The sequence listing that is contained in the file named “UTFBP1057WO_SeqListing.txt”, which is 33 KB (as measured in Microsoft Windows®) and was created on Nov. 4, 2016, is filed herewith by electronic submission and is incorporated by reference herein.
The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns methods for selectively degrading target proteins, such as proteins involved in disease.
There are at least four reasons to remove a protein from cells: 1) to determine the function of a protein as part of biomedical research; 2) to validate a therapeutic target; 3) to stop the action of a disease-causing proteins, such as an oncoprotein or a protein causing neurodegeneration; 4) to enhance degradation a protein that accumulates during aging. The current state-of-the-art methodology for controlling cellular protein concentrations is to reduce protein synthesis using RNA interference (RNAi). RNAi technology is based on small RNA molecules that basepair to specific messenger RNAs to inhibit their translation or induce their hydrolysis. This method is well-understood and can be implemented broadly1.
While RNAi has proven to be a powerful tool and revolutionized experimental biology, it also has several disadvantages. For one, inhibiting protein synthesis means that protein depletion depends on the target's intrinsic degradation rate. Naturally long-lived proteins are difficult to deplete with RNAi, requiring several days of repeated application to decrease protein concentration by more than 50% of the original protein population. Also, RNAi cannot distinguish between different forms of post-translational modification (PTM) on proteins. Another complication is the challenge of delivering highly charged RNA agents into cells. When implemented as a research tool, RNAi precursors are typically transfected directly into cells by lentivirus infection, which can overload the cellular RNA processing machinery and lead to pleiotropic side effects2,3. The delivery challenge has largely prevented therapeutic use of RNAi.
In a first embodiment, the present disclosure provides a recombinant polypeptide comprising a proteasome-binding domain (e.g., a ubiquitin-like domain) and a target-binding domain, wherein said target binding domain specifically binds to a target polypeptide. In some aspects, target polypeptide is a mammalian polypeptide. In certain aspects, the target polypeptide is an intracellular mammalian polypeptide.
In certain aspects, the target polypeptide is a polypeptide associated with a disease or with aging. In some aspects, the target polypeptide is not a reporter protein. In certain aspects, the target-binding domain does not bind to the Huntington protein (HTT). In other aspects, the target-binding domain does not bind to protein having a poly-Q sequence.
In some aspects, the target-binding domain is positioned N-terminally relative to the proteasome-binding domain. In other aspects, the target-binding domain is positioned C-terminally relative to the proteasome-binding domain. For example, the proteasome-binding domain comprises a domain from HPV E7, gankyrin, Rad 23 or Rad23b. In certain aspects, the proteasome-binding domain comprises a domain from human protein. For example, the proteasome-binding domain comprises a domain from human Rad23b. In particular, the proteasome-binding domain comprises amino acids 1-83 of human Rad23b. In still further aspects, the proteasome-binding domain can be from a yeast Rad23 (e.g., amino acids 1-77 of yeast Rad23).
In some aspects, the target-binding domain and the proteasome-binding domain are separated by a linker. In certain aspects, the linker is a sequence from Rad23b. In still further aspects, a polypeptide of the embodiments further comprises a cell penetrating peptide (CPP) sequence or a cellular receptor-binding sequence (e.g., for receptor mediated cell penetration). For example, in some aspects, a cellular receptor-binding sequence can be a portion of the sequence of a ligand for the cellular receptor. In some aspects, the CPP sequence (or the cellular receptor-binding sequence) can be positioned N-terminally relative to the target binding domain and/or the proteasome-binding domain. In further aspects, the CPP sequence (or cellular receptor-binding sequence) can be positioned C-terminally relative to the target-binding domain and/or the proteasome-binding domain.
In further aspects, the target-binding domain comprises a monobody or scFv, or another antibody derivative, or a designed or evolved affinity domain that binds to a polypeptide. For example, the target-binding domain comprises the HA4 monobody, the Nsa1 monobody, and/or the Cs1 monobody.
In certain aspects, the target-binding domain binds to a prion, a viral polypeptide, a cellular polypeptide having a disease-associated mutation or the product of an oncogene. In other aspects, the target-binding domain binds to the product of an oncogene. For example, the oncogene is Abl and/or Shp2. In particular, the target-binding domain binds to the SH2 domain of Abl, the N-terminal SH2 domain of Shp2, and/or the C-terminal SH2 domain of Shp2. In some aspects, the target-binding domain binds to misfolded beta-amyloid. In some aspects the target-binding domain recognizes a post-translationally modified protein.
In a further embodiment, there is provided a nucleic acid molecule encoding the polypeptide provided herein comprising a target-binding domain and a proteasome-binding domain, wherein said target-binding domain specifically binds to a target polypeptide. In some aspects, the nucleic acid molecule is a DNA. In other aspects, the nucleic acid molecule is a RNA.
In another embodiment, there is provided an expression vector comprising a sequence encoding the polypeptide provided herein, operably linked to a promoter. In some aspects, the promoter is an inducible or a repressible promoter. In other aspects, the promoter is a tissue or cell type specific promoter. In certain aspects, the vector is a plasmid, a viral vector or an episomal vector. In further aspects, the expression vector further comprises an inducible expression cassette for a suicide gene.
In a further embodiments, there is provided a pharmaceutical composition comprising a polypeptide provided herein the polypeptide comprising a target-binding domain and a proteasome-binding domain, wherein said target-binding domain specifically binds to a target polypeptide. In further aspects, there is provided aa vector comprising a nucleic acid sequence encoding a polypeptide of the embodiments.
In an even further embodiment, there is provided a method of treating a subject having a disease comprising administering an effective amount of the pharmaceutical composition provided herein, wherein said target-binding domain of the polypeptide in the pharmaceutical composition (or encoded by the vector in the pharmaceutical composition) binds to a polypeptide associate with the disease.
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Embodiments of the present disclosure provide methods and compositions to deplete specific proteins from cells by targeting them directly to the proteasome for destruction, even without the need for ubiquitination (see, e.g.,
In studies detailed herein, artificial proteasome adaptors, referred to as degradons, comprising a proteasome-binding domain fused to a target recognition domain were built. The proteasome-binding domain comes from the ubiquitin-like (UbL) domain in the human homologue of yeast Rad23, hHR23b. hHR23b belongs to the class of UbL-UBA proteins found in all eukaryotic organisms27,28, which enhance the degradation of certain proteins by shuttling them to the proteasome28. In cells, hHR23b binds to the proteasome through an N-terminal ubiquitin-like (UbL) domain and to the target protein through a C-terminal ubiquitin-associated (UBA) domain and thus delivers the protein directly to the substrate entrance of the proteasome30,31. The hHR23b protein itself escapes degradation and recycles to deliver the next protein for destruction32,33.
The degradons recognize their substrates through a target-binding domain, such as an antibody mimic called a monobody (also known as Adnectins, see reference 34-35, the contents of which are incorporated herein by reference). Monobodies resemble the antigen-binding domain of antibodies and can be evolved against a wide range of targets36,37. They interact with their ligands through larger interfaces than small molecules, which allows them to recognize their targets with high affinity and sufficient specificity to discriminate between members of homologous protein families38-40. Exemplary degradons provided herein were built around the HA4 monobody, that recognizes the Src homology 2 (SH2) domain of the tyrosine kinase Abl1, as well as an oncogenic derivative of Abl, BCR-Abl (see reference 38-39, the contents of which are incorporated herein by reference). BCR-Abl causes chronic myelogenous leukemia (CML) and is the target of the small molecule tyrosine kinase inhibitor imatinib (Gleevec)43. Other monobodies, called Nsa1 and Cs1 used here target the SH2 domain of Shp2 (see reference 40, the contents of which are incorporated herein by reference).
Using both in vitro and in-cell techniques, it has been demonstrated that the degradons of the embodiments efficiently promote the degradation of the target protein. Thus, the degradons provided herein can be used in a novel strategy for depleting cellular proteins, which can be applied as a research tool to study intracellular protein function and has the potential for therapeutic use.
As used herein the term “degradon” refers to a recombinant polypeptide comprising a target binding domain and a proteasome-binding domain (e.g., a ubiquitin-like domain). A degradon can comprise from N-terminus to C-terminus (a) the target binding domain and (b) proteasome binding domain or from N-terminus to C-terminus (a) proteasome binding domain and (b) the target binding domain. Optionally, the degradon can comprise a linker sequence positioned between the target binding domain and proteasome binding domain.
The degradon systems of the embodiments provide a method to deplete specific proteins from cells by targeting them to the proteasome for destruction through synthetic adaptor proteins called degradons. Generally, degradons comprise a recognition element to select the target protein and a carrier element (a ubiquitin-like domain) that shuttles the target to the proteasome for proteolysis. This strategy has several advantages over current methods. First, degradons act directly on the target protein, so depletion is not limited by the intrinsic turnover rates of the protein and the data suggest destruction can be rapid and substantial or even complete. Also, the ubiquitin proteasome system (UPS) is robust and designed to degrade hundreds of regulatory proteins in addition to misfolded and damaged proteins in the cytosol and nucleus of eukaryotic cells27, and the proteasome's power can be harnessed for the purposes of this technology. In addition, degradons can be derived from natural proteasome substrate receptors and redesigned to function as specific proteasome adaptor molecules or degradons. In some aspects, degradons link the target directly to the proteasome, circumventing the ubiquitination pathway. Avoiding the ubiquitination step may allow degradons reduce off-target effects.
Further, degradons of the embodiments are versatile and customizable. They can be evolved or designed to target almost any protein and to recognize specific PTMs. The degradons tested in the examples recognize their targets by protein affinity agents derived from the tenth domain of fibronectin type III called monobodies which are easy to synthesize and contain simple sequences that can be evolved against a wide range of targets36,37.
Finally, degradons have therapeutic potential. The degradons of the examples, for instance, contain monobodies, which in themselves are therapeutically relevant, and several are currently in phase II clinical trials35,42. Thus far, most monobodies act by simply blocking protein-protein interactions. This not only limits the processes that can be targeted, but also means that monobodies can only act on one target molecule at a time, thus requiring high dosages to be effective. These degradons are designed to escape proteasomal degradation32,33,53,54, which allow them recycle and to act catalytically. This could potentially increase their power by orders of magnitude.
The degradons described in the examples bind to the proteasome through the UbL domain of Rad23 or hHR23b. Other proteasome interaction domains may be effective too. For example, HPV E7 binds to both the target protein Rb57 and the S4 subunit of the proteasome58 with high affinity, which allows direct proteasome targeting, bypassing the ubiquitination machinery18,59. The interactions of E7 with the proteasome58 and Rb57 have been defined and E7's proteasome binding domain could be used in degradons. Similarly, the protein gankyrin binds to Rb60 and the proteasome subunit S661,62 and gankyrin's proteasome binding domain could be incorporated into degradons. Other binding modalities will also be explored in the future.
The degradons described in the examples were built using monobodies as the substrate targeting head. Other targeting strategies are feasible and in the future, degradons constructed from additional types of targeting domains will be tested. Antibodies, represent a fast growing class of drugs41. In most current implementations they are directed against cell surface molecules to block their action. Human antibodies offer an attractive avenue for therapeutics because they can be raised to bind tightly (low picomolar to nanomolar affinities) to many targets and have inherently low immunogenicity63. Smaller derivatives of antibodies, such as single chain antibodies (scFvs) and Fab fragments, may be more suitable for some applications, especially those where Fc-mediated effects are undesirable64. Two specifically designed target recognition domains will be tested. Omomyc is a Myc-binding domain derived from Myc's binding partner Max and mutated to increase the specificity and affinity of Omomyc-Myc dimer formation65. Generalizable computational protein design strategies have been developed to engineer protein—protein interaction surfaces66. Affinity reagents that recognize Mdm2 or MdmX with high affinity and specificity will also be tested. The tightest binders recognize Mdm2 with sub-nanomolar dissociation constants and discriminate between Mdm2 and MdmX by a thousand-fold67. Incorporating any of these affinity reagents into degradons may turn them into catalytically acting agents, greatly enhancing their power.
In the examples, degradons were delivered into cells by transfection, which is a viable delivery method for a research tool. Current protein drugs work extracellularly but several strategies have been developed to allow proteins to enter cells: the attachment of cell-penetrating peptides68-70, receptor-mediated delivery systems71, and mRNA delivery72,73. In the future, all three delivery strategies will be tested on degradons. Gene therapy, the delivery of genes to somatic cells in animals, is making significant progress74,75 and may eventually become a feasible delivery approach for degradons.
Thus, embodiments detailed herein provide system has been developed that specifically targets and destroys the Abl or the Shp2 SH2 domain by using adaptors, or degradons, to shuttle it to the proteasome. Using both in vitro and in-cell techniques, it has been demonstrated that these degradons efficiently promote the degradation of our target proteins. The system has also been used to destroy Shp2 protein in cells. Together, these results describe a novel strategy for depleting cellular proteins, which can be applied as a research tool to study intracellular protein function and has the potential for therapeutic use.
Targeting domains for use in degradons of the embodiments can include antibodies or fragments thereof, such as monoclonal antibodies, single chain antibodies (scFvs), Fv fragments or Fab fragments. Monoclonal antibodies to a target molecule can be made using standard methods such as, for example, hybridoma-based methods, genetically altered and transgenic mouse-based methods, recombinant methods, and display methods. Human antibodies can be made using methods such as, for example, transgenic mice comprising human heavy chain and light chain loci, human B-lymphocytes, recombinant methods, and display methods. In certain embodiments, monoclonal antibodies may be manipulated by recombinant techniques. In certain such embodiments, nucleic acid(s) encoding the heavy chain and light chain of the monoclonal antibody chains may be isolated and cloned from the cell expressing the antibody. For example, RNA can be prepared from cells expressing the desired antibody, such as mature B-cells or hybridoma cells, using standard methods. The RNA can then be used to make cDNA using standard methods, and the cDNA can be amplified, for example, by PCR, using specific oligonucleotide primers.
In certain embodiments, human or non-human antibodies are chimerized. In certain embodiments, mouse monoclonal antibodies are chimerized by replacing the mouse Fc with a human Fc. In certain embodiments, human monoclonal antibodies are chimerized by replacing the human Fc with a non-human animal Fc. In certain embodiments, the human Fc is replaced with a mouse Fc. Certain exemplary methods for making chimeric antibodies are provided, for example, in Morrison et al. (1984) Proc. Nat'l Acad. Sci. USA 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454; and U.S. Pat. Nos. 6,075,181 and 5,877,397.
In certain embodiments, non-human antibodies are “humanized.” As a non-limiting example, a mouse monoclonal antibody that specifically binds the target molecule may be humanized in order to reduce immunogenicity (e.g., reduced human anti-mouse antibody (HAMA) response) when administered to a human. In certain embodiments, a humanized antibody has a similar binding affinity for the target molecule as the non-humanized parent antibody. In certain embodiments, a humanized antibody has increased binding affinity for the target molecule when compared to the non-humanized parent antibody. Certain exemplary humanization methods include, but are not limited to, CDR grafting and human engineering, as described in detail below.
In certain embodiments, one or more complementarity determining regions (CDRs) from the light chain and/or heavy chain variable regions of an antibody with the desired binding specificity (the “donor” antibody) are grafted onto human framework regions (FRs) of the light and/or heavy chain of an “acceptor” antibody in order to create a humanized antibody with the binding specificity of the donor antibody.
In certain embodiments, cDNA encoding a heavy chain and/or light chain can be modified in order to modify the expressed heavy and/or light chain. For example, in certain embodiments, the constant region of a mouse heavy or light chain can be replaced with the constant region of a human heavy or light chain. In this manner, in certain embodiments, a chimeric antibody can be produced which possesses human antibody constant regions but retains the binding specificity of a mouse antibody. Alternatively, the constant region of a human heavy or light chain can be replaced with the constant region of a non-human animal heavy or light chain. In this case, a chimeric antibody can be produced which possesses non-human animal antibody constant regions, e.g., for expression in a non-human animal model, but retains the binding specificity of the human antibody.
Degradon targets can include proteins that are involved in a disease. For example, they include mutant proteins, such a proteins encoded by genes that are inherited. In further aspects, the target proteins are encoded by oncogenes such as BCL2, c-MYC, Ras and HER2. Other exemplary oncogenes included, but are not limited to, BCR/ABL, ABL1/BCR, ABL, BCL1, CD24, CDK4, EGFR/ERBB-1, HSTF1, INT1/WNT1, INT2, MDM2, MET, MYB, MYC, MYCN, MYCL1, RAF1, NRAS, REL, AKT2, APC, BCL2-ALPHA, BCL2-BETA, BCL3, BCR, BRCA1, BRCA2, CBL, CCND1, CDKN1A, CDKN1C, CDKN2A, CDKN2B, CRK, CRK-II, CSF1R/FMS, DBL, DDOST, DCC, DPC4/SMAD4, E-CAD, E2F1/RBAP, ELK1, ELK3, EPH, EPHA1, E2F1, EPHA3, ERG, ETS1, ETS2, FER, FGR, FLI1/ERGB2, FOS, FPS/FES, FRA1, FRA2, FYN, HCK, HEK, HER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2, INK4A, INK4B, JUN, JUNB, JUND, KIP2, KIT, KRAS2A, KRAS2B, LCK, LYN, MAS, MAX, MCC, MLH1, MOS, MSH2, MYBA, MYBB, NF1, NF2, P53, PDGFB, PIM1, PTC, RB1, RET, ROS1, SKI, SRC1, TAL1, TGFBR2, THRA1, THRB, TIAM1, TRK, VAV, VHL, WAF1, WNT2, WT1, YES1, ALK/NPM1, AMI1, AXL, FMS, GIP, GLI, GSP, HOX11, HST, IL3, INT2, KS3, K-SAM, LBC, LMO-1, LMO-2, L-MYC, LYL1, LYT-10, MDM-2, MLH1, MLL, MLM, N-MYC, OST, PAX-5, PMS-1, PMS-2, PRAD-1, RAF, RHOM-1, RHOM-2, SIS, TAL2, TAN1, TIAM1, TSC2, TRK, TSC1, STK11, PTCH, MEN1, MEN2, P57/KIP2, PTEN, HPC1, ATM, XPA/XPG, BCL6, DEK, AKAP13, CDH1, BLM, EWSR1/FLI1, FES, FGF3, FGF4, FGF6, FANCA, FLI1/ERGB2, FOSL1, FOSL2, GLI, HRAS1, HRX/MLLT1, HRX/MLLT2, KRAS2, MADH4, MAS1, MCF2, MLLT1/MLL, MLLT2/HRX, MTG8/RUNX1, MYCLK1, MYH11/CBFB, NFKB2, NOTCH1, NPM1/ALK, NRG/REL, NTRK1, PBX1/TCF3, PML/RARA, PRCA1, RUNX1, RUNX1/CBFA2T1, SET, SHP2, TCF3/PBX1, TGFB1, TLX1, P53, WNT1, WNT2, WT1, αv-β3, PKCα, TNFα, Clusterin, Survivin, TGFβ, c-fos, c-SRC, and INT-1.
Furthermore, in certain aspects, degradons can include segments of sequence that encode a cell penetrating peptide (CPP) As used herein the terms “cell penetrating peptide” refers to segments of polypeptide sequence that allow a polypeptide to cross the cell membrane (e.g., the plasma membrane in the case a eukaryotic cell). Examples of CPP segments include, but are not limited to, segments derived from HIV Tat (e.g., GRKKRRQRRRPPQ (SEQ ID NO: 16)), herpes virus VP22, the Drosophila Antennapedia homeobox gene product, protegrin I, Penetratin (RQIKIWFQNRRMKWKK (SEQ ID NO: 17)) or melittin (GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 18)). In certain aspects the CPP comprises the T1 (TKIESLKEHG (SEQ ID NO: 19)), T2 (TQIENLKEKG (SEQ ID NO: 20)), 26 (AALEALAEALEALAEALEALAEAAAA (SEQ ID NO: 21)) or INF7 (GLFEAIEGFIENGWEGMIEGWYGCG (SEQ ID NO: 22)) CPP sequence.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
To test whether degradons are able to induce the degradation of proteins by the proteasome, a reliable readout to easily measure target protein abundance was needed. The target proteins, Abl and Shp2 SH2 domains, were attached to green fluorescent protein (GFP) both because its intrinsic protein fluorescence is directly proportional to the concentration of GFP, and because it has been used extensively in vitro and in vivo47,48. In addition, because GFP has a long halftime in vitro and in vivo and is challenging for the proteasome to degrade49,50, successfully degrading GFP should demonstrate the system's robustness. Therefore, for the target protein, the SH2 domain of human c-Abl and either the N-terminal or C-terminal SH2 domain of Shp2 was fused to the N-terminus of GFP. A 35 amino acid long disordered sequence was added to the C-terminus of GFP to create the final substrate protein SH2-GFP-tail (
To target the model protein for degradation in vitro, a set of degradons was constructed consisting of a monobody (mB) domain attached through a flexible linker to the UbL domain of S. cerevisiae Rad23 (
In Vitro Degradation of Proteins is Induced by Degradons.
The degradation of substrate protein (
The Degradons can Act Catalytically.
Next, it was tested whether degradons can act catalytically, where one degradon molecule induces the degradation of several target proteins. To do so, the depletion of different concentrations of the target protein by a fixed concentration of degradon (10 nM) was assayed and the amount of degraded substrate was quantified (
Degradon Transfection Leads to Substrate Degradation in Cells.
Next, an experiment was performed to determine whether the system developed in vitro could be applied to cells. First, stable HEK293 FlpIn™ cells expressing the three substrates (SH2Abl-GFP-35ΔK, N-SH2Shp2-GFP-35ΔK, and C-SH2Shp2-GFP-35ΔK) through genomic integration were set up (
Adaptor Transfection Leads to Endogenous Shp2 Depletion in Cells.
The next step was to determine whether the degradons were able to degrade full-length, endogenous substrate in cells. The Shp2 degradons were used to deplete endogenous Shp2, which is readily detectable in 293T cells (
Substrate and Degradon Plasmid Design.
In vitro substrates and degradons were created using Gibson Assembly PCR. Constructs were cloned into the second multiple cloning site in a pETDUET vector with a C-terminal Chitin binding domain tag followed by a 6× Histidine tag and expressed from the T7 promoter in E. coli strains BL21(DE3)pLysS or Rosetta(DE3)pLysS (Novagen).
The target protein consisted of the SH2 domain of Abl1 (amino acids 112-232 of human Abl1), the N-terminal SH2 domain of Shp2 (amino acids 2-103), or the C-terminal SH2 domain of Shp2 (amino acids 112-217 of human Shp2) followed by a 6 amino acid long Gly-Gly-Ser-Gly-Ser linker, the entire sequence of monomeric superfolder GFP, and finally a C-terminal 35 amino acid long sequence corresponding to amino acids 2 to 36 of yeast cytochrome b2 with all lysine residues mutated to arginine (called 35ΔK).
The proteasome adaptors (degradons) consisted of the UbL domains of yeast Rad23 (amino acids 1-77 of Rad23) for the in vitro experiments or of human Rad23b (amino acids 1-83 of Rad23b) for the cell culture experiments fused to full length HA4, Nsa1, or Cs1 monobodies38. In the constructs with the longer linkers an additional 69 amino acids from Rad23 following the UbL domain were included in the degradon.
Protein Purification.
Yeast proteasome was purified from S. cerevisiae strain YYS40 (MATa rpn11::RPN11 3×FLAG-HIS3 leu2 his3 trp1 ade2 can1 ssd1) by immunoaffinity chromatography using FLAG antibodies (M2 agarose affinity beads, Sigma) as previously described56. Proteasome preparations were analyzed by SDS-PAGE and compared to published compositions. Each proteasome preparation was checked for activity by testing degradation of the positive control proteasome substrate UbL-GFP-tail.
The substrates and degradons used for the in vitro degradation assays were overexpressed in and purified from E. coli using standard methodologies. Bacterial strains were grown in 1 L of 2XYT media at 37° C. to an optical density of 0.6-1.0. Protein expression was induced with 0.25-1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubation continued overnight at 16° C. Cells were harvested by centrifugation, resuspended in ice-cold NPI-10 buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl, and 10 mM imidazole) and homogenized using the Avestin Emulsiflex C3 (2-3 passes) at 15,000 psi. The cell lysate was incubated with Ni-NTA metal affinity beads (GE Healthcare) and nutated for 30 min at 4° C. This mixture was applied to a gravity column, and washed with five column volumes of NPI-10 buffer, then five column volumes NPI-20 buffer (NPI-10 with 20 mM imidazole). The protein was eluted from the resin with NPI-250 buffer (NPI-10 with 250 mM imidazole). The elution was desalted by PD-10 desalting column (GE Healthcare) and into Chitin-column binding buffer (CBB) (20 mM Tris pH 7.4, 300 mM NaCl, and 0.5 mM EDTA). This was bound to Chitin beads (NEB) then washed with 50 column volumes of CBB. The protein was eluted after an overnight incubation with Chitin cleavage buffer (CBB with 100 mM dithiothreitol) and buffer exchanged into storage buffer (20 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol). Protein concentration was measured using absorbance at 280 and the extinction coefficient from the proteins' sequence (ExPASy's ProtParam). The identity and purity of purified proteins was confirmed by SDS-PAGE.
Substrate Degradation Assay.
The degradation of fluorescent substrate (SH2-GFP-tail) in vitro was monitored by fluorescence intensity and performed in 384-well plate on a Tecan plate reader (Infinite® M1000 PRO, Tecan). Assays were carried out at 30° C. by adding fluorescent substrates to 25-40 nM of purified yeast proteasome in a reaction buffer containing a creatine-phosphate/creatine kinase/ATP regenerating system54. Fluorescence intensity was read every minute at the excitation wavelength of 388 nm and the emission wavelength of 420 nm for 75 minutes. Protein amount was calculated based on fluorescence intensity of the reaction in each well and the standard curves describing the correlation between fluorescence intensity and protein concentration.
Each assay was repeated at least two times. Initial degradation rates are given by the slope of the decay curves at time zero and are calculated as the product of the amplitude and the rate constant of the decay curve determined by nonlinear fitting to a single exponential decay in the software package Kaleidagraph (version 4.1, Synergy Software).
Mammalian Construct Design.
The substrate of Abl or Shp2 SH2-GFP-tail was cloned into pcDNA5/FRT/TO using the In Fusion® cloning kit (Clontech). The binding degradon, consisting of human Rad23b UbL domain fused N-terminally to the monobody (HA4, Nsa1, or Cs1), was cloned using restriction enzymes into a pcDNA3 vector containing an N-terminal FLAG tag and C-terminal Internal Ribosome Entry Site (IRES) followed by mCherry. The resulting constructs were FLAG-hUbL-mB-IRES-mCherry. The non-binding degradon was created by changing HA4 Tyr87 to Ala using the QuikChange Site-Directed Mutagenesis kit (Agilent) to generate FLAG-hUbL-mBHA4(Y87A)-IRES-mCherry.
Stable Cell Generation.
Stable HEK293 Flp-In™ cells expressing the substrate (Shp2 or Abl SH2-GFP-tail) were generated using the Flp-In™ system (Life Technologies) according to manufacturer's instructions. Briefly, Flp-In™-293 host cells were transfected with 1 μg substrate DNA in pcDNA5/FRT/TO plasmid and 9 μg pOG44 plasmid encoding Flp recombinase in OPTI-MEM (Life Technologies) with Lipofectamine® 2000 in a 6-well plate. 24 hours later, OPTI-MEM was replaced with Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (pen/strep) and cells were allowed to recover overnight. Cells were trypsinized the next day and plated on 10 cm2 plates in fresh medium. Medium was replaced 24 hours later with DMEM supplemented with 10% FBS, 1% pen/strep, and 200 μg/mL hygromycin B (Gibco). Medium was replaced every two to three days with fresh selective medium until colonies formed. All colonies were considered to be isogenic and the whole plate was trypsinized and plated into fresh selective medium in a 75 cm2 dish, then subcultured according to manufacturer's instructions. Aliquots of cells were frozen in FBS supplemented with 10% DMSO by cooling at −1° C./min at −80° C. and stored in a −150° C. freezer until further use.
Transfections and Assays.
HEK293 Flp-In™ host and SH2-GFP-tail stable cells were transfected with either binding (FLAG-hUbL-mB-IRES-mCherry) or non-binding degradon (FLAG-hUbL-mBHA4(Y87A)-IRES-mCherry) or water (vehicle) with Lipofectamine® 2000 reagent according to manufacturer's instructions in 6-well plates. For immunoblot analysis, 48 hours after transfection, cells were harvested and lysed in whole-cell extract buffer (50 mM Tris pH8.0, 280 mM NaCl, 10% glycerol, 0.5% NP-40, 0.2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1 mM orthovanadate, and protease inhibitor cocktail from Roche) and cleared by centrifugation. ˜50-100 μg of cellular lysate was used for Western blotting. Antibodies used were: primary rabbit anti-Actin (Sigma), mouse anti-Shp2 (Santa Cruz, clone B-1), mouse anti-FLAG (Sigma), secondary IR dye 800 goat anti-mouse (Rockland), and Alexa Fluor 680 goat anti-rabbit (Invitrogen).
For Fluorescence Activated Cell Sorting (FACS), 48 hours after transfection, cells were trypsinized and neutralized in DMEM without phenol red supplemented with 10% FBS and 1% pen/strep. FACS data were collected on a BD Fortessa flow cytometer. Data were analyzed using FlowJo software (version 3.0).
For endogenous protein immunostaining, 72 hours post transfection, cells were trypsinized and neutralized in DMEM without phenol red (Gibco) supplemented with 10% FBS. Cells were washed once with D-PBS (Gibco), then fixed with 0.25% formaldehyde (from Formalin containing 4% formaldehyde, Sigma) for 20 minutes on ice. Cells were washed with PBS then permeabilized with 90% methanol in PBS for 30 minutes on ice. After washing with PBS, cells were blocked for one hour with PBS plus 0.5% BSA. Cells were washed and primary mouse anti-Shp2 antibody (Santa Cruz, clone B-1) was added at a 1:500 concentration and incubated overnight at 4° C. Cells were washed twice and then secondary goat anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen) was added at a 1:2500 concentration. Cells were washed and then analyzed via FACS as above.
SDS-PAGE fluorescence analysis was carried out on unboiled samples with a Typhoon FLA 7000 (GE Healthcare) with filters set at 488 nm (GFP) and 555 nm (mCherry). Images were quantified using ImageJ software.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims the benefit of U.S. Provisional Patent Application No. 62/252,472, filed Nov. 7, 2015, the entirety of which is incorporated herein by reference.
This invention was made with government support under Grant No. R01 GM063004 and R21 CA196456 awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2016/060787 | 11/7/2016 | WO | 00 |
Number | Date | Country | |
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62252472 | Nov 2015 | US |