on various mitochondrial substrates (Geisler et al., Nat. Cell Biol., 12: 119-131 (2010)). To examine the polyubiquitin chain topology on Tom20 and Miro, we repeated the ubiquitination assays with HA-ubiquitin mutants where all seven lysine residues were individually replaced with arginine (single K-to-R mutants), or with mutants where a single lysine was left intact and all other six lysines were replaced with arginine. We compared the amount of CCCP-induced Tom20 and Miro ubiquitination afforded by these ubiquitin mutants to wild-type ubiquitin. Among all “single K-to-R mutants”, only the K27R mutation blocked the CCCP-induced ubiquitination of Tom20, whereas the other K-to-R mutants (K6R, K11R, K29R, K33R, K48R, K63R) supported normal Tom20 ubiquitination (FIGS. 6C, D). Conversely, normal Tom20 ubiquitination was only supported by ubiquitin with K27 intact (all other lysines mutated), whereas all other single lysine mutants (K6, K11, K29, K33, K48, K63) had impaired Tom20 ubiquitination (FIGS. 6E, F). Thus, K27 on ubiquitin is both necessary and sufficient for building polyubiquitin chains on Tom20, suggesting the primary polyubiquitin topology on Tom20 is K27-type chains. Similar to Tom20, Miro also required K27 (and not the other lysines on ubiquitin) for its normal ubiquitination (FIGS. 6C, D). Although the ubiquitin mutant that contains only K27 supported Miro ubiquitination the best, significantly less ubiquitin was attached on Miro as compared to wild-type ubiquitin (˜65% of wild-type ubiquitin), suggesting that Miro accumulates other chain-types in addition to K27 (FIGS. 6E, F). Our data are consistent with Parkin's ability to assembly K27-linked chains on other substrates (Geisler et al., Nat. Cell Biol., 12: 119-131 (2010)).


Beyond ubiquitination, does USP30 regulate protein turnover in addition to ubiquitination? Published evidence suggests that Parkin mediates the degradation of multiple mitochondrial outer membrane proteins (Chan et al., Human Mol. Genet., 20: 1726-1737 (2011)). Consistent with this, all of the several outer membrane proteins examined (MIRO, MFN-1, TOM70, VDAC, Tom20) showed significant drop in protein level during the 6 hours of CCCP treatment (5 μM) of GFP-Parkin stable cell lines (FIGS. 6G, H). Tom20 levels were also reduced but to a lesser extent than MIRO and other outer membrane proteins (10+/−1% decrease with CCCP at t=6 h, p<0.01) (FIGS. 6G, H). In contrast to the outer membrane proteins, mitochondrial matrix protein HSP60 and inner membrane protein TIMM8A were unchanged by CCCP within this time frame. Overexpression of USP30 in GFP-Parkin stable cells greatly attenuated or abolished the CCCP-induced depletion of MIRO and Tom20 (FIGS. 6G, H). Stabilization by USP30 overexpression appeared to be relatively specific for MIRO and Tom20, since CCCP-induced degradation of other mitochondria membrane proteins (MFN-1, TOM70, VDAC) was unaffected (FIGS. 6G, H). Unlike wildtype USP30, the inactive C77A- or C77S-USP30 mutants did not inhibit degradation of MIRO or Tom20 induced by CCCP, implying requirement for DUB activity (FIGS. 6G, H). These data indicate USP30 can specifically counteract degradation of MIRO and Tom20 without affecting the turnover of other mitochondrial proteins.


Since MIRO and Tom20 degradation accompanies mitophagy (FIGS. 6G, H and (Chan et al., Human Mol. Genet., 20: 1726-1737 (2011)) and USP30 knockdown enhances mitophagy (FIGS. 5C, E) and ubiquitination of MIRO and Tom20 (FIG. 7), it was speculated that the depletion of these proteins might trigger mitophagy. In this model, overexpression of these proteins would block mitophagy induced by USP30-knockdown. Instead, it was found that overexpression of MIRO or Tom20 in neurons—even by themselves—led to a robust increase in mitophagy in the mt-Keima assay (FIGS. 8B, C, D, E), an effect similar to USP30 knockdown. It was therefore hypothesized that it is the ubiquitination of MIRO and Tom20 that serves as the signal for mitophagy (rather than their degradation, which occurs secondary to ubiquitination), and that overexpression of MIRO and Tom20 promotes mitophagy by increasing the pool of these substrates available for ubiquitination.


MS analysis of ubiquitinated peptides derived from Tom20 identified 3 lysine residues (K56, K61 and K68) whose ubiquitination increased upon CCCP or USP30 knockdown, and that increased even further in response to the combination of CCCP treatment+USP30 knockdown (FIG. 9). To confirm ubiquitination on these particular sites, the three lysine residues in Tom20 were mutated to arginine (“3KR-Tom20” (K56R, K61R, K68R mutant)). In GFP-Parkin overexpressing cells, myc-tagged wildtype Tom20 exhibited an increase in ubiquitination with coexpression of enzymatically-inactive USP30-C77S (FIG. 8A), similar to endogenous Tom20 (FIGS. 7A, C). In contrast, 3KR-Tom20 showed less basal ubiquitination than wild-type Tom20 and additionally it was unaffected by the dominant negative USP30-C77S (FIG. 8A), indicating that these three lysine residues are the major USP30 target residues on Tom20.


In the mt-Keima assay in neurons, overexpression of wild-type Tom20 enhanced mitophagy, whereas the 3KR-Tom20 mutant failed to do so (FIGS. 8B, D); thus Tom20 is sufficient to drive mitophagy, but this ability depends on its ubiquitination. Moreover, 3KR-Tom20 blocked the increase in mitophagy induced by USP30-C77S (FIGS. 8B, D), implying that the increased mitophagic flux caused by dominant-negative USP30 requires Tom20 ubiquitination. Alternatively, overexpressed 3KR-Tom20 may be able to oppose USP30-C77S-induced mitophagy by physically associating with USP30 in a non-catalytic manner.


Mass spectrometry identified nine lysine-ubiquitination sites on MIRO regulated by USP30 and Parkin, some of which are known to be required for normal MIRO function (e.g. K427 required for GTPase activity (Fransson et al., Bioch. Biophys. Res. Comm., 344: 500-510 (2006)). Thus, instead of a pursuing a combinatorial mutagenesis, the effect of USP30 on MIRO's ability to induce mitophagy was studied since USP30 knockdown increases MIRO ubiquitination (FIGS. 7D, E). Consistent with the idea that MIRO ubiquitination drives mitophagy, USP30 knockdown further enhanced mitophagy beyond what was observed following MIRO overexpression alone (FIGS. 8C, E). Taken together, these data indicate that ubiquitination of MIRO or Tom20 can drive mitophagy in neurons, and that inhibition of mitophagy by USP30 can be explained at least in part by deubiquitination of these proteins.


Example 6
USP30 Knockdown Rescues Mitophagy Defect Associated with PD Mutations

If mitochondrial degradation defects associated with PD-linked mutations of Parkin are due to impaired ubiquitination of damaged mitochondria, and USP30 indeed functions as a biochemical and functional antagonist of Parkin, then inhibiting USP30 should restore mitochondria ubiquitination and degradation. To test this hypothesis, we focused on PD-linked Parkin pathogenic mutants, such as G430D and K161N, that display attenuated ligase activity (Sriram et al., Human Mol. Genet., 14: 2571-2586 (2005)) with accompanying defects in mitophagy (Geisler et al., Nat. Cell Biol., 12: 119-131 (2010); Lee et al., J. Cell Biol., 189: 671-680 (2010)) were studied (e.g. G430D and K161N).


In SH-SY5Y cells transfected with pathogenic mutant GFP-Parkin-G430D and treated with CCCP, mitochondria fail to be cleared and form perinuclear clusters in association with the defective Parkin protein (FIG. 10A, first column). The same cells doubly transfected with Parkin-G430D and USP30 siRNA, which led to knockdown of USP30 protein by ˜60% (FIG. 11A), showed a 60% decrease in mitochondria (as measured by total Tom20 fluorescence) compared to cells transfected with Parkin-G430D and control siRNA (FIG. 10A, quantified in B). This result shows that siRNA knockdown of USP30 protein level can largely rescue mitophagy in the face of defective Parkin. Mitochondria degradation was not rescued by knockdown of other DUBs (USP6, USP14) (FIG. 11B-D). Re-introduction of an RNAi-resistant wildtype USP30 (rat USP30 cDNA), but not the inactive rat USP30-C77S mutant, prevented the rescue of mitochondrial degradation by USP30 siRNA (FIGS. 10A, B). Rescue of mitochondrial degradation was correlated with loss of perinuclear clusters of mutant G430D Parkin (usually associated with mitochondria) and appearance of smaller dispersed Parkin-containing puncta throughout the cytoplasm (FIGS. 10A, C; see also FIGS. 11B, C). In CCCP-treated GFP-Parkin-G430D expressing cells, USP30 knockdown not only led to loss of Tom20 immunoreactivity but also decreased staining for matrix protein HSP60 suggesting that USP30 suppression restored degradation of the whole mitochondrion (FIGS. 11E, G). The mitochondrial degradation defect associated with another PD-associated Parkin mutant (K161N) was similarly rescued with USP30 siRNA knockdown (FIGS. 11F, H). In neurons, reduced mitophagy associated with Parkin knockdown (as measured in the mt-Keima assay) was also rescued with dominant-negative USP30-C77A (FIGS. 10D, E). Thus, suppressing the expression or activity of USP30 allows cells to overcome defective Parkin or Parkin knockdown and restore the clearance of damaged mitochondria.


While not intending to be bound by any particular theory, since Parkin ligase activity marks mitochondria through ubiquitination, some residual ligase activity present in Parkin mutants may be needed in order for USP30 knockdown to rescue mitophagy. It is currently unknown whether USP30 knockdown would be effective with complete loss of Parkin activity. It is possible, however that there are other E3s that have overlapping substrates or that can compensate for lack of Parkin.


Example 7
USP30 is a Parkin Substrate

Since Parkin has broad activity towards outer mitochondrial membrane proteins, we wondered whether Parkin ubiquitinates USP30, which also resides at this mitochondrial compartment. Supporting this possibility, we identified USP30-derived ubiquitinated peptides in proteomics experiments in GFP-Parkin expressing cells treated with CCCP (fold change in ubiquitination of USP30 in ‘GFP-Parkin+CCCP’ over ‘DMSO’=27.23, p<0.001). To confirm USP30 ubiquitination by Parkin, we repeated the ubiquitination assay in cells overexpressing GFP-Parkin and HA-ubiquitin, and found GFP-Parkin induced ubiquitination of endogenous USP30 following CCCP treatment (20 μM, 2 hours, FIGS. 9C, D). The ubiquitination sites of USP30 (K235 and K289) identified by mass spectrometry were not required for its ubiquitination suggesting other lysine residues in USP30 can accept ubiquitin (data not shown). CCCP treatment (20 μM) also induced a significant drop in USP30 levels in GFP-Parkin expressing cells (FIGS. 9E, F). Importantly, Parkin with pathogenic mutations G430D or K161N were not able to ubiquitinate (FIGS. 9C, D) or degrade USP30 (FIG. 9E-F). These data indicate that Parkin ubiquitinates and degrades USP30, thus removing the brake on mitophagy.


Example 8
USP30 Knockdown Decreases Oxidative Stress and Provides Protection In Vivo

Whether USP30 knockdown provides functional benefit to mitochondria and cells was examined next. ROS—which largely derive from mitochondria—is associated with neurodegenerative disorders and mitochondria dysfunction may contribute to increased oxidative stress in PD (Lee et al., Biochem. J., 441: 523-540 (2012)). To measure oxidative stress in mitochondria, mitochondria matrix-targeted ro-GFP (mito-roGFP), a redox-sensitive fluorescent protein that allows quantitative ratiometric imaging of mitochondrial redox potential was used (Dooley et al., J. Biol. Chem. 279: 22284-22293 (2004)). Following measurement of ratiometric mito-roGFP signal in individual cells, the dynamic range of the probe was calibrated by treating cultures sequentially with DTT (1 mM) to fully reduce the probe and aldrithiol (100 μM) to fully oxidize the probe (Guzman et al., Nature, 468: 696-700 (2010). Ratios of mito-roGFP measured after DTT and aldrithiol were set to 0 and 1, respectively, to calibrate the relative oxidation index. In control cells transfected with control luciferase shRNA, neurons had a mean relative oxidation index of ˜0.6 (FIGS. 17A, B). USP30 knockdown dropped the relative oxidation index to ˜0.4, suggesting that suppression of USP30 protein led to a reduction in mitochondrial oxidative stress.


To test whether knocking down USP30 would provide protection under stress conditions in vivo, we used Drosophila, which has emerged as an effective model system for studying PD molecular pathogenesis (Guo, Cold Spring Harb. Perspect. Med. 2(11) pii: a009944 (2012)). To knock down fly USP30 (CG3016, hereafter called dUSP30), we employed the GAL4/UAS system (Brand et al., Development, 118: 401-415 (1993)). We crossed an Actin-GAL4 driver line with a UAS-dUSP30RNAi transgenic line, which allows expression of dUSP30 RNAi under the control of the Actin promoter (this Actin-GAL4>dUSP30RNAi line is referred to as ‘dUSP30 knockdown’ line). Activation of UAS-dUSP30RNAi by Actin-GAL4 led to a ˜90% reduction of dUSP30 mRNA by quantitative RT-PCR, compared to control parental lines containing only Actin-GAL4 or only UAS-dUSP30RNAi (FIG. 17C). To test the protective effects of dUSP30 knockdown, we crossed the ‘dUSP30 knockdown’ line with parkin mutant flies (park25) (Greene et al., Proc. Natl. Acad. Sci. USA, 100: 4078-4083 (2003)). Flies lacking parkin show severe defects in mitochondrial morphology in their indirect flight muscles (IFMs), with mitochondria that are malformed with sparse, disorganized cristae, giving rise to a “pale” appearance of mitochondria under EM (FIG. 12A, and Greene et al., Proc. Natl. Acad. Sci. USA, 100: 4078-4083 (2003)). In contrast, wild-type flies have many dark-staining mitochondria evenly packed with cristae (FIG. 12A). To determine the effect of USP30 inhibition on Parkin-deficient mitochondria, we crossed parkin mutants to dUSP30 knockdown flies. In the “parkin mutant; dUSP30 knockdown” flies, most of the IFM mitochondria were electron-dense and contained numerous cristae, although “pale” mitochondria with fragmented cristae were also occasionally found (FIG. 12A). Quantification of the percent area of mitochondria containing disorganized cristae over total mitochondria area revealed a strong improvement in mitochondrial integrity with dUSP30 knockdown (FIG. 12B—˜90% in parkin mutants versus ˜25% in “parkin mutant; dUSP30 knockdown”). “Parkin mutant; dUSP30 knockdown” flies also had less damaged mitochondria per muscle area (FIG. 12C). Thus, suppressing dUSP30 expression is able to largely restore morphological mitochondrial integrity in vivo in parkin-deficient Drosophila.


To examine the effect of suppressing USP30 in neurons that are relevant to PD, we used dopamine decarboxylase (Ddc)-GAL439 to drive dUSP30RNAi specifically in aminergic neurons of the fly nervous system. As a model of mitochondrial damage and PD, we treated flies with paraquat, a mitochondrial toxin linked to PD (Castello et al., J. Biol. Chem., 282: 14186-14193 (2007); Cocheme et al., J. Biol. Chem., 283: 1786-1798 (2008); Tanner et al., Environmental Health Perspect., 119: 866-872 (2011)). Following treatment with paraquat (10 mM, 48 hours), both the Ddc-GAL4 and UAS-dUSP30RNAi control fly lines showed reduced ability to climb up beyond 15 cm (FIG. 12D). This climbing defect was fully rescued by additional treatment with L-DOPA (FIG. 17D), showing that this behavioral deficit is likely due to depletion of dopamine. Consistently, in control fly lines (Ddc-GAL4 or UAS-dUSP30RNAi transgenics alone), paraquat treatment (10 mM, 48 hours) caused a 30-60% reduction in dopamine levels in fly heads without altering serotonin neurotransmitter levels (indicating specific toxicity of paraquat on the dopaminergic system in this model—FIG. 12F and FIG. 17E). Similar to L-DOPA, dUSP30 knockdown in Ddc-GAL4>UAS-dUSP30RNAi flies also completely rescued the paraquat-induced climbing impairment (FIG. 12D), indicating that complete protection against paraquat toxicity in this behavioral test can be afforded by suppression of USP30 specifically in aminergic neurons. A similar complete protection was also observed with whole body knockdown of USP30 in Actin-GAL4>UAS-dUSP30RNAi flies (FIG. 12E). Strikingly, USP30 knockdown in Ddc neurons (Ddc-GAL4>UAS-dUSP30RNAi flies) also prevented the paraquat-induced dopamine depletion (FIG. 12F). Since USP30 knockdown rescued both depletion of dopamine and motor impairment, these results show that suppression of USP30 can benefit dopaminergic neurons and the organism in both neurochemical and functional terms.


To test whether USP30 knockdown has an effect on organism survival, we monitored the percentage of live flies over prolonged treatment with paraquat (10 mM, 96 hours). Flies expressing dUSP30 RNAi survived significantly longer than controls (FIG. 12G). Only <15% of flies treated with paraquat were alive in Actin-GAL4 and UAS-dUSP30RNAi control groups at 96 hours whereas ˜45% of flies were alive in the Actin-GAL4>UAS-dUSP30RNAi (‘whole body dUSP30 knockdown’) group (FIG. 12G). We confirmed that the benefit of USP30 knockdown was not due to differences in exposure to paraquat since all three fly lines ingested roughly equal amounts of paraquat as measured by LC-MS/MS (average mass of paraquat per fly: UAS-dUSP30RNAi: 3.2 μg, Actin-GAL4: 2.7 μg, Actin-GAL4>UAS-dUSP30RNAi: 2.7 μg). Knockdown of other DUBs in flies (dUSP47 (CG5486) or dYOD1 (CG4603)) did not provide benefit in the survival assay; if anything, they exacerbated the rate of death in response to paraquat (FIG. 17F-H) Furthermore, introduction of a human USP30 cDNA into flies expressing dUSP30RNAi reversed the survival benefit provided by dUSP30RNAi (FIG. 171), demonstrating the specificity of the RNAi effect. Remarkably, USP30 knockdown specifically in Ddc neurons was sufficient to provide significant survival benefit, albeit less than the whole body USP30 knockdown (FIG. 12H). This result implies that a significant portion of the organismal benefit of USP30 suppression is mediated in dopaminergic neurons, and it further reinforces the idea that USP30 plays a critical role in dopaminergic neuron dysfunction.


Example 9
Discussion

Better understanding of the pathogenic mechanisms in PD would be helpful for rational design of disease-modifying therapies for this neurodegenerative disease. Impaired activity of oxidative phosphorylation enzymes (Schapira et al., Lancet, 1: 1269 (1989)), elevated levels of oxidative stress markers (Lee et al., Biochem. J., 441: 523-540 (2012)) and mtDNA mutations (Bender et al., Nature Genet., 38: 515-517 (2006); Kraytsberg et al., Nature Genet., 38: 518-520 (2006)) in PD suggest accumulation of defective mitochondria (Zheng et al., Science Transl. Med., 2: 52ra73 (2010)). PINK1/Parkin genetics further implicate aberrant mitochondrial biology and point to impaired mitochondrial quality control as a causative factor in the etiology of PD (Youle et al., J. Biol. Chem., 12: 9-23 (2011)). Uncleared damaged mitochondria can be a source of toxicity and “pollute” the mitochondrial network through fusion with healthy mitochondria (Tanaka et al., J. Cell. Biol., 191: 1367-1380 (2010)).


We have identified USP30, a DUB localized to mitochondria, as a negative regulator of mitophagy. USP30, through its deubiquitinase activity, opposes Parkin-mediated ubiquitination and degradation of mitochondrial proteins and reverses the marking of damaged mitochondria for mitophagy. Knockdown inhibition of USP30 accelerated mitophagy, and it restored CCCP-induced mitochondrial degradation in cells expressing PD-associated mutants of Parkin. USP30 knockdown improves mitochondrial integrity in parkin mutant flies, confirming that Parkin and USP30 have opposing actions on mitochondrial quality in vivo. USP30 knockdown also conferred motor behavior and survival benefits in wildtype flies treated with paraquat, further supporting the idea that USP30 inhibition might ameliorate the effects of mitochondrial damage.


Parkin, USP30 and Mitochondrial Quality Control

Although Parkin and PINK1 are identified as key players in mitophagy, a detailed mechanistic understanding of the mitophagy pathway, especially in mammalian cells, is lacking. The fact that basal mitophagy in neurons depends on Parkin and PINK1 (FIG. 3) suggests that these proteins actively monitor normally occurring mitochondrial damage. Mitochondria fission—which appears to be required for Parkin-mediated mitophagy (Tanaka et al., J. Cell. Biol., 191: 1367-1380 (2010))—may contribute to basal mitochondrial turnover by eliciting a transient drop in membrane potential in one of the two daughter mitochondria (Twig et al., EMBO J., 27: 433-446 (2008)). This transient drop in membrane potential creates an opportunity for PINK1 accumulation and Parkin recruitment, leading to eventual mitophagy if membrane potential is not quickly re-established. Thus under basal conditions, USP30 knockdown may accelerate mitophagy by favoring Parkin-mediated ubiquitination during the fission-associated drops in mitochondrial membrane potential. As damaged mitochondria are more likely to accumulate Parkin, it is expected that suppression of USP30 function will preferentially clear unhealthy mitochondria.


Since Parkin ligase activity marks mitochondria through ubiquitination, some residual ligase activity present in Parkin mutants is presumably required in order for USP30 knockdown to rescue mitophagy. It would be expected that with complete loss of Parkin activity, USP30 knockdown would be ineffective at rescuing clearance unless other E3 ligases have overlapping substrates and can compensate for lack of Parkin. The rescue of mitochondrial integrity with USP30 knockdown in parkin mutant flies, even though a large portion of parkin gene is missing, supports the latter possibility.


As part of normal turnover, the cleared mitochondria presumably need to be replaced through mitochondrial biogenesis. In culture cell lines, mitochondrial damage increases overall mitochondrial mass (Narendra et al., PLoS Biology, 8: e1000298 (2010)). In this context it is interesting to note that Parkin can also boost mitochondrial biogenesis by degrading negative transcriptional regulators (Shin et al., Cell 144: 689-702 (2011)). Further studies are required to determine whether USP30 also regulates the biogenesis pathway and whether mitophagy induced by USP30 inhibition is accompanied by new mitochondria production.


USP30 Versus Parkin on Common Substrates

Global ubiquitination site mapping experiments identified multiple substrates whose ubiquitination is affected by both Parkin overexpression and USP30 knockdown. Amongst these shared presumptive substrates, we confirmed that Miro and Tom20 have ubiquitination levels that are antagonistically regulated by Parkin and USP30, i.e. GFP-Parkin overexpression or USP30 knockdown increased ubiquitination induced by CCCP treatment. Interestingly, a subset of these shared substrates, exemplified by Tom20, was regulated by USP30 even under basal conditions. Mull, Asns and Fkbp8—but not Miro—were mitochondrial substrates that behaved similarly to Tom20, exhibiting a basal increase in ubiquitination with USP30 knockdown in the absence of CCCP. Thus, USP30 basally deubiquitinates this set of proteins, presumably by counterbalancing against a mitochondrial E3 ligase that is active in the absence of CCCP and that acts on Tom20 but not Miro. Following CCCP, USP30 also counteracts Parkin dependent ubiquitination of this set of substrates. On the other hand, proteins such as Tom70, Mat2b and Pth2, behaved similarly to Miro in that they exhibited enhanced ubiquitination with USP30 knockdown only following CCCP. This observation suggests that these set of proteins undergo low levels of basal ubiquitination in the absence of recruited Parkin (i.e. Parkin is their major E3 ligase), or that USP30 is inactive toward those proteins under basal conditions. Mitochondrial depolarization regulates Parkin's E3 ligase activity (Matsuda et al., J. Cell Biol., 189: 211-221 (2010)) possibly via PINK1-mediated phosphorylation (Kondapalli et al., Open Biology, 2: 120080 (2012); but see Vives-Bauza et al., Proc. Natl. Acad. Sci. USA, 107: 378-383 (2010)); it remains to be studied whether the activity of USP30—which is constitutively localized on mitochondria—is also regulated by mitochondrial damage.


Global ubiquitination analysis also revealed a series of non-mitochondrial proteins whose ubiquitination was inversely regulated by Parkin and USP30 (including nuclear proteins, metabolic enzymes and components of the ubiquitin-proteasome system (UPS)). This suggests that the antagonistic functional relationship between Parkin and USP30 may extend beyond mitochondria. These non-mitochondrial “substrates” may be indirectly regulated by Parkin and USP30, or may have subpopulations on mitochondria, but have not formally been assigned as having mitochondrial localization due to the strict filtering criteria employed by computational tools (Pagliarini et al., Cell, 134: 112-123 (2008)). MS also identified some proteins that showed enhanced ubiquitination with CCCP (under endogenous Parkin and USP30 levels) with no further increase in ubiquitination upon Parkin overexpression or USP30 knockdown (e.g. Ssbp, ZO1, Rab10, Vamp1), suggesting engagement of alternative UPS pathways following mitochondrial depolarization. It is appropriate to note that in some cases, elevated levels of ubiquitinated species can derive from increases in the level of the total protein substrate itself.


Interestingly, Parkin also ubiquitinates and degrades USP30, and pathogenic Parkin mutations blocked this ability to downregulate USP30. Failure to remove a negative regulator of mitophagy may exacerbate the inefficient ubiquitination associated with these Parkin mutants, and could also partly explain the rescue of mitophagy by USP30 knockdown.


Parkin, USP30 and Neurodegeneration

PD-linked mutations in Parkin may lead to decreased catalytic activity, enhanced aggregation and/or reduced expression (Hampe et al., Human Mol. Genet., 15: 2059-2075 (2006); Matsuda et al., J. Biol. Chem., 281: 3204-3209 (2006); Wang et al., J. Neurochem., 93: 422-431 (2005); Winklhofer et al., J. Biol. Chem. 278: 47199-47208 (2003)). In sporadic PD, Parkin activity can be also inhibited due to cellular stress (Corti et al., Physiol. Rev., 91: 1161-1218 (2011)). PD-linked PINK1 mutations impair translocation of Parkin to damaged mitochondria (Matsuda et al., J. Cell Biol., 189: 211-221 (2010); Narendra et al., PLoS Biology, 8: e1000298 (2010); Vives-Bauza et al., Proc. Natl. Acad. Sci. USA, 107: 378-383 (2010)). Thus, reduced function of Parkin in mitochondrial quality control is likely more prevalent in PD than as represented by rare Parkin mutations, providing further support for a possible utility of USP30 inhibition in idiopathic PD. Consistent with a benefit of USP30 inhibition, knockdown of USP30 restores mitophagy in cells expressing PD-associated mutant Parkin and reduces oxidative stress in neurons. In Drosophila parkin mutants, knockdown of USP30 improves mitochondrial integrity. Furthermore, USP30 knockdown provides a benefit in behavior and survival assays against paraquat, an oxidative stressor linked to mitochondria (Castello et al., J. Biol. Chem., 282: 14186-14193 (2007); Cocheme et al., J. Biol. Chem., 283: 1786-1798 (2008)). Since paraquat is a mitochondrial poison epidemiologically linked to PD (Tanner et al., Environmental Health Perspect., 119: 866-872 (2011)), our findings provide in vivo evidence that inhibition of USP30 might be helpful in diseases caused by mitochondrial damage and dysfunction.


In PD, mitochondrial dysfunction is not specific to substantia nigra neurons and is present systemically (Schapira et al, Parkinson's Dis., 2011: 159160 (2011)). Since USP30 expression seems to be widespread (Nakamura and Hirose, Mol. Biol. Cell, 19: 1903-1914 (2008)), USP30 inhibition has the potential to provide wide benefit by promoting clearance of damaged mitochondria. In addition to neurons, long-lived metabolically active cells such as cardiomyocytes also rely on an efficient mitochondrial quality control system (Gottlieb et al., Am. J. Physiol. Cell Physiol., 299: C203-210 (2010)). In this context, Parkin has been shown to protect cardiac myocytes against ischemia/reperfusion injury through activating mitophagy and clearing damaged mitochondria in response to ischemic stress (Huang et al., PLoS One, 6: e20975 (2011)). In inherited mitochondrial diseases, mtDNA mutations co-exist with wildtype mtDNA within the same cells, and mitochondrial dysfunction and disease ensue only when the proportion of mutated mtDNAs is high (Bayona-Bafaluy et al., Proc. Natl. Acad. Sci. USA, 102: 14392-14397 (2005)). Interestingly, Parkin overexpression eliminates mitochondria with deleterious mtDNA mutations and restores mitochondrial function, presumably by degrading mitochondria containing mutant mtDNA (Suen et al., Proc. Natl. Acad. Sci. USA, 107: 11835-11840 (2010)). Thus, USP30 inhibition has the potential to benefit diseases beyond PD by enhancing mitochondrial quality.


Example 10
Peptide Inhibitors of USP30

Two types of phage-displayed naïve peptide libraries, Linear-lib and Cyclic-lib, were cycled through rounds of binding selections with biotinylated USP30_cd (USP30 catalytic domain with C77A mutation) in solution as described previously (Stanger, et al., 2012, Nat. Chem. Bio., 7: 655-660). The selection identified peptide USP303 and USP308, which had moderate spot ELISA signals (signal/noise ratios of ˜5). USP30 and USP8 have the sequences:











USP30_3:
PLYCFYDLTYGYLCFY;
(SEQ ID NO: 1)





USP30_8:
VSRCYIFWNEMFCDVE.
(SEQ ID NO: 2)






USP303 and USP308 were then assayed for inhibition of USP30 and also inhibition of USP7, USP5, UCHL3, and USP2, to determine each peptide's specificity for USP30, as follows. USP30 peptide ligands at a concentration range of 0-100 μM (for USP303) and 0-500 μM (for USP308) were mixed with 250 nM ubiquitin-AMC (Boston Biochem., Boston, Mass.; Cat. No. U-550). A panel of DUBs at 5.6, 2, 2, 5 and 0.05 nM for USP30, USP7, USP2, USP5, and UCHL3, respectively, in PBS buffer containing 0.05% Tween20, 0.1% BSA and 1 mM DTT for 30 minutes were added to the ubiquitin-AMC/USP30 peptide ligands mixture and the initial velocity was immediately measured by monitoring fluorescence (excitation at 340 nm and emission at 465 nm) using SpectraMax®M5e (Molecular Device, Sunnyvale, Calif.). The initial rates were calculated based on slopes of increasing fluorescence signal. The velocity was normalized to the percentage of the rate when the peptide ligand concentration was zero, and the data was processed using KaleidaGraph by fitting to the following equation:






v
=


v
0

+



v
max

-

v
0



1
+


(

I

IC
50


)

n








in which v is the percentage of maximum rate; I is the concentration of inhibitor (USP30 peptide ligands); v0 and vmax are minimum and maximum percentage of the rate, respectively.


The results of that experiment are shown in FIG. 14. Peptide USP303 showed good specificity for USP30, with an IC50 of 8.0 μM. The IC50 of peptide USP303 for USP5 was more than 6-fold higher, at 49.4 μM, and for USP2 was more than 10-fold higher, at about 100 μM. The IC50 of peptide USP303 for UCHL3 and USP2 was >200 μM.


Peptide USP303 was selected for affinity maturation. To improve the affinity, a soft randomized library was constructed using the USP303 sequence as the targeted parent, and panned against USP30_cd in solution as described previously (Stanger, et al., 2012, Nat. Chem. Bio., 7: 655-660). After four rounds of solution panning, 20 peptides were identified that bound to USP30 catalytic domain with stronger spot phage ELISA signal than the parent USP303, manifested by an improvement in the signal/noise ratio of about 3-6 fold.



FIG. 15 shows a graph of residue probability by position in USP303 and the affinity matured peptides. The sequences for certain affinity matured peptides are shown below the graph, along with the signal to noise ratio (“S/N”), which is the ratio of the spot phage ELISA signal (“signal”) detected against biotinylated USP30 cd captured on NeutrAvidin-coated 384 well Maxisorb plates versus the ELISA signal against the NeutrAvidin-coated plate alone. FIG. 15 also shows the number of occurrences of each sequence in the selection (“n”), the total number of clones (“Total”; 66), and the number of unique sequences (“Uniq”; 20). All of the affinity matured peptides shown had signal to noise ratios of greater than 10 except for USP303.27 and USP30_3.62.


Certain peptides were then tested for specificity for USP30 versus other deubiquitinating enzymes. Binding was tested by ELISA. FIG. 16 shows the signal to noise ratio (“s/n ratio”) for binding of parent USP303 peptide and affinity matured peptides USP303.2, USP303.23, USP303.65, and USP303.88 to the catalytic domains of USP2, USP7, USP14, and USP30 (each with the active site cysteine mutated to alanine), and to the catalytic domains of UCHL1, UCHL3, and UCHL5. For the set of bar graphs for each peptide, the targets tested, from left to right, were USP2, USP7, USP14, USP30, UCHL1, UCHL3, and UCHL5.


Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.









TABLE 4







Table of Sequences









SEQ




ID




NO
Description
Sequence





26
Human
MLSSRAEAAM TAADRAIQRF LRTGAAVRYK VMKNWGVIGG IAAALAAGIY



ubiquitin-
VIWGPITERK KRRKGLVPGL VNLGNTCFMN SLLQGLSACP AFIRWLEEFT



specific
SQYSRDQKEP PSHQYLSLTL LHLLKALSCQ EVTDDEVLDA SCLLDVLRMY



peptidase 30
RWQISSFEEQ DAHELFHVIT SSLEDERDRQ PRVTHLFDVH SLEQQSEITP



(USP30);
KQITCRTRGS PHPTSNHWKS QHPFHGRLTS NMVCKHCEHQ SPVRFDTFDS



SwissProt
LSLSIPAATW GHPLTLDHCL HHFISSESVR DVVCDNCTKI EAKGTLNGEK



Q70CQ3.1
VEHQRTTFVK QLKLGKLPQC LCIHLQRLSW SSHGTPLKRH EHVQFNEFLM




MDIYKYHLLG HKPSQHNPKL NKNPGPTLEL QDGPGAPTPV LNQPGAPKTQ




IFMNGACSPS LLPTLSAPMP FPLPVVPDYS SSTYLFRLMA VVVHHGDMHS




GHFVTYRRSP PSARNPLSTS NQWLWVSDDT VRKASLQEVL SSSAYLLFYE




RVLSRMQHQS QECKSEE





27
Human
MVGRNSAIAA GVCGALFIGY CIYFDRKRRS DPNFKNRLRE RRKKQKLAKE



mitochondrial
RAGLSKLPDL KDAEAVQKFF LEEIQLGEEL LAQGEYEKGV DHLTNAIAVC



import receptor
GQPQQLLQVL QQTLPPPVFQ MLLTKLPTIS QRIVSAQSLA EDDVE



subunit 20




homolog




(Tom20);




GenBank




NP_055580.1






28
Human
MKKDVRILLV GEPRVGKTSL IMSLVSEEFP EEVPPRAEEI TIPADVTPER



MIRO1;
VPTHIVDYSE AEQSDEQLHQ EISQANVICI VYAVNNKHSI DKVTSRWIPL



SwissProt
INERTDKDSR LPLILVGNKS DLVEYSSMET ILPIMNQYTE IETCVECSAK



Q8IXI2.2
NLKNISELFY YAQKAVLHPT GPLYCPEEKE MKPACIKALT RIFKISDQDN




DGTLNDAELN FFQRICFNTP LAPQALEDVK NVVRKHISDG VADSGLTLKG




FLFLHTLFIQ RGRHETTWTV LRRFGYDDDL DLTPEYLFPL LKIPPDCTTE




LNHHAYLFLQ STFDKHDLDR DCALSPDELK DLFKVFPYIP WGPDVNNTVC




TNERGWITYQ GFLSQWTLTT YLDVQRCLEY LGYLGYSILT EQESQASAVT




VTRDKKIDLQ KKQTQRNVFR CNVIGVKNCG KSGVLQALLG RNLMRQKKIR




EDHKSYYAIN TVYVYGQEKY LLLHDISESE FLTEAEIICD VVCLVYDVSN




PKSFEYCARI FKQHFMDSRI PCLIVAAKSD LHEVKQEYSI SPTDFCRKHK




MPPPQAFTCN TADAPSKDIF VKLTTMAMYP HVTQADLKSS TFWLRASFGA




TVFAVLGFAM YKALLKQR





29
Human Parkin;
MIVFVRFNSS HGFPVEVDSD TSIFQLKEVV AKRQGVPADQ LRVIFAGKEL



GenBank
RNDWTVQNCD LDQQSIVHIV QRPWRKGQEM NATGGDDPRN AAGGCEREPQ



NP_004553.2
SLTRVDLSSS VLPGDSVGLA VILHTDSRKD SPPAGSPAGR SIYNSFYVYC




KGPCQRVQPG KLRVQCSTCR QATLTLTQGP SCWDDVLIPN RMSGECQSPH




CPGTSAEFFF KCGAHPTSDK ETSVALHLIA TNSRNITCIT CTDVRSPVLV




FQCNSRHVIC LDCFHLYCVT RLNDRQFVHD PQLGYSLPCV AGCPNSLIKE




LHHFRILGEE QYNRYQQYGA EECVLQMGGV LCPRPGCGAG LLPEPDQRKV




TCEGGNGLGC GFAFCRECKE AYHEGECSAV FEASGTTTQA YRVDERAAEQ




ARWEAASKET IKKTTKPCPR CHVPVEKNGG CMHMKCPQPQ CRLEWCWNCG




CEWNRVCMGD HWFDV





30
Human
MAVRQALGRG LQLGRALLLR FTGKPGRAYG LGRPGPAAGC VRGERPGWAA



PINK1;
GPGAEPRRVG LGLPNRLRFF RQSVAGLAAR LQRQFVVRAW GCAGPCGRAV



SwissProt
FLAFGLGLGL IEEKQAESRR AVSACQEIQA IFTQKSKPGP DPLDTRRLQG



Q9BXM7.1
FRLEEYLIGQ SIGKGCSAAV YEATMPTLPQ NLEVTKSTGL LPGRGPGTSA




PGEGQERAPG APAFPLAIKM MWNISAGSSS EAILNTMSQE LVPASRVALA




GEYGAVTYRK SKRGPKQLAP HPNIIRVLRA FTSSVPLLPG ALVDYPDVLP




SRLHPEGLGH GRTLFLVMKN YPCTLRQYLC VNTPSPRLAA MMLLQLLEGV




DHLVQQGIAH RDLKSDNILV ELDPDGCPWL VIADFGCCLA DESIGLQLPF




SSWYVDRGGN GCLMAPEVST ARPGPRAVID YSKADAWAVG AIAYEIFGLV




NPFYGQGKAH LESRSYQEAQ LPALPESVPP DVRQLVRALL QREASKRPSA




RVAANVLHLS LWGEHILALK NLKLDKMVGW LLQQSAATLL ANRLTEKCCV




ETKMKMLFLA NLECETLCQA ALLLCSWRAA L





31
USP30
TGCGGCCGCA GGTTCCGCTG TCTCGGGAAC CGTCGTATCC CTCGGTCCGG



mRNA;
CGGCGGCGGC GGCGGTAGCG GAGGAGACGG TTTCAGGCCT CCGGTGCGGC



GenBank
TGCAATGCTG AGCTCCCGGG CCGAGGCGGC GATGACCGCG GCCGACAGGG



NM_032663.3
CCATCCAGCG CTTCCTGCGG ACCGGGGCGG CCGTCAGATA TAAAGTCATG




AAGAACTGGG GAGTTATAGG TGGAATTGCT GCTGCTCTTG CAGCAGGAAT




ATATGTTATT TGGGGTCCCA TTACAGAAAG AAAGAAGCGT AGAAAAGGGC




TTGTGCCTGG CCTTGTTAAT TTAGGGAACA CCTGCTTCAT GAACTCCCTG




CTACAAGGCC TGTCTGCCTG TCCTGCTTTC ATCAGGTGGC TGGAAGAGTT




CACCTCCCAG TACTCCAGGG ATCAGAAGGA GCCCCCCTCA CACCAGTATT




TATCCTTAAC ACTCTTGCAC CTTCTGAAAG CCTTGTCCTG CCAAGAAGTT




ACTGATGATG AGGTCTTAGA TGCAAGCTGC TTGTTGGATG TCTTAAGAAT




GTACAGATGG CAGATCTCAT CATTTGAAGA ACAGGATGCT CACGAATTAT




TCCATGTCAT TACCTCGTCA TTGGAAGATG AGCGAGACCG CCAGCCTCGG




GTCACACATT TGTTTGATGT GCATTCCCTG GAGCAGCAGT CAGAAATAAC




TCCCAAACAA ATTACCTGCC GCACAAGAGG GTCACCTCAC CCTACATCCA




ATCACTGGAA GTCTCAACAT CCTTTTCATG GAAGACTCAC TAGTAATATG




GTCTGCAAAC ACTGTGAACA CCAGAGTCCT GTTCGATTTG ATACCTTTGA




TAGCCTTTCA CTAAGTATTC CAGCCGCCAC ATGGGGTCAC CCATTGACCC




TGGACCACTG CCTTCACCAC TTCATCTCAT CAGAATCAGT GCGGGATGTT




GTGTGTGACA ACTGTACAAA GATTGAAGCC AAGGGAACGT TGAACGGGGA




AAAGGTGGAA CACCAGAGGA CCACTTTTGT TAAACAGTTA AAACTAGGGA




AGCTCCCTCA GTGTCTCTGC ATCCACCTAC AGCGGCTGAG CTGGTCCAGC




CACGGCACGC CTCTGAAGCG GCATGAGCAC GTGCAGTTCA ATGAGTTCCT




GATGATGGAC ATTTACAAGT ACCACCTCCT TGGACATAAA CCTAGTCAAC




ACAACCCTAA ACTGAACAAG AACCCAGGGC CTACACTGGA GCTGCAGGAT




GGGCCGGGAG CCCCCACACC AGTTCTGAAT CAGCCAGGGG CCCCCAAAAC




ACAGATTTTT ATGAATGGCG CCTGCTCCCC ATCTTTATTG CCAACGCTGT




CAGCGCCGAT GCCCTTCCCT CTCCCAGTTG TTCCCGACTA CAGCTCCTCC




ACATACCTCT TCCGGCTGAT GGCAGTTGTC GTCCACCATG GAGACATGCA




CTCTGGACAC TTTGTCACTT ACCGACGGTC CCCACCTTCT GCCAGGAACC




CTCTCTCAAC TAGCAATCAG TGGCTGTGGG TCTCCGATGA CACTGTCCGC




AAGGCCAGCC TGCAGGAGGT CCTGTCCTCC AGCGCCTACC TGCTGTTCTA




CGAGCGCGTC CTTTCCAGGA TGCAGCACCA GAGCCAGGAG TGCAAGTCTG




AAGAATGACT GTGCCCTCCT GCAAGGCTAG AGCTGATGGC ACTGTCTGCA




CTGTCCAGGA AAAAAGTAAA ACTGTACTGT TGCGTGTGCA AGCGGCCCCA




CTAGAGCCTT CCAGCCTTCT GGTGTGTTCT AAGAGCAGGC TCCACCTGGG




AGCCAGCCCC AGTTCACACC AAACCAGGCT CCCTGAACAG TCCTGTTCAT




GTGTGTAGGT GGTTCTGTTG TGTTAAGAAA GCATTCATTA TGTCCGGAGT




GTCTTTTTAC TCATCTGATA CAGGTAATTA AAAGAACTCA GATTCTTGAA




GCCACCGTTT TCATATTGTA ATGTTAGGTG TTCTCAGAGG GGAGGTACCT




TTGTCTAATC AACGTTTCCA CTTAGATCTT TTATTTTTAA TAAGCAGGCC




CATAAAAATT GTTGACAAGA ATTAATGAAA TTATTAAAGG CAACAATTTA




GAAGAAAAAG TGCCTTTCAC TTTCGATTGC TTTTGTAGCA CGTCCATTGT




GAAATATTCC TTCCAGGCTA CTCAAAGGAT AGCAAGAGAA CAGGTAAATG




ATGCCTAAAG AACACCTTCC TTTTTCTATG CCTTTTCTAA TCTTTCAATT




CTTTCTATGG AGTAAAGGCT CATCTGCCAA ATCTGCCCCC TGGGGAAACT




CTTTCACTAC TTTGTCAGTT ATAAGTGAAG AGCTTACTTG TTGCTTTTAT




CTTTTGTATA TTGGACTGAG ATGTAATTAC ACTGTATTAT AAAACTCTGT




GAATAGCCAG AACTGAGCTG GATCTTTGCA ACACCTGATT CCTCTGCTCT




GTGGAAAACT TTTTCTTACA CAAGGATCCA CTGTGGACGG TTACTTTCAT




CTGTTTATTT ATTGCCCATG CAGAGCTCTT AAGGTTTACA GGTGGGAGCT




TGGGGCTGTA TAAAAAAATA ATCCCTGCCC TGAGTTGACA CCTGGCTTAG




GAAGGAAGGG CTGACTATGG GGCTGCAGTC TCTCTGAACC TCAGTTTCCT




CATTTGTGAA GTGAAGGGTT AGATTTGATG ACCACCAAAG TTCAGCCCTT




TTCACGAAAA GGAGAAAGCA GCTTTTGACT TTTTAAAAAA CATATAACTA




CAGCTGGCAT CTAGTATTGT CATGTTGCTC TAGGTCCATA TTCTGAATTT




ATTCATTTCC AATAGCCTAA TACAAAAAGT ATATATTGAG CACTTTCTTC




CCTTTTCAGG TAAGTCTCTG AATGCAGCCC AGGGCCAAAG GAATTTTGAT




GACACAGTAG TACCTATGTT TTAAGCTATA TTTTTAATTT AGAAAAATGG




ATACCAAATT CAAACCGACT CATCAGAGGT AAGATTTGGA ATCAGACCTT




TCCAAAAGGT CATCTGAGGT AAGGCTAAGA CCGCACTTCC TCTGCTGGGG




GTGAGCTGGC AGACACACCA AACAGTGCCT TGGCAGCAGC TCACAGTGCA




GGAAGCCCAG GTGATCACTC TTCTGCTGGG CCCAGGCTGC ACCCTGAGGA




CTCAGTAACT CACTCTCAAC AGAATATTCT GTGCAGGCTC TCCAGGCTCT




GGGCGTCAGG GTGCAAGGGG CAGCTTGAAC TGTACGGTCC GTCCTGCACT




CACCCGATGC AGACCTTGAC TTTGATGTTG AAATGAACAC ACTTGTTTTA




CCCAAGTCTG GTGGAACAAA TGCCCAATCA TGTGACCTTA AAGTGTACTG




CAAAGCTGTA GCTTTAAGTA ATTGCTGTTC TGCCACTGCT TACTCTGAAA




TCTACCATCA AAGAAAGATA GAGAAAAGGG GCTGAGCCTT GGAATATATG




GTTATAAGCA GATCTTTCTT TGGTCAGAGA CCAGGGTTTG AGCCAAGGCT




GTAAATGTGA ACAATAGCTG TGCAAAGCCT TTTAACCTGA CTTCTTCATT




TTGTAAATTA TTATGCATTA AGTAGCAGCC CAATAATCTG ATTTCTAGTT




TTATTTTCAA AGTAAGTAGC TTCTTTTGGG AAAAACCTAA GTTAAACTAG




TAGTTTTGCC ATAATAACTG CTGATTTATG TATTTGCTAA AGGTACTTTT




GTATCTGCTG TGTATTATAG CAATAAAATA ATCATTTTGT TAGAAAAAAA




TCAAAAAAAA AAAAAA





32
Human MUL1;
MESGGRPSLC QFILLGTTSV VTAALYSVYR QKARVSQELK GAKKVHLGED



SwissProt
LKSILSEAPG KCVPYAVIEG AVRSVKETLN SQFVENCKGV IQRLTLQEHK



Q969V5.1
MVWNRTTHLW NDCSKIIHQR TNTVPFDLVP HEDGVDVAVR VLKPLDSVDL




GLETVYEKFH PSIQSFTDVI GHYISGERPK GIQETEEMLK VGATLTGVGE




LVLDNNSVRL QPPKQGMQYY LSSQDFDSLL QRQESSVRLW KVLALVFGFA




TCATLFFILR KQYLQRQERL RLKQMQEEFQ EHEAQLLSRA KPEDRESLKS




ACVVCLSSFK SCVFLECGHV CSCTECYRAL PEPKKCPICR QAITRVIPLY




NS





33
Human ASNS;
MCGIWALFGS DDCLSVQCLS AMKIAHRGPD AFRFENVNGY TNCCFGFHRL



GenBank
AVVDPLFGMQ PIRVKKYPYL WLCYNGEIYN HKKMQQHFEF EYQTKVDGEI



NP_899199.2
ILHLYDKGGI EQTICMLDGV FAFVLLDTAN KKVFLGRDTY GVRPLFKAMT




EDGFLAVCSE AKGLVTLKHS ATPFLKVEPF LPGHYEVLDL KPNGKVASVE




MVKYHHCRDV PLHALYDNVE KLFPGFEIET VKNNLRILFN NAVKKRLMTD




RRIGCLLSGG LDSSLVAATL LKQLKEAQVQ YPLQTFAIGM EDSPDLLAAR




KVADHIGSEH YEVLFNSEEG IQALDEVIFS LETYDITTVR ASVGMYLISK




YIRKNTDSVV IFSGEGSDEL TQGYIYFHKA PSPEKAEEES ERLLRELYLF




DVLRADRTTA AHGLELRVPF LDHRFSSYYL SLPPEMRIPK NGIEKHLLRE




TFEDSNLIPK EILWRPKEAF SDGITSVKNS WFKILQEYVE HQVDDAMMAN




AAQKFPFNTP KTKEGYYYRQ VFERHYPGRA DWLSHYWMPK WINATDPSAR




TLTHYKSAVK A





34
Human
MASCAEPSEP SAPLPAGVPP LEDFEVLDGV EDAEGEEEEE EEEEEEDDLS



FKBP8;
ELPPLEDMGQ PPAEEAEQPG ALAREFLAAM EPEPAPAPAP EEWLDILGNG



SwissProt
LLRKKTLVPG PPGSSRPVKG QVVTVHLQTS LENGTRVQEE PELVFTLGDC



Q14318.2
DVIQALDLSV PLMDVGETAM VTADSKYCYG PQGRSPYIPP HAALCLEVTL




KTAVDGPDLE MLTGQERVAL ANRKRECGNA HYQRADFVLA ANSYDLAIKA




ITSSAKVDMT FEEEAQLLQL KVKCLNNLAA SQLKLDHYRA ALRSCSLVLE




HQPDNIKALF RKGKVLAQQG EYSEAIPILR AALKLEPSNK TIHAELSKLV




KKHAAQRSTE TALYRKMLGN PSRLPAKCPG KGAWSIPWKW LFGATAVALG




GVALSVVIAA RN





35
Human
MAASKPVEAA VVAAAVPSSG SGVGGGGTAG PGTGGLPRWQ LALAVGAPLL



TOM70;
LGAGAIYLWS RQQRRREARG RGDASGLKRN SERKTPEGRA SPAPGSGHPE



SwissProt
GPGAHLDMNS LDRAQAAKNK GNKYFKAGKY EQAIQCYTEA ISLCPTEKNV



O94826.1
DLSTFYQNRA AAFEQLQKWK EVAQDCTKAV ELNPKYVKAL FRRAKAHEKL




DNKKECLEDV TAVCILEGFQ NQQSMLLADK VLKLLGKEKA KEKYKNREPL




MPSPQFIKSY FSSFTDDIIS QPMLKGEKSD EDKDKEGEAL EVKENSGYLK




AKQYMEEENY DKIISECSKE IDAEGKYMAE ALLLRATFYL LIGNANAAKP




DLDKVISLKE ANVKLRANAL IKRGSMYMQQ QQPLLSTQDF NMAADIDPQN




ADVYHHRGQL KILLDQVEEA VADFDECIRL RPESALAQAQ KCFALYRQAY




TGNNSSQIQA AMKGFEEVIK KFPRCAEGYA LYAQALTDQQ QFGKADEMYD




KCIDLEPDNA TTYVHKGLLQ LQWKQDLDRG LELISKAIEI DNKCDFAYET




MGTIEVQRGN MEKAIDMFNK AINLAKSEME MAHLYSLCDA AHAQTEVAKK




YGLKPPTL





36
Human
MVGREKELSI HFVPGSCRLV EEEVNIPNRR VLVTGATGLL GRAVHKEFQQ



MAT2B;
NNWHAVGCGF RRARPKFEQV NLLDSNAVHH IIHDFQPHVI VHCAAERRPD



SwissProt
VVENQPDAAS QLNVDASGNL AKEAAAVGAF LIYISSDYVF DGTNPPYREE



Q9NZL9.1
DIPAPLNLYG KTKLDGEKAV LENNLGAAVL RIPILYGEVE KLEESAVTVM




FDKVQFSNKS ANMDHWQQRF PTHVKDVATV CRQLAEKRML DPSIKGTFHW




SGNEQMTKYE MACAIADAFN LPSSHLRPIT DSPVLGAQRP RNAQLDCSKL




ETLGIGQRTP FRIGIKESLW PFLIDKRWRQ TVFH





37
Human
MAAAVGRLLR ASVARHVSAI PWGISATAAL RPAACGRTSL TNLLCSGSSQ



PRDX3;
AKLFSTSSSC HAPAVTQHAP YFKGTAVVNG EFKDLSLDDF KGKYLVLFFY



SwissProt
PLDFTFVCPT EIVAFSDKAN EFHDVNCEVV AVSVDSHFSH LAWINTPRKN



P30048.3
GGLGHMNIAL LSDLTKQISR DYGVLLEGSG LALRGLFIID PNGVIKHLSV




NDLPVGRSVE ETLRLVKAFQ YVETHGEVCP ANWTPDSPTI KPSPAASKEY




FQKVNQ





38
Human IDE;
MRYRLAWLLH PALPSTFRSV LGARLPPPER LCGFQKKTYS KMNNPAIKRI



SwissProt
GNHITKSPED KREYRGLELA NGIKVLLISD PTTDKSSAAL DVHIGSLSDP



P14735.4
PNIAGLSHFC EHMLFLGTKK YPKENEYSQF LSEHAGSSNA FTSGEHTNYY




FDVSHEHLEG ALDRFAQFFL CPLFDESCKD REVNAVDSEH EKNVMNDAWR




LFQLEKATGN PKHPFSKFGT GNKYTLETRP NQEGIDVRQE LLKFHSAYYS




SNLMAVCVLG RESLDDLTNL VVKLFSEVEN KNVPLPEFPE HPFQEEHLKQ




LYKIVPIKDI RNLYVTFPIP DLQKYYKSNP GHYLGHLIGH EGPGSLLSEL




KSKGWVNTLV GGQKEGARGF MFFIINVDLT EEGLLHVEDI ILHMFQYIQK




LRAEGPQEWV FQECKDLNAV AFRFKDKERP RGYTSKIAGI LHYYPLEEVL




TAEYLLEEFR PDLIEMVLDK LRPENVRVAI VSKSFEGKTD RTEEWYGTQY




KQEAIPDEVI KKWQNADLNG KFKLPTKNEF IPTNFEILPL EKEATPYPAL




IKDTAMSKLW FKQDDKFFLP KACLNFEFFS PFAYVDPLHC NMAYLYLELL




KDSLNEYAYA AELAGLSYDL QNTIYGMYLS VKGYNDKQPI LLKKIIEKMA




TFEIDEKRFE IIKEAYMRSL NNFRAEQPHQ HAMYYLRLLM TEVAWTKDEL




KEALDDVTLP RLKAFIPQLL SRLHIEALLH GNITKQAALG IMQMVEDTLI




EHAHTKPLLP SQLVRYREVQ LPDRGWFVYQ QRNEVHNNCG IEIYYQTDMQ




STSENMFLEL FCQIISEPCF NTLRTKEQLG YIVFSGPRRA NGIQGLRFII




QSEKPPHYLE SRVEAFLITM EKSIEDMTEE AFQKHIQALA IRRLDKPKKL




SAECAKYWGE IISQQYNFDR DNTEVAYLKT LTKEDIIKFY KEMLAVDAPR




RHKVSVHVLA REMDSCPVVG EFPCQNDINL SQAPALPQPE VIQNMTEFKR




GLPLFPLVKP HINFMAAKL





39
Human
MAVPPTYADL GKSARDVFTK GYGFGLIKLD LKTKSENGLE FTSSGSANTE



VDAC1;
TTKVTGSLET KYRWTEYGLT FTEKWNTDNT LGTEITVEDQ LARGLKLTFD



SwissProt
SSFSPNTGKK NAKIKTGYKR EHINLGCDMD FDIAGPSIRG ALVLGYEGWL



P21796.2
AGYQMNFETA KSRVTQSNFA VGYKTDEFQL HTNVNDGTEF GGSIYQKVNK




KLETAVNLAW TAGNSNTRFG IAAKYQIDPD ACFSAKVNNS SLIGLGYTQT




LKPGIKLTLS ALLDGKNVNA GGHKLGLGLE FQA





44
Human
MATHGQTCAR PMCIPPSYAD LGKAARDIFN KGFGFGLVKL DVKTKSCSGV



VDAC2;
EFSTSGSSNT DTGKVTGTLE TKYKWCEYGL TFTEKWNTDN TLGTEIAIED



SwissProt
QICQGLKLTF DTTFSPNTGK KSGKIKSSYK RECINLGCDV DFDFAGPAIH



P45880.2
GSAVFGYEGW LAGYQMTFDS AKSKLTRNNF AVGYRTGDFQ LHTNVNDGTE




FGGSIYQKVC EDLDTSVNLA WTSGTNCTRF GIAAKYQLDP TASISAKVNN




SSLIGVGYTQ TLRPGVKLTL SALVDGKSIN AGGHKVGLAL ELEA





45
Human
MCNTPTYCDL GKAAKDVFNK GYGFGMVKID LKTKSCSGVE FSTSGHAYTD



VDAC3;
TGKASGNLET KYKVCNYGLT FTQKWNTDNT LGTEISWENK LAEGLKLTLD



SwissProt
TIFVPNTGKK SGKLKASYKR DCFSVGSNVD IDFSGPTIYG WAVLAFEGWL



Q9Y277.1
AGYQMSFDTA KSKLSQNNFA LGYKAADFQL HTHVNDGTEF GGSIYQKVNE




KIETSINLAW TAGSNNTRFG IAAKYMLDCR TSLSAKVNNA SLIGLGYTQT




LRPGVKLTLS ALIDGKNFSA GGHKVGLGFE LEA





40
Human IPO5;
MAAAAAEQQQ FYLLLGNLLS PDNVVRKQAE ETYENIPGQS KITFLLQAIR



SwissProt
NTTAAEEARQ MAAVLLRRLL SSAFDEVYPA LPSDVQTAIK SELLMIIQME



O00410.4
TQSSMRKKVC DIAAELARNL IDEDGNNQWP EGLKFLFDSV SSQNVGLREA




ALHIFWNFPG IFGNQQQHYL DVIKRMLVQC MQDQEHPSIR TLSARATAAF




ILANEHNVAL FKHFADLLPG FLQAVNDSCY QNDDSVLKSL VEIADTVPKY




LRPHLEATLQ LSLKLCGDTS LNNMQRQLAL EVIVTLSETA AAMLRKHTNI




VAQTIPQMLA MMVDLEEDED WANADELEDD DFDSNAVAGE SALDRMACGL




GGKLVLPMIK EHIMQMLQNP DWKYRHAGLM ALSAIGEGCH QQMEGILNEI




VNFVLLFLQD PHPRVRYAAC NAVGQMATDF APGFQKKFHE KVIAALLQTM




EDQGNQRVQA HAAAALINFT EDCPKSLLIP YLDNLVKHLH SIMVLKLQEL




IQKGTKLVLE QVVTSIASVA DTAEEKFVPY YDLFMPSLKH IVENAVQKEL




RLLRGKTIEC ISLIGLAVGK EKFMQDASDV MQLLLKTQTD FNDMEDDDPQ




ISYMISAWAR MCKILGKEFQ QYLPVVMGPL MKTASIKPEV ALLDTQDMEN




MSDDDGWEFV NLGDQQSFGI KTAGLEEKST ACQMLVCYAK ELKEGFVEYT




EQVVKLMVPL LKFYFHDGVR VAAAESMPLL LECARVRGPE YLTQMWHFMC




DALIKAIGTE PDSDVLSEIM HSFAKCIEVM GDGCLNNEHF EELGGILKAK




LEEHFKNQEL RQVKRQDEDY DEQVEESLQD EDDNDVYILT KVSDILHSIF




SSYKEKVLPW FEQLLPLIVN LICPHRPWPD RQWGLCIFDD VIEHCSPASF




KYAEYFLRPM LQYVCDNSPE VRQAAAYGLG VMAQYGGDNY RPFCTEALPL




LVRVIQSADS KTKENVNATE NCISAVGKIM KFKPDCVNVE EVLPHWLSWL




PLHEDKEEAV QTFNYLCDLI ESNHPIVLGP NNTNLPKIFS IIAEGEMHEA




IKHEDPCAKR LANVVRQVQT SGGLWTECIA QLSPEQQAAI QELLNSA





41
Human PTH2;
MPSKSLVMEY LAHPSTLGLA VGVACGMCLG WSLRVCFGML PKSKTSKTHT



SwissProt
DTESEASILG DSGEYKMILV VRNDLKMGKG KVAAQCSHAA VSAYKQIQRR



Q9Y3E5.1
NPEMLKQWEY CGQPKVVVKA PDEETLIALL AHAKMLGLTV SLIQDAGRTQ




IAPGSQTVLG IGPGPADLID KVTGHLKLY





42
Human
MKDVPGFLQQ SQNSGPGQPA VWHRLEELYT KKLWHQLTLQ VLDFVQDPCF



PSD13;
AQGDGLIKLY ENFISEFEHR VNPLSLVEII LHVVRQMTDP NVALTFLEKT



SwissProt
REKVKSSDEA VILCKTAIGA LKLNIGDLQV TKETIEDVEE MLNNLPGVTS



Q9UNM6.2
VHSRFYDLSS KYYQTIGNHA SYYKDALRFL GCVDIKDLPV SEQQERAFTL




GLAGLLGEGV FNFGELLMHP VLESLRNTDR QWLIDTLYAF NSGNVERFQT




LKTAWGQQPD LAANEAQLLR KIQLLCLMEM TFTRPANHRQ LTFEEIAKSA




KITVNEVELL VMKALSVGLV KGSIDEVDKR VHMTWVQPRV LDLQQIKGMK




DRLEFWCTDV KSMEMLVEHQ AHDILT





43
Human
MQRRGALFGM PGGSGGRKMA AGDIGELLVP HMPTIRVPRS GDRVYKNECA



UBP13;
FSYDSPNSEG GLYVCMNTFL AFGREHVERH FRKTGQSVYM HLKRHVREKV



SwissProt
RGASGGALPK RRNSKIFLDL DTDDDLNSDD YEYEDEAKLV IFPDHYEIAL



Q92995.2
PNIEELPALV TIACDAVLSS KSPYRKQDPD TWENELPVSK YANNLTQLDN




GVRIPPSGWK CARCDLRENL WLNLTDGSVL CGKWFFDSSG GNGHALEHYR




DMGYPLAVKL GTITPDGADV YSFQEEEPVL DPHLAKHLAH FGIDMLHMHG




TENGLQDNDI KLRVSEWEVI QESGTKLKPM YGPGYTGLKN LGNSCYLSSV




MQAIFSIPEF QRAYVGNLPR IFDYSPLDPT QDFNTQMTKL GHGLLSGQYS




KPPVKSELIE QVMKEEHKPQ QNGISPRMFK AFVSKSHPEF SSNRQQDAQE




FFLHLVNLVE RNRIGSENPS DVFRFLVEER IQCCQTRKVR YTERVDYLMQ




LPVAMEAATN KDELIAYELT RREAEANRRP LPELVRAKIP FSACLQAFSE




PENVDDFWSS ALQAKSAGVK TSRFASFPEY LVVQIKKFTF GLDWVPKKFD




VSIDMPDLLD INHLRARGLQ PGEEELPDIS PPIVIPDDSK DRLMNQLIDP




SDIDESSVMQ LAEMGFPLEA CRKAVYFTGN MGAEVAFNWI IVHMEEPDFA




EPLTMPGYGG AASAGASVFG ASGLDNQPPE EIVAIITSMG FQRNQAIQAL




RATNNNLERA LDWIFSHPEF EEDSDFVIEM ENNANANIIS EAKPEGPRVK




DGSGTYELFA FISHMGTSTM SGHYICHIKK EGRWVIYNDH KVCASERPPK




DLGYMYFYRRIPS









APPENDIX A

















1433B_HUMAN
ABCE1_HUMAN
AGM1_HUMAN
AN32B_HUMAN


1433E_HUMAN
ABCF1_HUMAN
AGO1_HUMAN
AN32E_HUMAN


1433F_HUMAN
ABCF2_HUMAN
AGO2_HUMAN
ANFY1_HUMAN


1433G_HUMAN
ABCF3_HUMAN
AHNK_HUMAN
ANLN_HUMAN


1433T_HUMAN
ABHD2_HUMAN
AHSA1_HUMAN
ANM1_HUMAN


1433Z_HUMAN
ABT1_HUMAN
AIBP_HUMAN
ANM5_HUMAN


1A01_HUMAN
ACACA_HUMAN
AIF1L_HUMAN
ANR26_HUMAN


1A02_HUMAN
ACBD6_HUMAN
AIFM1_HUMAN
ANR46_HUMAN


1B07_HUMAN
ACBP_HUMAN
AIMP1_HUMAN
ANX11_HUMAN


1C07_HUMAN
ACHA5_HUMAN
AIMP2_HUMAN
ANXA1_HUMAN


2A5D_HUMAN
ACINU_HUMAN
AINX_HUMAN
ANXA2_HUMAN


2AAA_HUMAN
ACLY_HUMAN
AIP_HUMAN
ANXA5_HUMAN


2ABA_HUMAN
ACO13_HUMAN
AKA11_HUMAN
ANXA6_HUMAN


2ABD_HUMAN
ACOD_HUMAN
AKA12_HUMAN
AOFA_HUMAN


3BP5_HUMAN
ACSL1_HUMAN
AKAP1_HUMAN
AP1G1_HUMAN


41_HUMAN
ACSL3_HUMAN
AKAP9_HUMAN
AP1M1_HUMAN


4F2_HUMAN
ACSL4_HUMAN
AKIB1_HUMAN
AP2A1_HUMAN


5NT3_HUMAN
ACTA_HUMAN
AKP13_HUMAN
AP2A2_HUMAN


6PGD_HUMAN
ACTB_HUMAN
AKP8L_HUMAN
AP2A_HUMAN


6PGL_HUMAN
ACTN1_HUMAN
AKT2_HUMAN
AP2B1_HUMAN


A0PJ76_HUMAN
ACTN4_HUMAN
AL3A2_HUMAN
AP2M1_HUMAN


A2I9Y7_HUMAN
ACTZ_HUMAN
AL7A1_HUMAN
AP2S1_HUMAN


A4UCU2_HUMAN
ACV1B_HUMAN
AL9A1_HUMAN
AP3B1_HUMAN


A4_HUMAN
ACYP1_HUMAN
ALBU_HUMAN
AP3D1_HUMAN


A7UJ17_HUMAN
ADAM9_HUMAN
ALDOA_HUMAN
AP3M1_HUMAN


A8K781_HUMAN
ADCY3_HUMAN
ALDR_HUMAN
AP3S1_HUMAN


A8K7N0_HUMAN
ADCY9_HUMAN
ALG5_HUMAN
AP3S2_HUMAN


A8KAM7_HUMAN
ADDA_HUMAN
ALG6_HUMAN
APBA2_HUMAN


AAAS_HUMAN
ADHX_HUMAN
ALKB5_HUMAN
APC1_HUMAN


AAAT_HUMAN
ADNP_HUMAN
ALO17_HUMAN
APC4_HUMAN


AACS_HUMAN
ADPPT_HUMAN
AMD_HUMAN
APC5_HUMAN


AAKG1_HUMAN
ADRM1_HUMAN
AMOL1_HUMAN
APC7_HUMAN


AAMP_HUMAN
ADT1_HUMAN
AMOT_HUMAN
APC_HUMAN


AAPK2_HUMAN
ADT2_HUMAN
AMPL_HUMAN
APEX1_HUMAN


AATF_HUMAN
ADT3_HUMAN
AMPM2_HUMAN
API5_HUMAN


ABC3C_HUMAN
AES_HUMAN
AMRA1_HUMAN
APLP2_HUMAN


ABCA3_HUMAN
AF1Q_HUMAN
AN13A_HUMAN
APOL2_HUMAN


ABCB6_HUMAN
AFF4_HUMAN
AN13B_HUMAN
APOO_HUMAN


ABCBA_HUMAN
AGAL_HUMAN
AN13C_HUMAN
APR_HUMAN


ABCD3_HUMAN
AGK_HUMAN
AN32A_HUMAN
APT_HUMAN


AR6P1_HUMAN
AT12A_HUMAN
B3KPC1_HUMAN
BCCIP_HUMAN


AR6P4_HUMAN
AT131_HUMAN
B3KRI9_HUMAN
BCD1_HUMAN


ARF1_HUMAN
AT132_HUMAN
B3KTN8_HUMAN
BCLF1_HUMAN


ARF4_HUMAN
AT1A1_HUMAN
B4DE27_HUMAN
BCOR_HUMAN


ARF5_HUMAN
AT2A2_HUMAN
B4DIH6_HUMAN
BDH2_HUMAN


ARF6_HUMAN
AT2B1_HUMAN
B4DIM0_HUMAN
BET1L_HUMAN


ARFG1_HUMAN
AT2B4_HUMAN
B4DKA3_HUMAN
BET1_HUMAN


ARFG2_HUMAN
AT2C1_HUMAN
B4DKB3_HUMAN
BEX4_HUMAN


ARH40_HUMAN
AT5F1_HUMAN
B4DL94_HUMAN
BHLH9_HUMAN


ARHG7_HUMAN
ATAD1_HUMAN
B4DLR3_HUMAN
BI1_HUMAN


ARI1A_HUMAN
ATBD4_HUMAN
B4DMT9_HUMAN
BIEA_HUMAN


ARI1_HUMAN
ATF1_HUMAN
B4DNE0_HUMAN
BIRC2_HUMAN


ARI2_HUMAN
ATF2_HUMAN
B4DSP0_HUMAN
BIRC5_HUMAN


ARID2_HUMAN
ATF7_HUMAN
B4DW33_HUMAN
BIRC6_HUMAN


ARIP4_HUMAN
ATG3_HUMAN
B4E184_HUMAN
BL1S1_HUMAN


ARL1_HUMAN
ATLA2_HUMAN
B4E2Y0_HUMAN
BMP2K_HUMAN


ARL2_HUMAN
ATLA3_HUMAN
B7H6_HUMAN
BNI3L_HUMAN


ARL3_HUMAN
ATM_HUMAN
B7Z2A7_HUMAN
BNIP2_HUMAN


ARL6_HUMAN
ATOX1_HUMAN
B7Z4W9_HUMAN
BOD1L_HUMAN


ARL8B_HUMAN
ATP7B_HUMAN
B7Z613_HUMAN
BOD1_HUMAN


ARM10_HUMAN
ATPA_HUMAN
B7Z6F8_HUMAN
BOP1_HUMAN


ARMC1_HUMAN
ATPB_HUMAN
B7Z780_HUMAN
BOREA_HUMAN


ARMC6_HUMAN
ATPG_HUMAN
B7Z8Y4_HUMAN
BORG5_HUMAN


ARMC8_HUMAN
ATPO_HUMAN
B9D1_HUMAN
BPNT1_HUMAN


ARMX3_HUMAN
ATR_HUMAN
BABA1_HUMAN
BRAP_HUMAN


ARP19_HUMAN
ATX10_HUMAN
BACD3_HUMAN
BRAT1_HUMAN


ARP2_HUMAN
ATX2_HUMAN
BACH_HUMAN
BRCA1_HUMAN


ARP3_HUMAN
ATX3_HUMAN
BAF_HUMAN
BRCC3_HUMAN


ARP5L_HUMAN
AUP1_HUMAN
BAG2_HUMAN
BRAP_HUMAN


ARP8_HUMAN
AURKA_HUMAN
BAG5_HUMAN
BRD4_HUMAN


ARPC2_HUMAN
AURKB_HUMAN
BAG6_HUMAN
BRE1A_HUMAN


ARPC3_HUMAN
AVEN_HUMAN
BAP18_HUMAN
BRE1B_HUMAN


ARPC4_HUMAN
AZI1_HUMAN
BAP29_HUMAN
BRE_HUMAN


ARPC5_HUMAN
AZI2_HUMAN
BAP31_HUMAN
BRK1_HUMAN


ARV1_HUMAN
AZIN1_HUMAN
BARD1_HUMAN
BROX_HUMAN


ASB13_HUMAN
B1AK87_HUMAN
BASI_HUMAN
BRWD3_HUMAN


ASCC2_HUMAN
B1ALK7_HUMAN
BASP1_HUMAN
BSDC1_HUMAN


ASNA_HUMAN
B2CI53_HUMAN
BAX_HUMAN
BT2A1_HUMAN


ASNS_HUMAN
B2L12_HUMAN
BAZ1A_HUMAN
BT3L4_HUMAN


ASPP1_HUMAN
B2L13_HUMAN
BAZ1B_HUMAN
BTBD1_HUMAN


ASPP2_HUMAN
B2RDE1_HUMAN
BAZ2A_HUMAN
BTBD2_HUMAN


ASXL2_HUMAN
B3A2_HUMAN
BBS1_HUMAN
BTBDA_HUMAN


AT11C_HUMAN
B3KNS4_HUMAN
BBS2_HUMAN
BTF3_HUMAN


BUB1B_HUMAN
CBR3_HUMAN
CDC16_HUMAN
CFDP1_HUMAN


BUB1_HUMAN
CBS_HUMAN
CDC20_HUMAN
CG044_HUMAN


BUB3_HUMAN
CBWD1_HUMAN
CDC23_HUMAN
CG074_HUMAN


BYST_HUMAN
CBX1_HUMAN
CDC27_HUMAN
CGL_HUMAN


BZW1_HUMAN
CBX2_HUMAN
CDC37_HUMAN
CH055_HUMAN


BZW2_HUMAN
CBX3_HUMAN
CDC42_HUMAN
CH059_HUMAN


C19L1_HUMAN
CBX5_HUMAN
CDC45_HUMAN
CH10_HUMAN


C1QBP_HUMAN
CBX6_HUMAN
CDC5L_HUMAN
CH60_HUMAN


C1TC_HUMAN
CC037_HUMAN
CDC73_HUMAN
CHC10_HUMAN


C8AP2_HUMAN
CC038_HUMAN
CDC7_HUMAN
CHCH2_HUMAN


C99L2_HUMAN
CC075_HUMAN
CDIPT_HUMAN
CHCH3_HUMAN


CA031_HUMAN
CC104_HUMAN
CDK1_HUMAN
CHD1_HUMAN


CA043_HUMAN
CC138_HUMAN
CDK2_HUMAN
CHD4_HUMAN


CA052_HUMAN
CC167_HUMAN
CDK4_HUMAN
CHD8_HUMAN


CA055_HUMAN
CC85B_HUMAN
CDK5_HUMAN
CHIC1_HUMAN


CA124_HUMAN
CCD14_HUMAN
CDKAL_HUMAN
CHIC2_HUMAN


CAB39_HUMAN
CCD22_HUMAN
CDV3_HUMAN
CHK1_HUMAN


CAB45_HUMAN
CCD47_HUMAN
CDYL1_HUMAN
CHM1A_HUMAN


CACO2_HUMAN
CCD50_HUMAN
CE025_HUMAN
CHM1B_HUMAN


CADH2_HUMAN
CCD58_HUMAN
CE170_HUMAN
CHM2A_HUMAN


CADM1_HUMAN
CCD72_HUMAN
CE192_HUMAN
CHM2B_HUMAN


CAF1A_HUMAN
CCD86_HUMAN
CE290_HUMAN
CHM4B_HUMAN


CAF1B_HUMAN
CCD94_HUMAN
CEBPZ_HUMAN
CHMP5_HUMAN


CAH2_HUMAN
CCD97_HUMAN
CEGT_HUMAN
CHRD1_HUMAN


CAH8_HUMAN
CCDB1_HUMAN
CELF1_HUMAN
CHSP1_HUMAN


CALD1_HUMAN
CCDC6_HUMAN
CENPB_HUMAN
CHTOP_HUMAN


CALM_HUMAN
CCDC8_HUMAN
CENPF_HUMAN
CI040_HUMAN


CALU_HUMAN
CCNA2_HUMAN
CENPH_HUMAN
CI041_HUMAN


CALX_HUMAN
CCNB1_HUMAN
CENPL_HUMAN
CI064_HUMAN


CAN1_HUMAN
CCNB2_HUMAN
CENPN_HUMAN
CI078_HUMAN


CAN7_HUMAN
CCND1_HUMAN
CENPQ_HUMAN
CIB1_HUMAN


CANB1_HUMAN
CCNK_HUMAN
CEP44_HUMAN
CING_HUMAN


CAND1_HUMAN
CCZ1L_HUMAN
CEP55_HUMAN
CIP2A_HUMAN


CAP1_HUMAN
CD032_HUMAN
CEP78_HUMAN
CIR1A_HUMAN


CAPR1_HUMAN
CD11A_HUMAN
CERS2_HUMAN
CISD1_HUMAN


CAPZB_HUMAN
CD123_HUMAN
CETN1_HUMAN
CISD2_HUMAN


CARM1_HUMAN
CD151_HUMAN
CETN2_HUMAN
CISY_HUMAN


CASC3_HUMAN
CD276_HUMAN
CF072_HUMAN
CJ032_HUMAN


CASC5_HUMAN
CD2AP_HUMAN
CF106_HUMAN
CK046_HUMAN


CAV1_HUMAN
CD320_HUMAN
CF115_HUMAN
CK067_HUMAN


CAZA1_HUMAN
CD81_HUMAN
CF130_HUMAN
CK5P2_HUMAN


CBPD_HUMAN
CD97_HUMAN
CF192_HUMAN
CK5P3_HUMAN


CBR1_HUMAN
CD99_HUMAN
CF211_HUMAN
CKAP2_HUMAN


CKAP5_HUMAN
COPG_HUMAN
CSN4_HUMAN
CYBP_HUMAN


CKS1_HUMAN
COPZ1_HUMAN
CSN5_HUMAN
CYBP_HUMAN


CL023_HUMAN
COQ2_HUMAN
CSN6_HUMAN
CYC_HUMAN


CL16A_HUMAN
COR1B_HUMAN
CSN7A_HUMAN
CYFP1_HUMAN


CLCA_HUMAN
COR1C_HUMAN
CSN7B_HUMAN
CYFP2_HUMAN


CLCB_HUMAN
COX17_HUMAN
CSPG5_HUMAN
CYLD_HUMAN


CLCC1_HUMAN
COX41_HUMAN
CSTF2_HUMAN
CYTB_HUMAN


CLH1_HUMAN
CP013_HUMAN
CSTF3_HUMAN
CYTSA_HUMAN


CLIC1_HUMAN
CP072_HUMAN
CSTFT_HUMAN
CYTSB_HUMAN


CLIC4_HUMAN
CP080_HUMAN
CT004_HUMAN
D3DQ69_HUMAN


CMIP_HUMAN
CP110_HUMAN
CT011_HUMAN
D3VVH3_HUMAN


CN166_HUMAN
CP135_HUMAN
CT030_HUMAN
D6RDG3_HUMAN


CN37_HUMAN
CP250_HUMAN
CTBP1_HUMAN
DACH1_HUMAN


CNBP_HUMAN
CP51A_HUMAN
CTBP2_HUMAN
DAD1_HUMAN


CND1_HUMAN
CPIN1_HUMAN
CTCF_HUMAN
DAG1_HUMAN


CND2_HUMAN
CPNE1_HUMAN
CTNA1_HUMAN
DAXX_HUMAN


CND3_HUMAN
CPNE3_HUMAN
CTNB1_HUMAN
DAZP1_HUMAN


CNDG2_HUMAN
CPNE5_HUMAN
CTND1_HUMAN
DBLOH_HUMAN


CNN3_HUMAN
CPNE8_HUMAN
CTR1_HUMAN
DBNL_HUMAN


CNNM3_HUMAN
CPNS1_HUMAN
CTR2_HUMAN
DBPA_HUMAN


CNNM4_HUMAN
CPSF1_HUMAN
CU059_HUMAN
DC1L1_HUMAN


CNOT1_HUMAN
CPSF2_HUMAN
CUED2_HUMAN
DC1L2_HUMAN


CNOT8_HUMAN
CPSF3_HUMAN
CUL1_HUMAN
DCA13_HUMAN


CNOTA_HUMAN
CPSF5_HUMAN
CUL2_HUMAN
DCAF5_HUMAN


CNO_HUMAN
CPSF6_HUMAN
CUL3_HUMAN
DCAF6_HUMAN


CO038_HUMAN
CPSF7_HUMAN
CUL4A_HUMAN
DCAF7_HUMAN


CO044_HUMAN
CPT1A_HUMAN
CUL4B_HUMAN
DCAF8_HUMAN


CO057_HUMAN
CR021_HUMAN
CUL5_HUMAN
DCAKD_HUMAN


COBL1_HUMAN
CREB5_HUMAN
CUL7_HUMAN
DCAM_HUMAN


COF1_HUMAN
CRIPT_HUMAN
CUL9_HUMAN
DCK_HUMAN


COF2_HUMAN
CRKL_HUMAN
CUTA_HUMAN
DCNL1_HUMAN


COG2_HUMAN
CRNL1_HUMAN
CUTC_HUMAN
DCNL5_HUMAN


COG4_HUMAN
CS010_HUMAN
CWC15_HUMAN
DCPS_HUMAN


COMD1_HUMAN
CS043_HUMAN
CWC22_HUMAN
DCTN1_HUMAN


COMD4_HUMAN
CSDE1_HUMAN
CWC27_HUMAN
DCTN2_HUMAN


COMD9_HUMAN
CSK21_HUMAN
CX026_HUMAN
DCTN4_HUMAN


COMT_HUMAN
CSK22_HUMAN
CX056_HUMAN
DCTP1_HUMAN


COPA_HUMAN
CSK2B_HUMAN
CX057_HUMAN
DCUP_HUMAN


COPB2_HUMAN
CSKP_HUMAN
CX6B1_HUMAN
DCXR_HUMAN


COPB_HUMAN
CSK_HUMAN
CX7A2_HUMAN
DD19A_HUMAN


COPD_HUMAN
CSN1_HUMAN
CXA1_HUMAN
DDB1_HUMAN


COPE_HUMAN
CSN2_HUMAN
CY561_HUMAN
DDB2_HUMAN


COPG2_HUMAN
CSN3_HUMAN
CYB5B_HUMAN
DDHD2_HUMAN


DDI1_HUMAN
DHX40_HUMAN
DRG1_HUMAN
EDRF1_HUMAN


DDI2_HUMAN
DHX57_HUMAN
DRG2_HUMAN
EEA1_HUMAN


DDIT4_HUMAN
DHX9_HUMAN
DRS7B_HUMAN
EF1A1_HUMAN


DDTL_HUMAN
DHYS_HUMAN
DSC3_HUMAN
EF1A2_HUMAN


DDX17_HUMAN
DIAP1_HUMAN
DSCR3_HUMAN
EF1B_HUMAN


DDX18_HUMAN
DICER_HUMAN
DSG2_HUMAN
EF1D_HUMAN


DDX1_HUMAN
DIDO1_HUMAN
DSRAD_HUMAN
EF1G_HUMAN


DDX20_HUMAN
DIM1_HUMAN
DTL_HUMAN
EF2K_HUMAN


DDX21_HUMAN
DIP2B_HUMAN
DUS3L_HUMAN
EF2_HUMAN


DDX23_HUMAN
DJC11_HUMAN
DUS3_HUMAN
EFHD1_HUMAN


DDX24_HUMAN
DJC21_HUMAN
DUT_HUMAN
EFNB1_HUMAN


DDX27_HUMAN
DKC1_HUMAN
DVL1L_HUMAN
EFTU_HUMAN


DDX3X_HUMAN
DLL1_HUMAN
DVL2_HUMAN
EHD4_HUMAN


DDX41_HUMAN
DLRB1_HUMAN
DX39A_HUMAN
EHMT1_HUMAN


DDX46_HUMAN
DMD_HUMAN
DX39B_HUMAN
EHMT2_HUMAN


DDX47_HUMAN
DMKN_HUMAN
DYH7_HUMAN
EI2BA_HUMAN


DDX59_HUMAN
DNA2L_HUMAN
DYHC1_HUMAN
EI2BB_HUMAN


DDX5_HUMAN
DNJA1_HUMAN
DYHC2_HUMAN
EI2BD_HUMAN


DDX6_HUMAN
DNJA2_HUMAN
DYL1_HUMAN
EID1_HUMAN


DEK_HUMAN
DNJB1_HUMAN
DYL2_HUMAN
EIF1A_HUMAN


DEN4C_HUMAN
DNJB2_HUMAN
DYLT1_HUMAN
EIF1_HUMAN


DENR_HUMAN
DNJB3_HUMAN
DYM_HUMAN
EIF3A_HUMAN


DEP1A_HUMAN
DNJB4_HUMAN
DYN1_HUMAN
EIF3B_HUMAN


DESM_HUMAN
DNJB6_HUMAN
DYN2_HUMAN
EIF3C_HUMAN


DESP_HUMAN
DNJC7_HUMAN
DYR_HUMAN
EIF3D_HUMAN


DEST_HUMAN
DNJC8_HUMAN
DZIP3_HUMAN
EIF3E_HUMAN


DFFA_HUMAN
DNJC9_HUMAN
E2AK2_HUMAN
EIF3F_HUMAN


DHAK_HUMAN
DNLI1_HUMAN
E41L2_HUMAN
EIF3G_HUMAN


DHB11_HUMAN
DNLI3_HUMAN
E41L5_HUMAN
EIF3H_HUMAN


DHB12_HUMAN
DNM1L_HUMAN
E7EW20_HUMAN
EIF3I_HUMAN


DHB4_HUMAN
DNMT1_HUMAN
E9PDP1_HUMAN
EIF3K_HUMAN


DHB7_HUMAN
DOCK7_HUMAN
E9PHA7_HUMAN
EIF3L_HUMAN


DHC24_HUMAN
DP13A_HUMAN
E9PIE5_HUMAN
EIF3M_HUMAN


DHCR7_HUMAN
DPM1_HUMAN
EAA1_HUMAN
ELAV1_HUMAN


DHRS1_HUMAN
DPOA2_HUMAN
EAPP_HUMAN
ELAV2_HUMAN


DHRS3_HUMAN
DPOD1_HUMAN
EBP2_HUMAN
ELMD2_HUMAN


DHRS4_HUMAN
DPOE1_HUMAN
ECH1_HUMAN
ELOB_HUMAN


DHRS7_HUMAN
DPOE2_HUMAN
ECHA_HUMAN
ELOC_HUMAN


DHSO_HUMAN
DPOE3_HUMAN
ECHM_HUMAN
ELP1_HUMAN


DHX15_HUMAN
DPOLA_HUMAN
ECM29_HUMAN
ELP2_HUMAN


DHX30_HUMAN
DPY30_HUMAN
EDC3_HUMAN
ELP3_HUMAN


DHX32_HUMAN
DPYL2_HUMAN
EDC4_HUMAN
EM55_HUMAN


DHX36_HUMAN
DREB_HUMAN
EDF1_HUMAN
EMAL3_HUMAN


EMAL4_HUMAN
EXOS9_HUMAN
FBX42_HUMAN
FWCH2_HUMAN


EMD_HUMAN
EZRI_HUMAN
FBXL3_HUMAN
FXR1_HUMAN


ENAH_HUMAN
F10A1_HUMAN
FBXL4_HUMAN
FYV1_HUMAN


ENOA_HUMAN
F115A_HUMAN
FCF1_HUMAN
FZD1_HUMAN


ENOPH_HUMAN
F120A_HUMAN
FCHO2_HUMAN
FZR_HUMAN


ENPLL_HUMAN
F125A_HUMAN
FCL_HUMAN
G2E3_HUMAN


ENPL_HUMAN
F127A_HUMAN
FDFT_HUMAN
G3BP1_HUMAN


ENSA_HUMAN
F127B_HUMAN
FEM1A_HUMAN
G3BP2_HUMAN


EP15R_HUMAN
F136A_HUMAN
FEM1B_HUMAN
G3P_HUMAN


EP400_HUMAN
F175B_HUMAN
FEN1_HUMAN
G6PI_HUMAN


EPCAM_HUMAN
F188A_HUMAN
FETUA_HUMAN
GA45A_HUMAN


EPHA2_HUMAN
F195B_HUMAN
FHL1_HUMAN
GAK_HUMAN


EPHA7_HUMAN
F208A_HUMAN
FHL3_HUMAN
GANAB_HUMAN


EPIPL_HUMAN
F263_HUMAN
FIBP_HUMAN
GAPD1_HUMAN


EPN1_HUMAN
F6XY72_HUMAN
FIP1_HUMAN
GAR1_HUMAN


EPN2_HUMAN
F8VZ13_HUMAN
FIS1_HUMAN
GASP2_HUMAN


EPN4_HUMAN
F92A1_HUMAN
FKB1A_HUMAN
GATL1_HUMAN


EPS15_HUMAN
FA40A_HUMAN
FKBP3_HUMAN
GBB1_HUMAN


ERBB4_HUMAN
FA49B_HUMAN
FKBP4_HUMAN
GBB2_HUMAN


ERC6L_HUMAN
FA50A_HUMAN
FKBP5_HUMAN
GBB4_HUMAN


ERCC2_HUMAN
FA54A_HUMAN
FKBP8_HUMAN
GBF1_HUMAN


ERCC3_HUMAN
FA54B_HUMAN
FL2D_HUMAN
GBG12_HUMAN


ERCC5_HUMAN
FA63A_HUMAN
FLII_HUMAN
GBG5_HUMAN


ERCC6_HUMAN
FA98A_HUMAN
FLNA_HUMAN
GBLP_HUMAN


ERF1_HUMAN
FABP5_HUMAN
FLNB_HUMAN
GBRAP_HUMAN


ERF3A_HUMAN
FACD2_HUMAN
FLOT1_HUMAN
GBRL2_HUMAN


ERG1_HUMAN
FACE1_HUMAN
FLOT2_HUMAN
GCC2_HUMAN


ERG7_HUMAN
FACR1_HUMAN
FLVC1_HUMAN
GCF_HUMAN


ERH_HUMAN
FADS2_HUMAN
FMR1_HUMAN
GCN1L_HUMAN


ERI3_HUMAN
FAF1_HUMAN
FNBP1_HUMAN
GCP2_HUMAN


ESPL1_HUMAN
FAF2_HUMAN
FOPNL_HUMAN
GCP4_HUMAN


ESTD_HUMAN
FAIM1_HUMAN
FOXC1_HUMAN
GCP60_HUMAN


ESYT1_HUMAN
FAKD1_HUMAN
FPPS_HUMAN
GDAP1_HUMAN


ETFA_HUMAN
FANCA_HUMAN
FRYL_HUMAN
GDAP2_HUMAN


ETUD1_HUMAN
FANCI_HUMAN
FTM_HUMAN
GDE_HUMAN


EWS_HUMAN
FANCJ_HUMAN
FTO_HUMAN
GDIA_HUMAN


EXD2_HUMAN
FAS_HUMAN
FUBP1_HUMAN
GDIB_HUMAN


EXOC1_HUMAN
FBRL_HUMAN
FUBP2_HUMAN
GDIR1_HUMAN


EXOC2_HUMAN
FBX21_HUMAN
FUBP3_HUMAN
GDPD1_HUMAN


EXOC4_HUMAN
FBX28_HUMAN
FUMH_HUMAN
GDS1_HUMAN


EXOS5_HUMAN
FBX32_HUMAN
FUND1_HUMAN
GEMI4_HUMAN


EXOS6_HUMAN
FBX38_HUMAN
FUND2_HUMAN
GEMI5_HUMAN


EXOS8_HUMAN
FBX3_HUMAN
FUS_HUMAN
GEMI6_HUMAN


GEMI_HUMAN
GORS2_HUMAN
H2B1A_HUMAN
HES1_HUMAN


GFPT1_HUMAN
GOSR1_HUMAN
H2B1B_HUMAN
HEXI1_HUMAN


GFRP_HUMAN
GOT1B_HUMAN
H2B1C_HUMAN
HGB1A_HUMAN


GGA1_HUMAN
GPAA1_HUMAN
H2B1D_HUMAN
HGS_HUMAN


GGA3_HUMAN
GPAT1_HUMAN
H2B1H_HUMAN
HIF1N_HUMAN


GGCT_HUMAN
GPHRA_HUMAN
H2B1J_HUMAN
HINT1_HUMAN


GGPPS_HUMAN
GPI8_HUMAN
H31T_HUMAN
HINT3_HUMAN


GIPC1_HUMAN
GPKOW_HUMAN
H33_HUMAN
HIP1_HUMAN


GKAP1_HUMAN
GPM6B_HUMAN
H4_HUMAN
HLTF_HUMAN


GLCNE_HUMAN
GPTC4_HUMAN
H90B2_HUMAN
HM13_HUMAN


GLMN_HUMAN
GPTC8_HUMAN
H90B3_HUMAN
HMCS1_HUMAN


GLNA_HUMAN
GRB2_HUMAN
HACD2_HUMAN
HMDH_HUMAN


GLO2_HUMAN
GRHL2_HUMAN
HACD3_HUMAN
HMG3M_HUMAN


GLOD4_HUMAN
GRHPR_HUMAN
HAP28_HUMAN
HMGB1_HUMAN


GLP3L_HUMAN
GRK6_HUMAN
HAT1_HUMAN
HMGB2_HUMAN


GLPK3_HUMAN
GRP75_HUMAN
HAUS1_HUMAN
HMGB3_HUMAN


GLPK5_HUMAN
GRP78_HUMAN
HAUS3_HUMAN
HMGN1_HUMAN


GLPK_HUMAN
GRSF1_HUMAN
HAUS5_HUMAN
HMGN2_HUMAN


GLRX3_HUMAN
GSHR_HUMAN
HAUS6_HUMAN
HMGN3_HUMAN


GLTP_HUMAN
GSK3A_HUMAN
HAUS7_HUMAN
HMGN4_HUMAN


GLYC_HUMAN
GSTA4_HUMAN
HAUS8_HUMAN
HMGN5_HUMAN


GLYR1_HUMAN
GSTM3_HUMAN
HAX1_HUMAN
HMOX2_HUMAN


GMFB_HUMAN
GSTO1_HUMAN
HBS1L_HUMAN
HN1_HUMAN


GMPPB_HUMAN
GSTP1_HUMAN
HCD2_HUMAN
HNRCL_HUMAN


GNA11_HUMAN
GTF2I_HUMAN
HCFC1_HUMAN
HNRDL_HUMAN


GNA13_HUMAN
GTPB1_HUMAN
HDAC1_HUMAN
HNRH1_HUMAN


GNA1_HUMAN
GTR1_HUMAN
HDAC2_HUMAN
HNRH2_HUMAN


GNAI1_HUMAN
GUAA_HUMAN
HDDC2_HUMAN
HNRH3_HUMAN


GNAI3_HUMAN
GWL_HUMAN
HDGF_HUMAN
HNRL1_HUMAN


GNAL_HUMAN
GYS1_HUMAN
HDGR2_HUMAN
HNRL2_HUMAN


GNAQ_HUMAN
H11_HUMAN
HD_HUMAN
HNRLL_HUMAN


GNAS1_HUMAN
H12_HUMAN
HEAT1_HUMAN
HNRPC_HUMAN


GNAS2_HUMAN
H1X_HUMAN
HEAT2_HUMAN
HNRPD_HUMAN


GNAZ_HUMAN
H2A1A_HUMAN
HEAT3_HUMAN
HNRPF_HUMAN


GNL3_HUMAN
H2A1B_HUMAN
HECD1_HUMAN
HNRPG_HUMAN


GNPAT_HUMAN
H2A1D_HUMAN
HECD3_HUMAN
HNRPK_HUMAN


GNPI1_HUMAN
H2A2B_HUMAN
HELC1_HUMAN
HNRPL_HUMAN


GOGA5_HUMAN
H2A2C_HUMAN
HELLS_HUMAN
HNRPM_HUMAN


GOGA7_HUMAN
H2AV_HUMAN
HEM3_HUMAN
HNRPQ_HUMAN


GOGB1_HUMAN
H2AW_HUMAN
HERC1_HUMAN
HNRPR_HUMAN


GOLI_HUMAN
H2AX_HUMAN
HERC2_HUMAN
HNRPU_HUMAN


GOLP3_HUMAN
H2AY_HUMAN
HERC3_HUMAN
HOIL1_HUMAN


GOPC_HUMAN
H2AZ_HUMAN
HERC5_HUMAN
HOOK1_HUMAN


HPBP1_HUMAN
IF2P_HUMAN
IQGA2_HUMAN
KAP0_HUMAN


HPRT_HUMAN
IF4A1_HUMAN
IQGA3_HUMAN
KAP2_HUMAN


HPS3_HUMAN
IF4A2_HUMAN
IR3IP_HUMAN
KAPCA_HUMAN


HS105_HUMAN
IF4A3_HUMAN
IRAK1_HUMAN
KAT5_HUMAN


HS71L_HUMAN
IF4B_HUMAN
IREB2_HUMAN
KBRS2_HUMAN


HS74L_HUMAN
IF4E2_HUMAN
IRF3_HUMAN
KC1A_HUMAN


HS902_HUMAN
IF4E_HUMAN
IRS4_HUMAN
KC1D_HUMAN


HS904_HUMAN
IF4G1_HUMAN
ISOC2_HUMAN
KC1G1_HUMAN


HS905_HUMAN
IF4G2_HUMAN
IST1_HUMAN
KC1G3_HUMAN


HS90A_HUMAN
IF4H_HUMAN
ITB1_HUMAN
KCC2B_HUMAN


HS90B_HUMAN
IF5A1_HUMAN
ITCH_HUMAN
KCC2D_HUMAN


HSBP1_HUMAN
IF5_HUMAN
ITFG3_HUMAN
KCMF1_HUMAN


HSDL1_HUMAN
IFT27_HUMAN
ITM2B_HUMAN
KCRB_HUMAN


HSF2_HUMAN
IFT43_HUMAN
ITM2C_HUMAN
KCT2_HUMAN


HSP71_HUMAN
IGBP1_HUMAN
ITPA_HUMAN
KCTD3_HUMAN


HSP72_HUMAN
IKKB_HUMAN
ITPR2_HUMAN
KCTD5_HUMAN


HSP74_HUMAN
ILF2_HUMAN
ITPR3_HUMAN
KCTD9_HUMAN


HSP7C_HUMAN
ILF3_HUMAN
ITSN1_HUMAN
KDIS_HUMAN


HSPB1_HUMAN
ILKAP_HUMAN
ITSN2_HUMAN
KDM1A_HUMAN


HTAI2_HUMAN
ILK_HUMAN
IWS1_HUMAN
KDM3A_HUMAN


HTR5A_HUMAN
ILVBL_HUMAN
JAK1_HUMAN
KDM3B_HUMAN


HTSF1_HUMAN
IMA2_HUMAN
JAM1_HUMAN
KDM4A_HUMAN


HUWE1_HUMAN
IMA3_HUMAN
JIP4_HUMAN
KDM4B_HUMAN


HXB9_HUMAN
IMB1_HUMAN
JMJD6_HUMAN
KDM5C_HUMAN


HXK1_HUMAN
IMDH1_HUMAN
JOS1_HUMAN
KDM6A_HUMAN


HXK2_HUMAN
IMDH2_HUMAN
JUN_HUMAN
KEAP1_HUMAN


HYOU1_HUMAN
IMMT_HUMAN
K0090_HUMAN
KHDR1_HUMAN


I2BP1_HUMAN
IMPCT_HUMAN
K0195_HUMAN
KHNYN_HUMAN


I2BP2_HUMAN
INAR1_HUMAN
K0664_HUMAN
KI20A_HUMAN


ICAL_HUMAN
INGR1_HUMAN
K0889_HUMAN
KI67_HUMAN


ICLN_HUMAN
INO1_HUMAN
K1328_HUMAN
KIF11_HUMAN


ID4_HUMAN
INT3_HUMAN
K1797_HUMAN
KIF14_HUMAN


IDE_HUMAN
INT7_HUMAN
K1967_HUMAN
KIF1A_HUMAN


IDHC_HUMAN
IPO11_HUMAN
K1C18_HUMAN
KIF1B_HUMAN


IDI1_HUMAN
IPO4_HUMAN
K1C19_HUMAN
KIF22_HUMAN


IF1AX_HUMAN
IPO5_HUMAN
K2C8_HUMAN
KIF23_HUMAN


IF2A_HUMAN
IPO7_HUMAN
K6PF_HUMAN
KIF2A_HUMAN


IF2B1_HUMAN
IPO8_HUMAN
K6PL_HUMAN
KIF2C_HUMAN


IF2B2_HUMAN
IPO9_HUMAN
K6PP_HUMAN
KIF4A_HUMAN


IF2B3_HUMAN
IPYR2_HUMAN
KAD1_HUMAN
KIF5A_HUMAN


IF2B_HUMAN
IPYR_HUMAN
KAD2_HUMAN
KIF7_HUMAN


IF2GL_HUMAN
IQCB1_HUMAN
KAD6_HUMAN
KIFC1_HUMAN


IF2G_HUMAN
IQGA1_HUMAN
KAISO_HUMAN
KIN17_HUMAN


KINH_HUMAN
LIMS1_HUMAN
LZTL1_HUMAN
MD1L1_HUMAN


KIRR1_HUMAN
LIN7C_HUMAN
LZTR1_HUMAN
MD2L1_HUMAN


KLC1_HUMAN
LIPA1_HUMAN
M1IP1_HUMAN
MD2L2_HUMAN


KLH11_HUMAN
LIS1_HUMAN
M89BB_HUMAN
MDC1_HUMAN


KLH13_HUMAN
LITFL_HUMAN
MA7D1_HUMAN
MDHC_HUMAN


KLH15_HUMAN
LKHA4_HUMAN
MA7D3_HUMAN
MDHM_HUMAN


KLHL7_HUMAN
LLPH_HUMAN
MACOI_HUMAN
MDM2_HUMAN


KLHL9_HUMAN
LLR1_HUMAN
MAGD1_HUMAN
MDN1_HUMAN


KNTC1_HUMAN
LMAN1_HUMAN
MAGD2_HUMAN
MED10_HUMAN


KPCD_HUMAN
LMBD1_HUMAN
MAGD4_HUMAN
MED1_HUMAN


KPCI_HUMAN
LMBD2_HUMAN
MAGE1_HUMAN
MED22_HUMAN


KPRA_HUMAN
LMBL3_HUMAN
MALD2_HUMAN
MED25_HUMAN


KPRB_HUMAN
LMCD1_HUMAN
MAP1B_HUMAN
MED29_HUMAN


KPYM_HUMAN
LMNA_HUMAN
MAP4_HUMAN
MED4_HUMAN


KT3K_HUMAN
LMNB1_HUMAN
MARCS_HUMAN
MEIS1_HUMAN


KTN1_HUMAN
LMNB2_HUMAN
MARE1_HUMAN
MEIS2_HUMAN


KTNA1_HUMAN
LN28B_HUMAN
MARH5_HUMAN
MELK_HUMAN


L2GL1_HUMAN
LNP_HUMAN
MARH6_HUMAN
MERL_HUMAN


L2GL2_HUMAN
LPP3_HUMAN
MARK3_HUMAN
MERTK_HUMAN


LAMC1_HUMAN
LPPRC_HUMAN
MAT1_HUMAN
MET7A_HUMAN


LANC1_HUMAN
LRBA_HUMAN
MAT2B_HUMAN
METH_HUMAN


LANC2_HUMAN
LRC20_HUMAN
MATR3_HUMAN
METK2_HUMAN


LAP2A_HUMAN
LRC40_HUMAN
MAZ_HUMAN
MET_HUMAN


LAP2B_HUMAN
LRC41_HUMAN
MBB1A_HUMAN
MFA3L_HUMAN


LAP4A_HUMAN
LRC47_HUMAN
MBD3_HUMAN
MFAP1_HUMAN


LAR4B_HUMAN
LRC57_HUMAN
MBIP1_HUMAN
MFF_HUMAN


LARP1_HUMAN
LRC58_HUMAN
MBLC2_HUMAN
MFN1_HUMAN


LARP4_HUMAN
LRC59_HUMAN
MBNL1_HUMAN
MFN2_HUMAN


LAS1L_HUMAN
LRRC3_HUMAN
MBRL_HUMAN
MFSD1_HUMAN


LAT1_HUMAN
LRSM1_HUMAN
MCA3_HUMAN
MGAP_HUMAN


LAT3_HUMAN
LS14B_HUMAN
MCAF1_HUMAN
MGN2_HUMAN


LAT4_HUMAN
LSM12_HUMAN
MCES_HUMAN
MGRN1_HUMAN


LA_HUMAN
LSM4_HUMAN
MCL1_HUMAN
MIA3_HUMAN


LBR_HUMAN
LSM7_HUMAN
MCM10_HUMAN
MIA40_HUMAN


LC7L2_HUMAN
LSR_HUMAN
MCM2_HUMAN
MIB1_HUMAN


LC7L3_HUMAN
LST8_HUMAN
MCM3_HUMAN
MIB2_HUMAN


LCHN_HUMAN
LTOR1_HUMAN
MCM4_HUMAN
MICA3_HUMAN


LDHA_HUMAN
LTV1_HUMAN
MCM5_HUMAN
MID49_HUMAN


LDHB_HUMAN
LYN_HUMAN
MCM6_HUMAN
MIF_HUMAN


LEG8_HUMAN
LYPA1_HUMAN
MCM7_HUMAN
MIMIT_HUMAN


LEO1_HUMAN
LYPA2_HUMAN
MCM8_HUMAN
MINA_HUMAN


LGUL_HUMAN
LYPL1_HUMAN
MCMBP_HUMAN
MINT_HUMAN


LIFR_HUMAN
LYRIC_HUMAN
MCRS1_HUMAN
MIO_HUMAN


MIRO1_HUMAN
MRP_HUMAN
NAA15_HUMAN
NELFA_HUMAN


MIRO2_HUMAN
MRT4_HUMAN
NAA16_HUMAN
NEMF_HUMAN


MK01_HUMAN
MS18A_HUMAN
NAA25_HUMAN
NEMO_HUMAN


MK03_HUMAN
MSH2_HUMAN
NAA40_HUMAN
NEP1_HUMAN


MK14_HUMAN
MSH6_HUMAN
NAA50_HUMAN
NEUA_HUMAN


MK67I_HUMAN
MTA1_HUMAN
NACAD_HUMAN
NEUL4_HUMAN


MKLN1_HUMAN
MTA2_HUMAN
NACA_HUMAN
NEUL_HUMAN


MKRN1_HUMAN
MTAP_HUMAN
NACC1_HUMAN
NFIP1_HUMAN


MKRN2_HUMAN
MTBP_HUMAN
NADAP_HUMAN
NFIP2_HUMAN


MLL1_HUMAN
MTCH2_HUMAN
NAMPT_HUMAN
NFL_HUMAN


MLL2_HUMAN
MTFR1_HUMAN
NASP_HUMAN
NFX1_HUMAN


MMGT1_HUMAN
MTL13_HUMAN
NAT10_HUMAN
NFXL1_HUMAN


MMS19_HUMAN
MTL14_HUMAN
NB5R1_HUMAN
NFYC_HUMAN


MMS22_HUMAN
MTMR3_HUMAN
NB5R3_HUMAN
NGLY1_HUMAN


MMTA2_HUMAN
MTMR6_HUMAN
NBN_HUMAN
NH2L1_HUMAN


MO4L1_HUMAN
MTMR8_HUMAN
NBR1_HUMAN
NHP2_HUMAN


MO4L2_HUMAN
MTMR9_HUMAN
NC2A_HUMAN
NIBL1_HUMAN


MOB1A_HUMAN
MTOR_HUMAN
NCBP1_HUMAN
NIP7_HUMAN


MOC2A_HUMAN
MTPN_HUMAN
NCDN_HUMAN
NIPA_HUMAN


MOC2B_HUMAN
MTR1_HUMAN
NCKP1_HUMAN
NIPBL_HUMAN


MOES_HUMAN
MTRR_HUMAN
NCOAT_HUMAN
NISCH_HUMAN


MOFA1_HUMAN
MTX1_HUMAN
NDC1_HUMAN
NIT2_HUMAN


MON2_HUMAN
MTX2_HUMAN
NDK3_HUMAN
NKAPL_HUMAN


MORC3_HUMAN
MTX3_HUMAN
NDK8_HUMAN
NKAP_HUMAN


MORC4_HUMAN
MUL1_HUMAN
NDKA_HUMAN
NKRF_HUMAN


MOSC1_HUMAN
MXRA7_HUMAN
NDKB_HUMAN
NLTP_HUMAN


MOSC2_HUMAN
MYCBP_HUMAN
NDRG1_HUMAN
NMD3_HUMAN


MOT10_HUMAN
MYCBP_HUMAN
NDUA1_HUMAN
NMNA1_HUMAN


MOT1_HUMAN
MYC_HUMAN
NDUA4_HUMAN
NMT1_HUMAN


MOV10_HUMAN
MYH10_HUMAN
NDUA5_HUMAN
NOB1_HUMAN


MP2K1_HUMAN
MYH11_HUMAN
NDUA6_HUMAN
NOC2L_HUMAN


MP2K3_HUMAN
MYH9_HUMAN
NDUAD_HUMAN
NOL11_HUMAN


MP2K6_HUMAN
MYL6B_HUMAN
NDUA9_HUMAN
NOL9_HUMAN


MPCP_HUMAN
MYL6_HUMAN
NDUAD_HUMAN
NOLC1_HUMAN


MPI_HUMAN
MYO19_HUMAN
NDUB6_HUMAN
NOMO1_HUMAN


MPP6_HUMAN
MYO1B_HUMAN
NDUB8_HUMAN
NOMO2_HUMAN


MPRIP_HUMAN
MYO1C_HUMAN
NDUBA_HUMAN
NONO_HUMAN


MPRI_HUMAN
MYO1D_HUMAN
NDUC2_HUMAN
NOP56_HUMAN


MPZL1_HUMAN
MYO6_HUMAN
NDUS5_HUMAN
NOP58_HUMAN


MR1L1_HUMAN
MYPT1_HUMAN
NECP1_HUMAN
NOSIP_HUMAN


MRE11_HUMAN
MYSM1_HUMAN
NEDD8_HUMAN
NOTC3_HUMAN


MRP1_HUMAN
MZT1_HUMAN
NEK2_HUMAN
NP1L1_HUMAN


MRP4_HUMAN
NAA10_HUMAN
NEK9_HUMAN
NP1L4_HUMAN


NPA1P_HUMAN
NUP62_HUMAN
P66B_HUMAN
PDCD5_HUMAN


NPDC1_HUMAN
NUP85_HUMAN
P73_HUMAN
PDCL3_HUMAN


NPL4_HUMAN
NUP93_HUMAN
PA1B2_HUMAN
PDE12_HUMAN


NPM_HUMAN
NUP98_HUMAN
PA2G4_HUMAN
PDIA1_HUMAN


NPRL3_HUMAN
NVL_HUMAN
PAAF1_HUMAN
PDIA3_HUMAN


NRDC_HUMAN
NXT1_HUMAN
PABP1_HUMAN
PDIP3_HUMAN


NRP1_HUMAN
NYNRI_HUMAN
PABP2_HUMAN
PDK1L_HUMAN


NSD1_HUMAN
OBSL1_HUMAN
PABP4_HUMAN
PDLI1_HUMAN


NSD2_HUMAN
OCAD1_HUMAN
PACE1_HUMAN
PDLI5_HUMAN


NSDHL_HUMAN
OCLN_HUMAN
PACN3_HUMAN
PDPK1_HUMAN


NSE4A_HUMAN
OCRL_HUMAN
PAF1_HUMAN
PDRG1_HUMAN


NSF1C_HUMAN
ODFP2_HUMAN
PAF_HUMAN
PDS5A_HUMAN


NSF_HUMAN
ODPB_HUMAN
PAG16_HUMAN
PDXD1_HUMAN


NSMA3_HUMAN
OFD1_HUMAN
PAIP2_HUMAN
PDZ11_HUMAN


NSUN2_HUMAN
OGFD1_HUMAN
PAIRB_HUMAN
PEA15_HUMAN


NSUN5_HUMAN
OGFR_HUMAN
PALM_HUMAN
PEBP1_HUMAN


NT5D1_HUMAN
OGT1_HUMAN
PAMM_HUMAN
PEG10_HUMAN


NTCP4_HUMAN
OLA1_HUMAN
PANK3_HUMAN
PELO_HUMAN


NTF2_HUMAN
OPTN_HUMAN
PANX1_HUMAN
PEPD_HUMAN


NTM1A_HUMAN
ORC2_HUMAN
PAPOA_HUMAN
PERI_HUMAN


NTPCR_HUMAN
ORC5_HUMAN
PAPS1_HUMAN
PERQ2_HUMAN


NU107_HUMAN
ORN_HUMAN
PAPS2_HUMAN
PESC_HUMAN


NU133_HUMAN
OSB10_HUMAN
PAR12_HUMAN
PEX13_HUMAN


NU153_HUMAN
OSBL3_HUMAN
PAR1_HUMAN
PEX19_HUMAN


NU155_HUMAN
OSBL9_HUMAN
PARG_HUMAN
PEX3_HUMAN


NU160_HUMAN
OSGEP_HUMAN
PARK7_HUMAN
PEX5_HUMAN


NU188_HUMAN
OST48_HUMAN
PARP1_HUMAN
PFD2_HUMAN


NU205_HUMAN
OSTC_HUMAN
PAWR_HUMAN
PFD3_HUMAN


NUB1_HUMAN
OSTM1_HUMAN
PB1_HUMAN
PFDS_HUMAN


NUCKS_HUMAN
OTU1_HUMAN
PBX2_HUMAN
PFD6_HUMAN


NUCL_HUMAN
OTU6B_HUMAN
PCBP1_HUMAN
PGAM1_HUMAN


NUD19_HUMAN
OTUB1_HUMAN
PCBP2_HUMAN
PGAM5_HUMAN


NUDC1_HUMAN
OTUD5_HUMAN
PCGF6_HUMAN
PGES2_HUMAN


NUDC2_HUMAN
OXA1L_HUMAN
PCH2_HUMAN
PGK1_HUMAN


NUDC_HUMAN
OXR1_HUMAN
PCID2_HUMAN
PGM1_HUMAN


NUDT5_HUMAN
P121A_HUMAN
PCM1_HUMAN
PGM2_HUMAN


NUF2_HUMAN
P20D2_HUMAN
PCNA_HUMAN
PGP_HUMAN


NUFP2_HUMAN
P3C2A_HUMAN
PCNP_HUMAN
PGRC1_HUMAN


NUMA1_HUMAN
P3C2B_HUMAN
PCNT_HUMAN
PGRC2_HUMAN


NUP37_HUMAN
P4K2A_HUMAN
PCX3_HUMAN
PGTB2_HUMAN


NUP50_HUMAN
P4K2B_HUMAN
PDC10_HUMAN
PHB2_HUMAN


NUP53_HUMAN
P4R3A_HUMAN
PDC6I_HUMAN
PHB_HUMAN


NUP54_HUMAN
P53_HUMAN
PDCD4_HUMAN
PHC2_HUMAN


PHF10_HUMAN
PLST_HUMAN
PPME1_HUMAN
PRS8_HUMAN


PHF14_HUMAN
PLXA1_HUMAN
PPP5_HUMAN
PSA1_HUMAN


PHF5A_HUMAN
PLXA2_HUMAN
PPT1_HUMAN
PSA2_HUMAN


PHF6_HUMAN
PLXB2_HUMAN
PPWD1_HUMAN
PSA3_HUMAN


PHIP_HUMAN
PM14_HUMAN
PR38A_HUMAN
PSA4_HUMAN


PHLP_HUMAN
PMF1_HUMAN
PR38B_HUMAN
PSA5_HUMAN


PHP14_HUMAN
PMGE_HUMAN
PR40A_HUMAN
PSA6_HUMAN


PI42A_HUMAN
PML_HUMAN
PRAF1_HUMAN
PSA7L_HUMAN


PI42C_HUMAN
PMVK_HUMAN
PRAF3_HUMAN
PSA7_HUMAN


PI4KA_HUMAN
PNKP_HUMAN
PRAME_HUMAN
PSA_HUMAN


PI51A_HUMAN
PNMA1_HUMAN
PRC1_HUMAN
PSB1_HUMAN


PI51C_HUMAN
PNMA2_HUMAN
PRC2A_HUMAN
PSB2_HUMAN


PIAS1_HUMAN
PNML1_HUMAN
PRC2C_HUMAN
PSB3_HUMAN


PIBF1_HUMAN
PNO1_HUMAN
PRCC_HUMAN
PSB4_HUMAN


PICAL_HUMAN
PNPH_HUMAN
PRDX1_HUMAN
PSB5_HUMAN


PIGU_HUMAN
PO2F1_HUMAN
PRDX2_HUMAN
PSB7_HUMAN


PIMT_HUMAN
POGK_HUMAN
PRDX3_HUMAN
PSD10_HUMAN


PIN1_HUMAN
POLH_HUMAN
PRDX4_HUMAN
PSD11_HUMAN


PIN4_HUMAN
POLI_HUMAN
PRDX5_HUMAN
PSD12_HUMAN


PININ_HUMAN
POLK_HUMAN
PRDX6_HUMAN
PSD13_HUMAN


PIPNA_HUMAN
POMP_HUMAN
PREB_HUMAN
PSD7_HUMAN


PIPNB_HUMAN
POP1_HUMAN
PRI1_HUMAN
PSDE_HUMAN


PIPSL_HUMAN
POP7_HUMAN
PRI2_HUMAN
PSF1_HUMAN


PJA1_HUMAN
PP1A_HUMAN
PRKDC_HUMAN
PSIP1_HUMAN


PJA2_HUMAN
PP1G_HUMAN
PRKN2_HUMAN
PSMD1_HUMAN


PK3CA_HUMAN
PP1RA_HUMAN
PROF1_HUMAN
PSMD2_HUMAN


PKHA1_HUMAN
PP2AA_HUMAN
PROF2_HUMAN
PSMD3_HUMAN


PKHA7_HUMAN
PP2AB_HUMAN
PROSC_HUMAN
PSMD4_HUMAN


PKHH3_HUMAN
PP4C_HUMAN
PRP16_HUMAN
PSMD6_HUMAN


PKN1_HUMAN
PP4R2_HUMAN
PRP19_HUMAN
PSMD8_HUMAN


PKN2_HUMAN
PP6R3_HUMAN
PRP31_HUMAN
PSMD9_HUMAN


PKNX1_HUMAN
PPAC_HUMAN
PRP4_HUMAN
PSME1_HUMAN


PKP4_HUMAN
PPCEL_HUMAN
PRP6_HUMAN
PSME2_HUMAN


PLAK_HUMAN
PPCE_HUMAN
PRP8_HUMAN
PSME3_HUMAN


PLAP_HUMAN
PPDPF_HUMAN
PRPF3_HUMAN
PSMG1_HUMAN


PLCE_HUMAN
PPIA_HUMAN
PRPS1_HUMAN
PSMG2_HUMAN


PLCG1_HUMAN
PPIB_HUMAN
PRPS2_HUMAN
PSMG3_HUMAN


PLD3_HUMAN
PPID_HUMAN
PRR11_HUMAN
PTBP1_HUMAN


PLEC_HUMAN
PPIG_HUMAN
PRS10_HUMAN
PTBP2_HUMAN


PLIN3_HUMAN
PPIH_HUMAN
PRS4_HUMAN
PTH2_HUMAN


PLK1_HUMAN
PPIL4_HUMAN
PRS6A_HUMAN
PTK7_HUMAN


PLRG1_HUMAN
PPM1B_HUMAN
PRS6B_HUMAN
PTMA_HUMAN


PLSL_HUMAN
PPM1G_HUMAN
PRS7_HUMAN
PTMS_HUMAN


PTN11_HUMAN
QRIC1_HUMAN
RB39A_HUMAN
RER1_HUMAN


PTN23_HUMAN
QTRD1_HUMAN
RB3GP_HUMAN
RERE_HUMAN


PTN2_HUMAN
R13AX_HUMAN
RB6I2_HUMAN
RFA1_HUMAN


PTOV1_HUMAN
RA1L2_HUMAN
RBBP4_HUMAN
RFA2_HUMAN


PTPRA_HUMAN
RA51C_HUMAN
RBBP5_HUMAN
RFA3_HUMAN


PTPRF_HUMAN
RAB10_HUMAN
RBBP6_HUMAN
RFC2_HUMAN


PTPRG_HUMAN
RAB13_HUMAN
RBBP7_HUMAN
RFC3_HUMAN


PTPS_HUMAN
RAB14_HUMAN
RBGPR_HUMAN
RFC4_HUMAN


PTRF_HUMAN
RAB1A_HUMAN
RBM12_HUMAN
RFC5_HUMAN


PTSS1_HUMAN
RAB21_HUMAN
RBM14_HUMAN
RFIP1_HUMAN


PTTG1_HUMAN
RAB24_HUMAN
RBM15_HUMAN
RFWD3_HUMAN


PTTG_HUMAN
RAB2A_HUMAN
RBM22_HUMAN
RGAP1_HUMAN


PUF60_HUMAN
RAB34_HUMAN
RBM23_HUMAN
RHBD2_HUMAN


PUM1_HUMAN
RAB35_HUMAN
RBM26_HUMAN
RHBT3_HUMAN


PUR2_HUMAN
RAB3B_HUMAN
RBM27_HUMAN
RHEB_HUMAN


PUR6_HUMAN
RAB5A_HUMAN
RBM28_HUMAN
RHG05_HUMAN


PUR8_HUMAN
RAB5B_HUMAN
RBM39_HUMAN
RHG22_HUMAN


PUR9_HUMAN
RAB5C_HUMAN
RBM42_HUMAN
RHOA_HUMAN


PURA2_HUMAN
RAB7A_HUMAN
RBM4B_HUMAN
RHOU_HUMAN


PUS7_HUMAN
RAB8A_HUMAN
RBM4_HUMAN
RIF1_HUMAN


PVRL2_HUMAN
RABE1_HUMAN
RBMS1_HUMAN
RIFK_HUMAN


PVRL3_HUMAN
RABE2_HUMAN
RBP2_HUMAN
RING2_HUMAN


PWP1_HUMAN
RABP2_HUMAN
RBP56_HUMAN
RINI_HUMAN


PWP2_HUMAN
RABX5_HUMAN
RBX1_HUMAN
RIOK1_HUMAN


PYGB_HUMAN
RAC1_HUMAN
RB_HUMAN
RIOK2_HUMAN


PYGL_HUMAN
RAD18_HUMAN
RCC1_HUMAN
RIOK3_HUMAN


PYR1_HUMAN
RAD1_HUMAN
RCC2_HUMAN
RIR1_HUMAN


PYRG1_HUMAN
RAD21_HUMAN
RCCD1_HUMAN
RIR2B_HUMAN


Q13384_HUMAN
RAD50_HUMAN
RCD1_HUMAN
RIR2_HUMAN


Q59GX9_HUMAN
RADI_HUMAN
RCL1_HUMAN
RL10A_HUMAN


Q5FWY2_HUMAN
RAE1L_HUMAN
RCN1_HUMAN
RL10L_HUMAN


Q5JWE8_HUMAN
RAGP1_HUMAN
RCN2_HUMAN
RL10_HUMAN


Q5LJA5_HUMAN
RAI14_HUMAN
RD23A_HUMAN
RL11_HUMAN


Q6FG99_HUMAN
RALYL_HUMAN
RD23B_HUMAN
RL12_HUMAN


Q6IPH7_HUMAN
RALY_HUMAN
RDH11_HUMAN
RL13A_HUMAN


Q6IQ27_HUMAN
RANG_HUMAN
RDH14_HUMAN
RL13_HUMAN


Q7Z5V0_HUMAN
RAN_HUMAN
RECQ1_HUMAN
RL14_HUMAN


Q8NDP0_HUMAN
RAP1A_HUMAN
RED_HUMAN
RL15_HUMAN


Q9HBI2_HUMAN
RAP2B_HUMAN
REEP4_HUMAN
RL17_HUMAN


Q9ULW9_HUMAN
RASK_HUMAN
REEP5_HUMAN
RL18A_HUMAN


QCR2_HUMAN
RASN_HUMAN
REN3B_HUMAN
RL18_HUMAN


QCR9_HUMAN
RB11A_HUMAN
RENT1_HUMAN
RL19_HUMAN


QKI_HUMAN
RB11B_HUMAN
REPI1_HUMAN
RL1D1_HUMAN


RL21_HUMAN
RN122_HUMAN
RPC2_HUMAN
RS2_HUMAN


RL22_HUMAN
RN123_HUMAN
RPC4_HUMAN
RS30_HUMAN


RL23A_HUMAN
RN138_HUMAN
RPF1_HUMAN
RS3A_HUMAN


RL23_HUMAN
RN141_HUMAN
RPIA_HUMAN
RS3_HUMAN


RL24_HUMAN
RN146_HUMAN
RPN1_HUMAN
RS4X_HUMAN


RL26L_HUMAN
RN166_HUMAN
RPN2_HUMAN
RS5_HUMAN


RL27A_HUMAN
RN167_HUMAN
RPP29_HUMAN
RS6_HUMAN


RL27_HUMAN
RN168_HUMAN
RPP30_HUMAN
RS7_HUMAN


RL28_HUMAN
RN185_HUMAN
RPR1B_HUMAN
RS8_HUMAN


RL29_HUMAN
RN187_HUMAN
RPRD2_HUMAN
RS9_HUMAN


RL30_HUMAN
RN213_HUMAN
RRAGA_HUMAN
RSBNL_HUMAN


RL31_HUMAN
RN216_HUMAN
RRAGC_HUMAN
RSCA1_HUMAN


RL32_HUMAN
RN219_HUMAN
RRBP1_HUMAN
RSF1_HUMAN


RL34_HUMAN
RN220_HUMAN
RRMJ1_HUMAN
RSMB_HUMAN


RL35A_HUMAN
RNBP6_HUMAN
RRMJ3_HUMAN
RSPRY_HUMAN


RL35_HUMAN
RNF10_HUMAN
RRP12_HUMAN
RSRC2_HUMAN


RL36A_HUMAN
RNF12_HUMAN
RRP1B_HUMAN
RSSA_HUMAN


RL36_HUMAN
RNF13_HUMAN
RRP1_HUMAN
RSU1_HUMAN


RL37A_HUMAN
RNF25_HUMAN
RRP44_HUMAN
RT06_HUMAN


RL37_HUMAN
RNF31_HUMAN
RRP5_HUMAN
RT21_HUMAN


RL38_HUMAN
RNF4_HUMAN
RRS1_HUMAN
RT27_HUMAN


RL3L_HUMAN
RNF5_HUMAN
RS10L_HUMAN
RTC1_HUMAN


RL3_HUMAN
RNH2A_HUMAN
RS10_HUMAN
RTCB_HUMAN


RL40_HUMAN
RNPS1_HUMAN
RS11_HUMAN
RTF1_HUMAN


RL4_HUMAN
RNZ2_HUMAN
RS12_HUMAN
RTN3_HUMAN


RL5_HUMAN
RO60_HUMAN
RS13_HUMAN
RTN4_HUMAN


RL6_HUMAN
ROA0_HUMAN
RS14_HUMAN
RU17_HUMAN


RL7A_HUMAN
ROA1_HUMAN
RS15A_HUMAN
RU1C_HUMAN


RL7L_HUMAN
ROA2_HUMAN
RS15_HUMAN
RU2A_HUMAN


RL7_HUMAN
ROA3_HUMAN
RS16_HUMAN
RU2B_HUMAN


RL8_HUMAN
ROAA_HUMAN
RS17L_HUMAN
RUFY1_HUMAN


RL9_HUMAN
ROBO1_HUMAN
RS18_HUMAN
RUVB1_HUMAN


RLA0L_HUMAN
RPA1_HUMAN
RS19_HUMAN
RUVB2_HUMAN


RLA0_HUMAN
RPA49_HUMAN
RS20_HUMAN
RUXE_HUMAN


RLA1_HUMAN
RPAB1_HUMAN
RS21_HUMAN
RUXF_HUMAN


RLA2_HUMAN
RPAB5_HUMAN
RS23_HUMAN
RUXG_HUMAN


RM12_HUMAN
RPAP2_HUMAN
RS24_HUMAN
RWDD1_HUMAN


RM20_HUMAN
RPB11_HUMAN
RS25_HUMAN
RXRB_HUMAN


RM43_HUMAN
RPB1_HUMAN
RS26L_HUMAN
RYBP_HUMAN


RM53_HUMAN
RPB2_HUMAN
RS26_HUMAN
S10AA_HUMAN


RMD2_HUMAN
RPB7_HUMAN
RS27A_HUMAN
S10AB_HUMAN


RMD3_HUMAN
RPC10_HUMAN
RS28_HUMAN
S12A2_HUMAN


RN114_HUMAN
RPC1_HUMAN
RS29_HUMAN
S12A4_HUMAN


S12A6_HUMAN
SAS10_HUMAN
SETB1_HUMAN
SLK_HUMAN


S12A7_HUMAN
SAT1_HUMAN
SETD7_HUMAN
SLN11_HUMAN


S14L1_HUMAN
SATT_HUMAN
SET_HUMAN
SLU7_HUMAN


S15A4_HUMAN
SBDS_HUMAN
SF01_HUMAN
SMAD3_HUMAN


S18L2_HUMAN
SC11A_HUMAN
SF3A1_HUMAN
SMAD4_HUMAN


S19A1_HUMAN
SC11C_HUMAN
SF3A3_HUMAN
SMAP_HUMAN


S20A1_HUMAN
SC22B_HUMAN
SF3B1_HUMAN
SMC1A_HUMAN


S20A2_HUMAN
SC23A_HUMAN
SF3B2_HUMAN
SMC2_HUMAN


S22A5_HUMAN
SC23B_HUMAN
SF3B3_HUMAN
SMC3_HUMAN


S23A2_HUMAN
SC24C_HUMAN
SF3B5_HUMAN
SMC4_HUMAN


S23IP_HUMAN
SC31A_HUMAN
SFPQ_HUMAN
SMC6_HUMAN


S2546_HUMAN
SC5A3_HUMAN
SFR15_HUMAN
SMCA1_HUMAN


S2611_HUMAN
SC5D_HUMAN
SFR19_HUMAN
SMCA2_HUMAN


S26A6_HUMAN
SC6A8_HUMAN
SFSWA_HUMAN
SMCA4_HUMAN


S27A2_HUMAN
SCAFB_HUMAN
SFT2C_HUMAN
SMCA5_HUMAN


S29A1_HUMAN
SCAM1_HUMAN
SFXN1_HUMAN
SMCE1_HUMAN


S29A2_HUMAN
SCAM3_HUMAN
SGPL1_HUMAN
SMD1_HUMAN


S30BP_HUMAN
SCFD1_HUMAN
SGT1_HUMAN
SMD2_HUMAN


S35B2_HUMAN
SCLY_HUMAN
SGTA_HUMAN
SMD3_HUMAN


S35E1_HUMAN
SCML2_HUMAN
SGTB_HUMAN
SMG1_HUMAN


S38A1_HUMAN
SCO2_HUMAN
SH3G1_HUMAN
SMG8_HUMAN


S38A2_HUMAN
SCOC_HUMAN
SH3L1_HUMAN
SMHD1_HUMAN


S38A9_HUMAN
SCPDL_HUMAN
SH3L2_HUMAN
SMN_HUMAN


S39A6_HUMAN
SCRIB_HUMAN
SHIP1_HUMAN
SMOX_HUMAN


S39AA_HUMAN
SDC2_HUMAN
SHIP2_HUMAN
SMRC1_HUMAN


S39AE_HUMAN
SDCB1_HUMAN
SHKB1_HUMAN
SMRC2_HUMAN


S4A7_HUMAN
SDCG3_HUMAN
SHLB2_HUMAN
SMRCD_HUMAN


S61A1_HUMAN
SDSL_HUMAN
SHOT1_HUMAN
SMRD1_HUMAN


S6A15_HUMAN
SEC20_HUMAN
SHPK_HUMAN
SMU1_HUMAN


SAAL1_HUMAN
SEC62_HUMAN
SHPRH_HUMAN
SNAA_HUMAN


SAE1_HUMAN
SEC63_HUMAN
SHQ1_HUMAN
SNAG_HUMAN


SAE2_HUMAN
SEH1_HUMAN
SHRPN_HUMAN
SND1_HUMAN


SAFB1_HUMAN
SELR1_HUMAN
SIAS_HUMAN
SNF5_HUMAN


SAHH2_HUMAN
SENP3_HUMAN
SIN3A_HUMAN
SNF8_HUMAN


SAHH_HUMAN
SEP11_HUMAN
SIRT1_HUMAN
SNP23_HUMAN


SALL2_HUMAN
SEPT2_HUMAN
SIRT2_HUMAN
SNP29_HUMAN


SAM50_HUMAN
SEPT6_HUMAN
SIVA_HUMAN
SNP47_HUMAN


SAMH1_HUMAN
SEPT7_HUMAN
SK2L2_HUMAN
SNR40_HUMAN


SAP18_HUMAN
SEPT9_HUMAN
SKA2L_HUMAN
SNR48_HUMAN


SAR1A_HUMAN
SERA_HUMAN
SKIV2_HUMAN
SNRPA_HUMAN


SARM1_HUMAN
SERC1_HUMAN
SKI_HUMAN
SNTB2_HUMAN


SARNP_HUMAN
SERC_HUMAN
SKP1_HUMAN
SNUT1_HUMAN


SART3_HUMAN
SESN1_HUMAN
SKP2_HUMAN
SNW1_HUMAN


SNX12_HUMAN
SRPK2_HUMAN
STRUM_HUMAN
SYRC_HUMAN


SNX1_HUMAN
SRPRB_HUMAN
STT3A_HUMAN
SYSC_HUMAN


SNX27_HUMAN
SRRM1_HUMAN
STX10_HUMAN
SYTC_HUMAN


SNX2_HUMAN
SRRM2_HUMAN
STX12_HUMAN
SYVC_HUMAN


SNX32_HUMAN
SRRT_HUMAN
STX16_HUMAN
SYWC_HUMAN


SNX5_HUMAN
SRR_HUMAN
STX17_HUMAN
SYYC_HUMAN


SNX6_HUMAN
SRS11_HUMAN
STX18_HUMAN
T106B_HUMAN


SNX8_HUMAN
SRSF1_HUMAN
STX4_HUMAN
T22D3_HUMAN


SO4A1_HUMAN
SRSF2_HUMAN
STX5_HUMAN
T2AG_HUMAN


SOAT1_HUMAN
SRSF3_HUMAN
STX6_HUMAN
T2EB_HUMAN


SODC_HUMAN
SRSF4_HUMAN
STX7_HUMAN
T2FB_HUMAN


SON_HUMAN
SRSF5_HUMAN
STX8_HUMAN
T2H2L_HUMAN


SORCN_HUMAN
SRSF6_HUMAN
STXB1_HUMAN
TAB2_HUMAN


SP16H_HUMAN
SRSF7_HUMAN
STXB2_HUMAN
TACC3_HUMAN


SPA5L_HUMAN
SRSF9_HUMAN
STXB3_HUMAN
TADBP_HUMAN


SPAG7_HUMAN
SSA27_HUMAN
SUFU_HUMAN
TAF10_HUMAN


SPB6_HUMAN
SSBP_HUMAN
SUGT1_HUMAN
TAF1B_HUMAN


SPC24_HUMAN
SSF1_HUMAN
SUMO1_HUMAN
TAF5L_HUMAN


SPDLY_HUMAN
SSNA1_HUMAN
SUMO2_HUMAN
TAF7_HUMAN


SPEE_HUMAN
SSPN_HUMAN
SUMO3_HUMAN
TAF9B_HUMAN


SPF30_HUMAN
SSRA_HUMAN
SUN1_HUMAN
TAF9_HUMAN


SPF45_HUMAN
SSRD_HUMAN
SURF4_HUMAN
TAGL2_HUMAN


SPG20_HUMAN
SSRG_HUMAN
SUV91_HUMAN
TALDO_HUMAN


SPIT2_HUMAN
SSRP1_HUMAN
SUV92_HUMAN
TANC2_HUMAN


SPOP_HUMAN
SSU72_HUMAN
SUZ12_HUMAN
TAP2_HUMAN


SPRY7_HUMAN
ST1A1_HUMAN
SYAC_HUMAN
TARB1_HUMAN


SPSY_HUMAN
STABP_HUMAN
SYAP1_HUMAN
TATD1_HUMAN


SPT5H_HUMAN
STAG1_HUMAN
SYCC_HUMAN
TAXB1_HUMAN


SPT6H_HUMAN
STAG2_HUMAN
SYDC_HUMAN
TB10A_HUMAN


SPTA2_HUMAN
STAM1_HUMAN
SYEP_HUMAN
TB10B_HUMAN


SPTB2_HUMAN
STAM2_HUMAN
SYF1_HUMAN
TBA1A_HUMAN


SPTC1_HUMAN
STAT2_HUMAN
SYFA_HUMAN
TBA1B_HUMAN


SPTCS_HUMAN
STAT3_HUMAN
SYFB_HUMAN
TBA1C_HUMAN


SQSTM_HUMAN
STAU1_HUMAN
SYG_HUMAN
TBB2A_HUMAN


SR140_HUMAN
STEA3_HUMAN
SYHC_HUMAN
TBB3_HUMAN


SRC8_HUMAN
STIP1_HUMAN
SYIC_HUMAN
TBB4A_HUMAN


SREK1_HUMAN
STML2_HUMAN
SYJ2B_HUMAN
TBB4B_HUMAN


SRP09_HUMAN
STMN1_HUMAN
SYK_HUMAN
TBB5_HUMAN


SRP14_HUMAN
STPAP_HUMAN
SYLC_HUMAN
TBB6_HUMAN


SRP54_HUMAN
STRAP_HUMAN
SYMC_HUMAN
TBC15_HUMAN


SRP68_HUMAN
STRBP_HUMAN
SYMPK_HUMAN
TBC17_HUMAN


SRP72_HUMAN
STRN3_HUMAN
SYNC_HUMAN
TBCA_HUMAN


SRPK1_HUMAN
STRN4_HUMAN
SYQ_HUMAN
TBCB_HUMAN


TBCD4_HUMAN
TFG_HUMAN
TM7S3_HUMAN
TPC12_HUMAN


TBCD_HUMAN
TFR1_HUMAN
TM87A_HUMAN
TPD52_HUMAN


TBCE_HUMAN
TGFR1_HUMAN
TM9S3_HUMAN
TPD53_HUMAN


TBG1_HUMAN
TGS1_HUMAN
TM9S4_HUMAN
TPD54_HUMAN


TBL1R_HUMAN
THIC_HUMAN
TMCC1_HUMAN
TPIS_HUMAN


TBL2_HUMAN
THIO_HUMAN
TMCO1_HUMAN
TPM1_HUMAN


TBL3_HUMAN
THOC2_HUMAN
TMCO7_HUMAN
TPM4_HUMAN


TBP_HUMAN
THOC3_HUMAN
TMED4_HUMAN
TPP2_HUMAN


TCAL1_HUMAN
THOC4_HUMAN
TMED9_HUMAN
TPPC1_HUMAN


TCAL4_HUMAN
THOC6_HUMAN
TMEDA_HUMAN
TPPC3_HUMAN


TCAL8_HUMAN
THOP1_HUMAN
TMM31_HUMAN
TPPC4_HUMAN


TCEA1_HUMAN
THTM_HUMAN
TMM59_HUMAN
TPPC5_HUMAN


TCOF_HUMAN
THTPA_HUMAN
TMM66_HUMAN
TPPC8_HUMAN


TCP4_HUMAN
THUM3_HUMAN
TMOD3_HUMAN
TPR_HUMAN


TCPA_HUMAN
TIAR_HUMAN
TMUB1_HUMAN
TPX2_HUMAN


TCPB_HUMAN
TIF1A_HUMAN
TMUB2_HUMAN
TR10B_HUMAN


TCPD_HUMAN
TIF1B_HUMAN
TMX1_HUMAN
TR10D_HUMAN


TCPE_HUMAN
TIFA_HUMAN
TMX2_HUMAN
TR150_HUMAN


TCPG_HUMAN
TIGAR_HUMAN
TNKS1_HUMAN
TRA2A_HUMAN


TCPH_HUMAN
TIM10_HUMAN
TNKS2_HUMAN
TRA2B_HUMAN


TCPQ_HUMAN
TIM13_HUMAN
TNPO1_HUMAN
TRABD_HUMAN


TCPW_HUMAN
TIM50_HUMAN
TNPO2_HUMAN
TRAD1_HUMAN


TCPZ_HUMAN
TIM8A_HUMAN
TNPO3_HUMAN
TRAF2_HUMAN


TCRG1_HUMAN
TIM8B_HUMAN
TNR6_HUMAN
TRAF4_HUMAN


TCTP_HUMAN
TIM9_HUMAN
TOIP1_HUMAN
TRAF7_HUMAN


TDIF2_HUMAN
TIM_HUMAN
TOLIP_HUMAN
TRAP1_HUMAN


TDRKH_HUMAN
TIPIN_HUMAN
TOM1_HUMAN
TRI11_HUMAN


TE2IP_HUMAN
TIPRL_HUMAN
TOM20_HUMAN
TRI18_HUMAN


TEAN2_HUMAN
TITIN_HUMAN
TOM22_HUMAN
TRI25_HUMAN


TEBP_HUMAN
TKT_HUMAN
TOM34_HUMAN
TRI26_HUMAN


TECR_HUMAN
TLE1_HUMAN
TOM40_HUMAN
TRI27_HUMAN


TECT3_HUMAN
TLE3_HUMAN
TOM70_HUMAN
TRI32_HUMAN


TELO2_HUMAN
TLK2_HUMAN
TOM7_HUMAN
TRI33_HUMAN


TERA_HUMAN
TLN1_HUMAN
TOP1_HUMAN
TRI44_HUMAN


TES_HUMAN
TM115_HUMAN
TOP2A_HUMAN
TRI56_HUMAN


TF2B_HUMAN
TM165_HUMAN
TOP2B_HUMAN
TRI65_HUMAN


TF2H3_HUMAN
TM192_HUMAN
TOPB1_HUMAN
TRIM1_HUMAN


TF2H5_HUMAN
TM1L1_HUMAN
TOPK_HUMAN
TRIM4_HUMAN


TF3C1_HUMAN
TM1L2_HUMAN
TP4A1_HUMAN
TRIP4_HUMAN


TF3C3_HUMAN
TM209_HUMAN
TP4A2_HUMAN
TRIPB_HUMAN


TF3C4_HUMAN
TM237_HUMAN
TP4AP_HUMAN
TRIPC_HUMAN


TF3C5_HUMAN
TM41B_HUMAN
TPC10_HUMAN
TRM1L_HUMAN


TFDP1_HUMAN
TM45A_HUMAN
TPC11_HUMAN
TRM1_HUMAN


TRM6_HUMAN
UB2D3_HUMAN
UBP25_HUMAN
UTP18_HUMAN


TRRAP_HUMAN
UB2E1_HUMAN
UBP28_HUMAN
UTP23_HUMAN


TRUA_HUMAN
UB2G2_HUMAN
UBP2L_HUMAN
UTP6_HUMAN


TRXR1_HUMAN
UB2L3_HUMAN
UBP30_HUMAN
UTRO_HUMAN


TS101_HUMAN
UB2Q1_HUMAN
UBP33_HUMAN
UXT_HUMAN


TSC2_HUMAN
UB2R1_HUMAN
UBP34_HUMAN
VA0D1_HUMAN


TSN10_HUMAN
UB2R2_HUMAN
UBP36_HUMAN
VAMP1_HUMAN


TSNAX_HUMAN
UB2V1_HUMAN
UBP3_HUMAN
VAMP2_HUMAN


TSN_HUMAN
UB2V2_HUMAN
UBP48_HUMAN
VAMP4_HUMAN


TSR3_HUMAN
UBA1_HUMAN
UBP5_HUMAN
VAMP7_HUMAN


TSYL1_HUMAN
UBA3_HUMAN
UBP7_HUMAN
VAMP8_HUMAN


TSYL2_HUMAN
UBA6_HUMAN
UBQL1_HUMAN
VANG1_HUMAN


TTC12_HUMAN
UBAC1_HUMAN
UBQL2_HUMAN
VAPA_HUMAN


TTC26_HUMAN
UBAP1_HUMAN
UBR4_HUMAN
VAPB_HUMAN


TTC27_HUMAN
UBB_HUMAN
UBR5_HUMAN
VAS1_HUMAN


TTC32_HUMAN
UBC12_HUMAN
UBR7_HUMAN
VASP_HUMAN


TTC37_HUMAN
UBCP1_HUMAN
UBX2A_HUMAN
VAT1_HUMAN


TTC5_HUMAN
UBE2C_HUMAN
UBXN1_HUMAN
VATA_HUMAN


TTC9C_HUMAN
UBE2H_HUMAN
UBXN4_HUMAN
VATB2_HUMAN


TTF2_HUMAN
UBE2K_HUMAN
UBXN6_HUMAN
VATC1_HUMAN


TTK_HUMAN
UBE2N_HUMAN
UBXN7_HUMAN
VATF_HUMAN


TTL12_HUMAN
UBE2O_HUMAN
UBXN8_HUMAN
VATH_HUMAN


TULP3_HUMAN
UBE2S_HUMAN
UCHL1_HUMAN
VCIP1_HUMAN


TUT4_HUMAN
UBE2T_HUMAN
UCHL5_HUMAN
VDAC1_HUMAN


TX264_HUMAN
UBE3A_HUMAN
UCK2_HUMAN
VDAC2_HUMAN


TXD17_HUMAN
UBE3C_HUMAN
UCRIL_HUMAN
VDAC3_HUMAN


TXLNA_HUMAN
UBE4A_HUMAN
UEVLD_HUMAN
VIGLN_HUMAN


TXN4A_HUMAN
UBE4B_HUMAN
UFC1_HUMAN
VIME_HUMAN


TXN4B_HUMAN
UBF1_HUMAN
UFD1_HUMAN
VINC_HUMAN


TXND9_HUMAN
UBFD1_HUMAN
UHRF1_HUMAN
VIR_HUMAN


TXNIP_HUMAN
UBL4A_HUMAN
UIMC1_HUMAN
VP13A_HUMAN


TXNL1_HUMAN
UBL5_HUMAN
UK114_HUMAN
VP13C_HUMAN


TYDP2_HUMAN
UBL7_HUMAN
ULA1_HUMAN
VP13D_HUMAN


TYSY_HUMAN
UBP10_HUMAN
ULK3_HUMAN
VP26A_HUMAN


TYW1_HUMAN
UBP11_HUMAN
UMPS_HUMAN
VP33A_HUMAN


TYY1_HUMAN
UBP13_HUMAN
UN45A_HUMAN
VP33B_HUMAN


U2AF1_HUMAN
UBP14_HUMAN
UNC5C_HUMAN
VPP1_HUMAN


U2AF2_HUMAN
UBP16_HUMAN
UPK3L_HUMAN
VPP2_HUMAN


U520_HUMAN
UBP19_HUMAN
URB2_HUMAN
VPS16_HUMAN


U5S1_HUMAN
UBP1_HUMAN
USMG5_HUMAN
VPS29_HUMAN


UACA_HUMAN
UBP20_HUMAN
USO1_HUMAN
VPS35_HUMAN


UAP1_HUMAN
UBP22_HUMAN
USP9X_HUMAN
VPS36_HUMAN


UB2D1_HUMAN
UBP24_HUMAN
UTP15_HUMAN
VPS39_HUMAN


VPS45_HUMAN
XPO7_HUMAN
ZMAT2_HUMAN
##PYGL_HUMAN


VPS4A_HUMAN
XPOT_HUMAN
ZMYM1_HUMAN
##RL6_HUMAN


VPS4B_HUMAN
XPP1_HUMAN
ZMYM2_HUMAN
##SMC1A_HUMAN


VRK1_HUMAN
XRCC1_HUMAN
ZMYM3_HUMAN
##TCPQ_HUMAN


VRK3_HUMAN
XRCC4_HUMAN
ZN207_HUMAN
##TITIN_HUMAN


VTA1_HUMAN
XRCC5_HUMAN
ZN264_HUMAN
##TXND3_HUMAN


WAC_HUMAN
XRCC6_HUMAN
ZN281_HUMAN



WAP53_HUMAN
XRN2_HUMAN
ZN326_HUMAN



WASH1_HUMAN
XRP2_HUMAN
ZN330_HUMAN



WBP11_HUMAN
YAF2_HUMAN
ZN346_HUMAN



WBP2_HUMAN
YAP1_HUMAN
ZN451_HUMAN



WBS22_HUMAN
YBOX1_HUMAN
ZN460_HUMAN



WDHD1_HUMAN
YETS4_HUMAN
ZN503_HUMAN



WDR11_HUMAN
YI017_HUMAN
ZN598_HUMAN



WDR12_HUMAN
YIPF3_HUMAN
ZN622_HUMAN



WDR1_HUMAN
YKT6_HUMAN
ZN638_HUMAN



WDR26_HUMAN
YMEL1_HUMAN
ZN711_HUMAN



WDR36_HUMAN
YTHD1_HUMAN
ZN768_HUMAN



WDR41_HUMAN
YTHD2_HUMAN
ZNF24_HUMAN



WDR43_HUMAN
Z280C_HUMAN
ZNT1_HUMAN



WDR44_HUMAN
Z3H7A_HUMAN
ZO1_HUMAN



WDR48_HUMAN
ZBT10_HUMAN
ZO2_HUMAN



WDR59_HUMAN
ZC11A_HUMAN
ZPR1_HUMAN



WDR61_HUMAN
ZC3HE_HUMAN
ZRAB2_HUMAN



WDR67_HUMAN
ZC3HF_HUMAN
ZSWM6_HUMAN



WDR6_HUMAN
ZCCHV_HUMAN
ZUFSP_HUMAN



WDR74_HUMAN
ZCH10_HUMAN
ZW10_HUMAN



WDR75_HUMAN
ZCH12_HUMAN
ZWILC_HUMAN



WDR82_HUMAN
ZCHC2_HUMAN
ZWINT_HUMAN



WDR85_HUMAN
ZCHC3_HUMAN
ZYX_HUMAN



WDTC1_HUMAN
ZCHC8_HUMAN
ZZEF1_HUMAN



WIZ_HUMAN
ZDH13_HUMAN
##AHNK2_HUMAN



WLS_HUMAN
ZEB1_HUMAN
##AHNK_HUMAN



WPB5_HUMAN
ZF106_HUMAN
##BAP31_HUMAN



WRB_HUMAN
ZF161_HUMAN
##CENPF_HUMAN



WRIP1_HUMAN
ZFAN5_HUMAN
##CLH1_HUMAN



WRP73_HUMAN
ZFAN6_HUMAN
##CNTRL_HUMAN



WWP1_HUMAN
ZFN2B_HUMAN
##ENOA_HUMAN



XIAP_HUMAN
ZFR_HUMAN
##FAS_HUMAN



XPC_HUMAN
ZFX_HUMAN
##HUWE1_HUMAN



XPO1_HUMAN
ZFY16_HUMAN
##MCM7_HUMAN



XPO2_HUMAN
ZFY19_HUMAN
##NBN_HUMAN



XPO5_HUMAN
ZKSC1_HUMAN
##PRKDC_HUMAN








Claims
  • 1. A method of increasing mitophagy in a cell comprising contacting the cell with an inhibitor of USP30.
  • 2. A method of increasing mitochondrial ubiquitination in a cell comprising contacting the cell with an inhibitor of USP30.
  • 3. A method of increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or fourteen proteins selected from Tom20, MIRO, MUL1, ASNS, FKBP8, TOM70, MAT2B, PRDX3, IDE, VDAC1, VDAC2, VDAC3, IP05, PSD13, UBP13, and PTH2 in a cell comprising contacting the cell with an inhibitor of USP30.
  • 4. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, or three amino acids selected from K56, K61, and K68 of Tom 20.
  • 5. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K153, K187, K330, K427, K512, K535, K567, and K572 of MIRO.
  • 6. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, or three amino acids selected from K273, K299, and K52 of MUL1.
  • 7. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from K147, K168, K176, K221, K244, K275, K478, K504, and K556 of ASNS.
  • 8. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K249, K271, K273, K284, K307, K317, K334, and K340 of FKBP8.
  • 9. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K78, K120, K123, K126, K129, K148, K168, K170, K178, K185, K204, K230, K233, K245, K275, K278, K312, K326, K349, K359, K441, K463, K470, K471, K494, K501, K524, K536, K563, K570, K599, K600, and K604 of TOM70.
  • 10. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, or four amino acids selected from K209, K245, K316, and K326 of MAT2B.
  • 11. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, or five amino acids selected from K83, K91, K166, K241, and K253 of PRDX3.
  • 12. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K558, K657, K854, K884, K929, and K933 of IDE.
  • 13. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, or seven amino acids selected from K20, K53, K61, K109, K110, K266, and K274 of VDAC1.
  • 14. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, or six amino acids selected from K31, K64, K120, K121, K277, and K285 of VDAC2.
  • 15. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight amino acids selected from K20, K53, K61, K109, K110, K163, K266, and K274 of VDAC3.
  • 16. The method of claim 3, wherein the method comprises increasing ubiquitination of at at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K238, K353, K436, K437, K548, K556, K613, K678, K690, K705, K775, and K806 of IP05.
  • 17. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K2, K32, K99, K115, K122, K132, K161, K186, K313, K321, K347, K350, and K361 of PSD13.
  • 18. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids selected from K18, K190, K259, K326, K328, K401, K405, K414, K418, K435, K586, K587, and K640 of UBP13.
  • 19. The method of claim 3, wherein the method comprises increasing ubiquitination of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine amino acids selected from 47, 76, 81, 95, 106, 119, 134, 171, 177 of PTH2.
  • 20. The method of claim 1, wherein the cell is under oxidative stress.
  • 21. A method of reducing oxidative stress in a cell comprising contacting the cell with an inhibitor of USP30.
  • 22. The method of claim 1, wherein the cell comprises a pathogenic mutation in Parkin, a pathogenic mutation in PINK1, or a pathogenic mutation in Parkin and a pathogenic mutation in PINK1.
  • 23. The method of claim 22, wherein the cell comprises a pathogenic mutation in Parkin selected from the mutations in Table 1.
  • 24. The method of claim 22, wherein the cell comprises a pathogenic mutation in PINK1 selected from the mutations in Table 2.
  • 25. The method of claim 1, wherein the cell is selected from a neuron, a cardiac cell, and a muscle cell.
  • 26. The method of claim 1, wherein the cell is comprised in a subject.
  • 27. The method of claim 1, wherein the cell is ex vivo or in vitro.
  • 28. A method of treating a condition involving a mitochondrial defect in a subject comprising administering to the subject an effective amount of an inhibitor of USP30.
  • 29. The method of claim 28, wherein the condition involving a mitochondrial defect is selected from a condition involving a mitophagy defect, a condition involving a mutation in mitochondrial DNA, a condition involving mitochondrial oxidative stress, a condition involving a defect in mitochondrial shape or morphology, a condition involving a defect in mitochondrial membrane potential, and a condition involving a lysosomal storage defect.
  • 30. The method of claim 28, wherein the condition involving a mitochondrial defect is selected from a neurodegenerative disease; mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome; Leber's hereditary optic neuropathy (LHON); neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS); Danon disease; ischemic heart disease leading to myocardial infarction; multiple sulfatase deficiency (MSD); mucolipidosis II (ML II); mucolipidosis III (ML III); mucolipidosis IV (ML IV); GM1-gangliosidosis (GM1); neuronal ceroid-lipofuscinoses (NCL1); Alpers disease; Barth syndrome; Beta-oxidation defects; carnitine-acyl-carnitine deficiency; carnitine deficiency; creatine deficiency syndromes; co-enzyme Q10 deficiency; complex I deficiency; complex II deficiency; complex III deficiency; complex IV deficiency; complex V deficiency; COX deficiency; chronic progressive external ophthalmoplegia syndrome (CPEO); CPT I deficiency; CPT II deficiency; glutaric aciduria type II; Kearns-Sayre syndrome; lactic acidosis; long-chain acyl-CoA dehydrongenase deficiency (LCHAD); Leigh disease or syndrome; lethal infantile cardiomyopathy (LIC); Luft disease; glutaric aciduria type II; medium-chain acyl-CoA dehydrongenase deficiency (MCAD); myoclonic epilepsy and ragged-red fiber (MERRF) syndrome; mitochondrial recessive ataxia syndrome; mitochondrial cytopathy; mitochondrial DNA depletion syndrome; myoneurogastointestinal disorder and encephalopathy; Pearson syndrome; pyruvate carboxylase deficiency; pyruvate dehydrogenase deficiency; POLG mutations; medium/short-chain 3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) deficiency; and very long-chain acyl-CoA dehydrongenase (VLCAD) deficiency.
  • 31. The method of claim 30, wherein the neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia.
  • 32. A method of treating a neurodegenerative disease in a subject comprising administering to the subject an effective amount of an inhibitor of USP30.
  • 33. The method of claim 32, wherein the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), ischemia, stroke, dementia with Lewy bodies, and frontotemporal dementia.
  • 34. A method of treating Parkinson's disease in a subject comprising administering to the subject an effective amount of an inhibitor of USP30.
  • 35. The method of claim 28, wherein the subject comprises a pathogenic mutation in Parkin, a pathogenic mutation in PINK1, or a pathogenic mutation in Parkin and a pathogenic mutation in PINK1 in at least a portion of the subject's cells.
  • 36. The method of claim 35, wherein the pathogenic mutation in Parkin is selected from the mutations in Table 1.
  • 37. The method of claim 35, wherein the pathogenic mutation in PINK1 is selected from the mutations in Table 2.
  • 38. A method of treating a condition involving cells undergoing oxidative stress in a subject comprising administering to the subject an effective amount of an inhibitor of USP30.
  • 39. The method of claim 28, wherein the inhibitor of USP30 is administered orally, intramuscularly, intravenously, intraarterially, intraperitoneally, or subcutaneously.
  • 40. The method of claim 28, wherein the method comprises administering at least one additional therapeutic agent.
  • 41. The method of claim 40, wherein the at least one additional therapeutic agent is selected from levodopa, a dopamine agonist, a monoamino oxygenase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, amantadine, riluzole, a cholinesterase inhibitor, memantine, tetrabenazine, an antipsychotic, clonazepam, diazepam, an antidepressant, and an anti-convulsant.
  • 42. The method of claim 1, wherein the inhibitor of USP30 is an inhibitor of USP30 expression.
  • 43. The method of claim 42, wherein the inhibitor of USP30 expression is selected from an antisense oligonucleotide and a short interfering RNA (siRNA).
  • 44. The method of claim 1, wherein the inhibitor of USP30 is an inhibitor of USP30 activity.
  • 45. The method of claim 44, wherein the inhibitor of USP30 activity is selected from an antibody, a peptide, a peptibody, an aptamer, and a small molecule.
  • 46. A peptide comprising the amino acid sequence:
  • 47. The peptide of claim 46, wherein the 1050 of the peptide for at least one, at least two, or at least three peptidases selected from USP7, USP5, UCHL3, and USP2 is greater than 20 μM, greater than 30 μM, greater than 40 μM, or greater than 50 μM.
  • 48. The peptide of claim 46, wherein the peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 22.
  • 49. An antisense oligonucleotide comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to a region of USP30 mRNA and/or a region of USP30 pre-mRNA, wherein the region is at least at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides long.
  • 50. An siRNA comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to a region of USP30 mRNA and/or a region of USP30 pre-mRNA, wherein the region is at least at least 10, at least 15, at least 20, or at least 25 nucleotides long.
Provisional Applications (2)
Number Date Country
61809927 Apr 2013 US
61701963 Sep 2012 US
Continuations (1)
Number Date Country
Parent PCT/EP2013/006898 Sep 2013 US
Child 14659204 US