Patent Application
20110165560
Mitochondria are complex organelles that play various roles in many aspects of cellular function, including ATP production, intermediary metabolism, apoptosis, and many others. As a result, mitochondrial dysfunction is tightly coupled with a variety of human diseases, including cancer, type 2 diabetes, and various age-related pathologies. The link between mitochondrial metabolism and cancer is a particularly emergent area of interest and investigation. Cancer-related mitochondrial defects have been identified, such as altered expression and activity of respiratory chain and tricarboxylic acid cycle enzymes and mitochondrial DNA (mtDNA) mutations. Specific discussion can be found in Baysal et al., (2000), Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma, Science 287, 848-851; Chatterjee et al., (2006), Mitochondrial DNA mutations in human cancer, Oncogene 25, 4663-4674; and Yan et al., (2009), IDH1 and IDH2 mutations in gliomas, N Engl J Med 360, 765-773, each of which are incorporated herein by reference. The discovery of the central role of mitochondria in apoptosis further emphasizes the importance of mitochondria in cancer, which typically exhibits altered sensitivity to apoptotic cell death.
Before the present invention is disclosed and described, it is to be understood that this invention is not limited to the particular structures, process steps, or materials disclosed herein, but is extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.
It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a promoter” includes one or more of such promoters and reference to “the protein” includes reference to one or more of such proteins.
Definitions
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below.
As used herein, “subject” refers to an organism that includes SDH proteins.
As used herein, the term “biological sample” refers to any material of a biological nature obtained from a subject from which DNA can be obtained. Non-limiting examples include biological fluids and biological tissues such as blood, blood serum, saliva, semen, vaginal fluid, lymph, urine, lachrymal fluid, cancerous tissue, non-cancerous tissue, tumor tissue, skin tissue, and the like.
As used herein, the term “succinate dehydrogenase-related disease condition” refers to a disease condition that is linked in some way to defective functionality of succinate dehdrogenase. As an example, a succinate dehydrogenase-related cancer would be any cancer that is linked to defective functionality of succinate dehydrogenase.
As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is “substantially free of an ingredient or element may still actually contain such item as long as there is no measurable effect thereof.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint.
As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to about 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Invention
The present invention provides methods, compositions, and kits associated with succinate dehydrogenase-related disease conditions. In one aspect of the present invention, a method for screening a test subject to determine risk for developing a succinate dehydrogenase-related disease condition is provided. Such a method can include obtaining a biological sample from the test subject and identifying a mutation in gene hSDH5 from the biological sample of the test subject, wherein the mutation effectuates a decreased level of succinate dehydrogenase flavination in the test subject as compared to a level of succinate dehydrogenase flavination in a normal subject. It should be noted that one of ordinary skill in the art is capable of readily establishing the level of succinate dehdryogenase flavination in a normal subject. Additionally, obtaining biological samples and testing those samples for gene mutations are well known in the art, and one of ordinary skill in the art would be readily able to test for mutations of the present invention once in possession of the present disclosure.
In one aspect, the mutation is at hSDH5 Gly78 of gene hSDH5. In another aspect, the mutation is equivalent to an hSDH5 Gly78Arg substitution of gene hSDH5. Additionally, non-limiting examples of biological samples includes biological fluids, biopsies, tumors, cancerous tissue, noncancerous tissue, and combinations thereof. In another aspect, the decreased level of succinate dehydrogenase flavination is a substantially complete or complete absence of succinate dehydrogenase flavination. In yet another aspect, the decreased level of succinate dehydrogenase flavination is at least a 50% decrease as compared to a normal subject. In a further aspect, the decreased level of succinate dehydrogenase flavination is at least a 75% decrease as compared to a normal subject.
A variety of succinate dehydrogenase-related disease conditions are implicated and within the scope of the present invention. In one aspect, for example, the disease condition is a succinate dehydrogenase-related cancer. Other non-limiting examples of such disease conditions can include neuroendocrine tumors, paraganglioma tumors, gastrointestinal tumors, Carney-Stratakis syndrome, phenochromocytoma tumors, renal cell carcinomas, optic atrophy, ataxia, myopathies, neurodegeneration, and combinations thereof. In one specific aspect, the disease condition is a paraganglioma tumor.
The results of identifying a mutation in gene hSDH5 can be utilized for various purposes. Non-limiting examples can include predicting the disease condition risk, predicting the disease condition progression, predicting genetic inheritance risks associated with the disease condition, making a clinical diagnosis of the disease condition, providing information to affect the course of the disease condition, adjusting clinical therapy to treat the disease condition, and the like, including combinations thereof.
The present invention also provides compositions containing nucleotide constructs of hSDH5 mutants. In one aspect, such a composition can include a nucleotide construct of a mutant of hSDH5 and a member selected from the group consisting of a vector, RNA, a virus, and combinations thereof. In one aspect, the composition is a vector including the nucleotide construct of mutant hSDH5. In another aspect, the mutant hSDH5 encodes a Gly78 mutation. In yet another aspect, the mutant hSDH5 encodes a Gly78Arg substitution.
The present invention also provides kits for screening a test subject. In one aspect, for example, a kit for screening a test subject to determine whether the subject is at risk for developing a succinate dehydrogenase-related disease condition can include a kit housing containing an assay capable of identifying a mutation in gene hSDH5 from the biological sample of the test subject, and instructions describing how to use the assay to screen the test subject for the disease condition associated with succinate dehydrogenase flavination. In one specific aspect, the assay identifies the mutation as being at hSDH5 Gly78 of gene hSDH5. It should be understood that one of ordinary skill in the art would readily understand what components and ingredients to include in such a kit once in possession of the present disclosure.
In another aspect, for example, a method for screening a test subject to determine risk for developing a succinate dehydrogenase-related disease condition is provided, including identifying a decreased level of succinate dehydrogenase flavination in the test subject as compared to a level of succinate dehydrogenase flavination in a normal subject. In another aspect, the decreased level of succinate dehydrogenase flavination is a substantially complete or complete absence of succinate dehydrogenase flavination. In one specific aspect, identifying the decreased level of succinate dehydrogenase flavination includes detecting a decrease in SDH1-FAD conjugates in biological sample from the test subject as compared to a level of SDH1-FAD conjugates in a normal subject. In another specific aspect, the decrease in SDH1-FAD conjugates in biological sample from the test subject includes a substantially complete or complete absence of SDH1-FAD conjugates.
It is contemplated that various factors can contribute to a decreased level of succinate dehydrogenase flavination. In one aspect, for example, the decreased level of succinate dehydrogenase flavination is characterized by a mutation in gene SDH5. In another aspect, the mutation is at hSDH5 Gly78 of gene SDH5. In yet another aspect, the mutation is equivalent to hSDH5 Gly78Arg in gene SDH5.
Succinate dehydrogenase (SDH) is an enzyme complex that is bound to the inner mitochondrial membrane in eukaryotic organisms and in some prokaryotes. SDH is known to participate in both the electron transport chain and the citric acid cycle. In the citric acid cycle, SDH catalyzes the oxidation of succinate to fumarate with the reduction of ubiquinone to ubiquinol as part of the energy production processes of the mitochondria. The SDH complex includes at least 4 major subunits, SDHA-D. It should be noted that subunits SDHA-D are synonymously known as Sdh1-4 (orthologs in yeast), and as such, these terms can be used interchangeably. SDH also contains multiple cofactors that play roles in the function of the complex. For example, an iron-sulfur cluster is present in SDHB and a heme is shared by SDHC and SDHD. Subunit SDHA includes flavin adenine dinucleotide (FAD) inserted covalently into SDHA in a process known as flavination, without which the succinate to fumarate oxidation does not occur.
A project was initiated to understand the function of a subset of previously uncharacterized mitochondrial proteins. Yeast was utilized as the primary model system for at least three reasons. First, the yeast S. cerevisiae is a tractable and facile genetic and biochemical system. Second, mitochondrial biology is cell-autonomous and highly conserved from yeast to humans. Finally, yeast can survive the complete loss of respiration, due to their ability to generate ATP from fermentation. This is important for genetic analysis of proteins that are essential for respiration, the deletion of which would be lethal in other organisms. This project focused on the protein EMI5/YOL071W. EMI5 (Early Meiotic Induction 5) was so named because it was initially identified in screens for sporulation (meiosis) mutants, but no additional work has been described. Mitochondrial proteins may affect sporulation due to the necessity of respiration in this process. Through this project, a novel protein interaction between the previously uncharacterized EMI5 protein and the SDH complex was discovered that may allow for the identification of subjects at risk for SDH-related disease conditions, as well as the potential diagnosis, prognosis, and treatment of such diseases.
Based on the molecular function that was discovered, EMI5 was renamed to Sdh5. As is shown in
The human Sdh5 ortholog similarly interacts with human SDHA (Sdh1 ortholog) and is able to complement the yeast sdh5Δ mutant phenotype and rescue Sdh1 flavination, suggesting functional conservation. SDH deficiency in humans has been linked to paraganglioma (PGL), neuroendocrine tumors derived from the extra-adrenal paraganglia of the autonomic nervous system. Familial PGL syndromes have been mapped to four loci, designated PGL1-4. Mutations in three of the SDH structural subunits, SDHB, SDHC and SDHD, have been associated with PGL4, PGL3 and PGL1, respectively. The PGL2 gene was mapped to an interval on chromosome 11 in a Dutch lineage, but the affected gene eluded identification. That interval includes human Sdh5, and the inventors have now identified an hSdh5 G78R mutation that segregates with disease in PGL2 patients and causes loss of SDHA flavination. Using cultured human cells and taking advantage of hSdh5 complementation of the yeast sdh5Δ mutant phenotype, it is now shown that this mutation causes a loss of function. Additionally, the G78R mutant of hSdh5 was inactive in functional studies and tumors derived from PGL2 patients exhibited a near complete loss of SDHA flavination. Thus, starting with an uncharacterized yeast protein, the inventors have determined Sdh5's molecular function and the causal role of its human ortholog in a familial neuroendocrine tumor syndrome. Further discussion of PGL1-4 can be found in Favier et al., (2005), Hereditary paraganglioma/pheochromocytoma and inherited succinate dehydrogenase deficiency, Horm Res 63, 171-179; and in Mariman et al., (1993), Analysis of a second family with hereditary non-chromaffin paragangliomas locates the underlying gene at the proximal region of chromosome 11q, Hum Genet 91, 357-361; both of which are incorporated herein by reference.
Thus, in one aspect of the present invention a method for screening a test subject to determine risk for developing a succinate dehydrogenase-related disease condition is provided. Such a method can include obtaining a biological sample from the test subject, and identifying a mutation in gene hSDH5 from the biological sample. It should be noted that gene hSDH5 may also be referred to as gene SDHAF2. In this case, the mutation effectuates a decreased level of succinate dehydrogenase flavination in the test subject as compared to a level of succinate dehydrogenase flavination in a normal subject. Thus subjects can be screened for mutations in hSDH5 as part of any genetic testing regime. This can be performed in subjects due to a suspected risk, as would be the case for subjects from families known to carry the mutant gene, or it can be performed as part of standard genetic testing.
Positive results indicating the presence of the mutant gene can be utilized for a variety of uses. For example, a subject testing positive for the mutant gene can be more closely monitored so that treatment can begin quickly once a disease condition is initiated. In many cases, particularly those involving cancers, frequent testing can identify a disease condition long before it becomes apparent to the subject. Such early monitoring and detection can greatly improve the prognosis of the disease condition, and in many cases increase the likelihood of survival for the affected subjects. In some cases, pretreatment for the disease condition can also be initiated. Other uses can include, without limitation, predicting the progression of a disease condition, predicting genetic inheritance risks associated with the disease condition, making a clinical diagnosis of the disease condition, providing information to affect the course of the disease condition, adjusting clinical therapy to treat the disease condition, and the like.
Sdh5 is a Soluble Mitochondrial Matrix Protein
To confirm the mitochondrial localization of Sdh5 by fluorescence microcopy and subcellular fractionation, strains of yeast were generated wherein deletion of the endogenous SDH5 gene (sdh5Δ, JRY609) was complemented by plasmids that express C-terminally GFP-tagged or HA-tagged forms of Sdh5 under the control of the native SDH5 promoter (pSdh5-GFP, pSdh5-HA). Both forms of tagged Sdh5 are functional, as they complement the sdh5Δ mutant phenotype (
The sub-mitochondrial localization of Sdh5 was examined by proteinase K (PK) protection (
To assess whether Sdh5 is a soluble matrix protein or associates with the inner membrane, isolated mitochondria were sonicated in detergent-free buffer and separated into soluble and insoluble membrane fractions by ultracentrifugation. The majority of Sdh5-HA and aconitase (a soluble matrix protein) were found in the soluble fraction, while the peripheral membrane protein Sdh1 was found predominantly in the membrane fraction. Together, these data show that Sdh5 is a soluble mitochondrial matrix protein.
Sdh5 is Required for Respiration
One function of mitochondria across phylogeny is to generate ATP for cellular activities and growth through aerobic respiration. Yeast have the ability to generate ATP and grow in the absence of respiration via fermentative metabolism. As a result, respiratory-deficient mutants are viable on fermentable carbon sources such as glucose, but are inviable on non-fermentable carbon sources such as glycerol. To investigate whether Sdh5 is required for mitochondrial respiration, the growth of wild-type (WT) and sdh5Δ mutant yeast were compared on glucose and glycerol medium (
It was also discovered that the sdh5Δ mutant strain had the respiration-related phenotypes of increased sensitivity to hydrogen peroxide and decreased chronological lifespan (
To directly assess respiration, the rate of oxygen consumption was measured and found to be decreased more than five-fold in the sdh5Δ mutant compared to wild-type (
Mutation or loss of the mitochondrial genome (mtDNA) is one common cause of respiratory deficiency in yeast. This explanation is unlikely in the present case because the sdh5Δ mutant phenotype is rescued by subsequent ectopic expression of Sdh5, and as such, this rescue is inconsistent with irreversible mtDNA mutation or loss. However, the integrity and function of mtDNA in sdh5Δ haploids was specifically tested by mating with rho0 haploids (
Sdh5 Interacts with Sdh1
To understand the role of Sdh5 in respiration, physical interactor(s) of Sdh5 by tandem affinity purification (TAP) were identified. An SDH5 allele was generated expressing a C-terminal polyhistidine and dual HA tagged Sdh5 fusion from the native SDH5 promoter (JRY597). This strategy has been shown to enable a similar degree of purification to the classical CBP/ProteinA TAP tag (See Honey et al., (2001), A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex, Nucleic Acids Res 29, E24, incorporated herein by reference). The Sdh5-His-HA fusion protein was purified from 50mg of isolated mitochondria, solubilized with 0.1% NP-40 detergent, using standard nickel chromatography followed by anti-HA affinity chromatography, eluting with HA peptide. Silver staining of the final elution samples showed a number of bands that were present in both the Sdh5-His-HA and untagged control samples. The 22 kD tagged Sdh5, the identity of which was confirmed by anti-HA immunoblot, is shown in
The SDH complex plays a role in both the TCA cycle and the electron transport chain (ETC), where it is often referred to as Complex II. The SDH complex oxidizes succinate to fumarate, coupled with electron transfer to Coenzyme Q and along the ETC eventually to oxygen. During this process a proton gradient is established across the inner membrane, which drives the synthesis of ATP through Complex V. The SDH complex is a heterotetramer and is highly conserved throughout eukaryotes (Sdh1-4 in yeast and SDHA-D in mammals). As illustrated in
Sdh1 was an intriguing potential binding protein due to the similarity between the sdh1Δ and sdh5Δ mutant phenotypes. Specifically, both phenotypes are unable to grow on glycerol medium, but are able to grow weakly with ethanol as the carbon source (
Sdh5 is Required for SDH Activity
To examine the functional significance of the Sdh1-Sdh5 interaction, SDH activity in sdh5Δ mutant mitochondria was measured. There was no detectable SDH activity in the sdh5Δ mutant (
SDH is a complex assembled from four distinct nuclear-encoded subunits. It may be hypothesized that Sdh5 might be required for the synthesis or assembly of Complex II. To test this hypothesis, respiratory complex formation was examined by Coomassie blue staining after BN-PAGE, and it was discovered that the band corresponding to Complex II in wild-type mitochondria is absent in sdh5Δ mutant mitochondria (
One explanation for the absence of Complex II in sdh5Δ mutant mitochondria is that Sdh5 is a necessary component of the SDH complex that has somehow eluded detection previously. To address this possibility, the Sdh5-containing complex was visualized by BN-PAGE followed by immunoblot as described above. As shown in
As Complex II is either not assembled or is unstable in sdh5Δ mutant mitochondria, the steady-state levels of each subunit were determined. All four SDH subunits were significantly decreased in the sdh5Δ mutant (
Sdh5 is necessary and sufficient for flavination of Sdh1 Multiple cofactors are required for the function of the SDH complex: FAD in Sdh1, an iron-sulfur cluster in Sdh2, and heme shared by Sdh3 and Sdh4. The cofactor attachment in Complex II was examined to see if this could be a reason for the impaired Complex II activity and stability in the sdh5Δ mutant. Due to the direct interaction between Sdh1 and Sdh5, the FAD attachment (flavination) of Sdh1 was examined. Unlike most cofactors, FAD is inserted covalently in Sdh1 and, therefore, can be detected fluorometrically after separation of mitochondrial proteins by SDS-PAGE. Deletion of SDH5 caused a complete loss of the Sdh1-FAD conjugate, just as was observed in sdh1Δ mutant mitochondria (
To address the sufficiency of Sdh5 for Sdh1 flavination, Sdh5 was overexpressed in wild-type cells using a high-copy 2μ plasmid, but an increase in Sdh1 flavination was not observed (
To further demonstrate the sufficiency of Sdh5 for Sdh1 flavination, Sdh1 was expressed in E. coli, either alone or in the presence of Sdh2 or Sdh5, and the flavination state of Sdh1 was examined. As shown in
Functional Conservation of Human SDH5 and Association with Paraganglioma
The amino acid sequence of yeast Sdh5 is 44% identical (from residue 33-158 of 163 total residues) to its human ortholog (SDH5), encoded by a previously uncharacterized gene Cllorf79. Cllorf79 will be referred to hereafter as hSDH5 to reflect its relationship with yeast SDH5. Based on the degree of sequence conservation, it can be hypothesized that hSDH5 will similarly be required for flavination of SDHA (the human ortholog of Sdh1) and thus for SDH activity.
Loss of SDH activity has been implicated in several neurological disorders and tumors, including paraganglioma (PGL). PGLs are neuroendocrine tumors derived from the extra-adrenal paraganglia of the autonomic nervous system. Four distinct familial PGL syndromes (PGL1-4) have been identified genetically, and PGL1, PGL3 and PGL4 have been associated with mutations in SDHD, SDHC and SDHB, respectively. The gene for PGL2 (OMIM accession# %601650) remains unidentified but was localized to a chromosomal region between the polymorphic markers D11S956 and PYGM, at chromosome 11q12.1 and 11q13.1, respectively. The hSDH5 gene is located almost exactly in the middle of this interval (NCBI Build 36.1 of the March 2006 human reference sequence) and is therefore a strong candidate for the PGL2 gene given its presumed role in SDH assembly and function.
Mutation analysis was conducted in three affected PGL2 individuals from different branches of the previously described Dutch lineage by DNA sequencing of all four exons and exon-intron boundaries of hSDH5. A single nucleotide change was found in all three individuals, c.232G>A in exon 2 (
The mutated G78 is part of an arginine-glycine dipeptide that is conserved in all eukaryotic and prokaryotic species for which sequence information is available and is in the most highly conserved region of the protein (
Constructs expressing wild-type and G78R hSDH5 were generated as C-terminal GFP and Myc fusions. In human HEK293 cells, both the wild-type and mutant GFP-fusion proteins localized exclusively to mitochondria, showing that neither expression nor mitochondrial localization is compromised by this mutation (
The hSDH5 G78R is a loss of Function Mutation
To further understand the nature of the G78R mutation in hSDH5, the ability of wild-type and G78R mutant hSDH5, both expressed from the yeast SDH5 promoter, to complement the glycerol growth defect of the sdh5Δ mutant strain was assessed. As shown in
The Sdh1 flavination in the same five strains used for the above glycerol growth test was then directly measured. As shown in
Further Discussion
The molecular mechanism of covalent FAD insertion into proteins has previously proven difficult to elucidate. Other covalent cofactors are inserted into proteins by specialized enzymes, such as cytochrome c heme lyase for heme attachment to apocytochrome c. Until now, it appears that no similar enzyme has been identified for covalent FAD attachment. Covalent FAD attachment to Sdh1 appears to be dependent on additional protein factors. Using in vitro translated Sdh1 precursor and differentially treated mitochondria, Sdh1 flavination was shown to require matrix import, ATP and at least one additional protein (See Robinson et a., (1996), A requirement for matrix processing peptidase but not for mitochondrial chaperonin in the covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein, J Biol Chem 271, 4061-4067, incorporated herein by reference.) It is possible that Sdh5 is the necessary matrix protein suggested by these earlier experiments.
The question remains, however, whether Sdh5 actually participates in the chemistry of FAD attachment (enzymatic function) or simply maintains Sdh1 in a conformation that is susceptible to autocatalytic FAD attachment (chaperone function). Four observations would suggest that Sdh5 does not act simply as a general chaperone. First, in addition to the myriad of native chaperones in both the mitochondrial matrix of S. cerevisiae and in E. coli, co-expression of Sdh2, a clear Sdh1 binding partner, is unable to support flavination of Sdh1 in either yeast or bacteria (
Whatever the exact role of Sdh5 in Sdh1 flavination, three observations suggest that it plays that role very specifically. First, as shown in
A surprising observation that has been consistently corroborated is that paraganglioma is associated with mutations in SDHB, SDHC and SDHD, but not with SDHA. SDHA mutations, but not SDHB, SDHC and SDHD mutations, are associated with Leigh syndrome. If these proteins function only when together as a complex, why should their mutation lead to different outcomes? One intriguing possibility is suggested by three observations shown in
There are immediate clinical implications of this work. It has been estimated that roughly 70% of familial cases of head and neck PGL are due to germline mutations in SDHB, SDHC or SDHD (See Baysal et al., (2002), Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas, J Med Genet 39, 178-183, incorporated herein by reference). It is likely that germline mutations in hSDH5 are causative in many of the remaining 30% of cases. Similarly, recent studies found that 10% of sporadic PGL was associated with mutations in SDH complex subunits (6% SDHB, 4% SDHD) (See Schiavi et al., (2005), Predictors and prevalence of paraganglioma syndrome associated with mutations of the SDHC gene, Jama 294, 2057-2063; and Timmers et al., (2009), Clinical aspects of SDHx-related pheochromocytoma and paraganglioma, Endocr Relat Cancer.; both of which are incorporated herein by reference). It is likely that hSDH5 mutations will also be found in a subset of the remaining 90% of sporadic PGL. Recently, the Carney-Stratakis syndrome has been identified, implicating mutations in SDH complex genes (SDHB, SDHC and SDHD) in gastrointestinal stromal tumors in addition to PGL (See McWhinney et al., (2007), Familial gastrointestinal stromal tumors and germ-line mutations, N Engl J Med 357, 1054-1056; and Pasini et al., (2008), Clinical and molecular genetics of patients with the Carney-Stratakis syndrome and germline mutations of the genes coding for the succinate dehydrogenase subunits SDHB, SDHC, and SDHD, Eur J Hum Genet 16, 79-88; both of which are incorporated herein by reference). Mutations in hSDH5 might also be causal in this new syndrome. Defects in SDH5 might also be involved in the development of pheochromocytomas, which are often associated with mutations in SDHB, SDHC and SDHD (Timmers et al., 2009). The identification of a new gene, the mutation of which is likely causal in a subset of patients, enables more comprehensive genetic testing. It has been suggested that the clinical management of PGL, even in the absence of evidence for familiality, should involve genetic testing (Cascon et. Al., (2009), Rationalization of Genetic Testing in Patients with Apparently Sporadic Pheochromocytoma/Paraganglioma, Horm Metab Res, incorporated herein by reference) and this genetic testing regime should now be modified to include hSDH5. This enables early identification and diagnosis of affected relatives, leading to improved morbidity and mortality outcomes. Other forms of cancer and a variety of other diseases may also be due to hSDH5 mutations (See Ricketts et al., (2008), Germline SDHB mutations and familial renal cell carcinoma, J Natl Cancer Inst 100, 1260-1262; and Birch-Machin et al., (2000), Late-onset optic atrophy, ataxia, and myopathy associated with a mutation of a complex II gene, Ann Neurol 48, 330-335; both of which are incorporated herein by reference).
Saccharomyces cerevisiae haploid strain JRY472 (W303a MATa his3 leu2 met15 trpl ura3) was used as the parental and wild-type strain. Deletion mutant strains were generated by KanMX4 disruption using homologous recombination in JRY472 except that sdh5Δ mutant haploid was derived from tetrad dissection of the heterozygous diploid. For all the double mutant strains, the sdh5Δ::KanMX4 mutant strain was first converted to NatMX4 by homologous recombination and then used for KanMX4 deletion of the second gene. The Sdh5-TAP strain was created by integration of the TAP tag in frame downstream of SDH5 in the genome of JRY472. All engineered strains were verified by PCR. Complete strain information is provided in Table 1. Yeast cells were transformed by the standard lithium acetate method and cultured in either rich (YP) medium or synthetic complete (SC) medium lacking the appropriate amino acids for plasmids selection, containing either 2% glucose, 2% raffinose or 3% glycerol carbon source as indicated.
HEK293 cells and HepG2 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FBS and antibiotics. HEK293 cells were transfected in suspension using Fugene 6 reagent (Roche) following the manufacturer's instructions. Frozen PGL tumor samples were obtained from affected patients with informed consent.
The family in which the PGL2 locus was identified (Mariman et al., (1995), Fine mapping of a putatively imprinted gene for familial non-chromaffin paragangliomas to chromosome 11q13.1: evidence for genetic heterogeneity, Hum Genet 95, 56-62; and Mariman et al., 1993; both of which are incorporated herein by reference) has been previously described (van Baars et al., (1982), Genetic aspects of nonchromaffin paraganglioma, Hum Genet 60, 305-309, incorporated herein by reference). A significant number of additional family members have been clinically evaluated in the Department of Otorhinolaryngology of the Radboud University Hospital and segregation of the disease haplotype performed as described by Mariman (Mariman et al., 1995). These studies were approved by local ethics committees for the Radboud University Nijmegen Medical Centre and the Leiden University Medical Centre.
All yeast expression plasmids used in this study were constructed by ligation of a PCR product containing the promoter and coding region of the relevant gene into a pRS416-based vector containing a C-terminal GFP, His-HA3 or His-Myc2 tag and a UGP1 terminator, or into pRS426 vector for overexpression as indicated. The integrity of the insert and functionality of the fusion protein were verified by DNA sequencing and rescue of the phenotype of the corresponding deletion strain, respectively. For bacterial expression of His-tagged yeast Sdh1, Sdh1 coding sequence was subcloned into pRSFDuet vector (Novagen) containing an N-terminal His tag. Sdh2-His-Myc and Sdh5-His-HA coding sequences were subcloned from yeast expression constructs into pCDFDuet and pETDuet, respectively. These pDuet vectors contain compatible replicons and drug resistance for co-expression. For expression of human SDH5 in yeast under control of the yeast SDH5 promoter, the yeast SDH5 promoter and human SDH5 coding sequence cloned from HepG2 cDNA were sequentially ligated into the Myc-tag containing pRS416 vector as described above. hSDH5 mammalian expression plasmids were generated by ligation of hSDH5 cDNA into a pcDNA3-based vector containing a C-terminal GFP or Myc tag. hSDH5 G78R mutants were generated from wild-type constructs using a QuickChange site-directed mutagenesis kit (Stratagene). pYX142-TPI-mtRFP yeast expression plasmid and pDsRed2-Mito mammalian expression plasmid used to visualize mitochondria were gifts of Dr. Janet Shaw and Dr. Kevin Flanigan at the University of Utah, respectively.
Intact mitochondria were isolated from yeast by differential centrifugation after cell wall removal by Zymolyase (US Biological) treatment and cell lysis by douncing as described previously (Diekert et al., (2001), Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae, Methods Cell Biol 65, 37-51, incorporated herein by reference). Mitochondrial protein concentration was determined by Advanced Protein Assay Reagent (Cytoskeleton, Inc). Proteinase K protection assay for sub-mitochondrial localization study was performed as previously described (Diekert et al., 2001). Mitochondrial membranes were isolated by sonication of mitochondria in 20mM Hepes buffer pH 7.4, followed by ultracentrifugation at 100,000 g for 30 min for the membrane association assay. The enzymatic activities of succinate dehydrogenase and malate dehydrogenase were measured spectrophotometrically with isolated mitochondria as described (Chen et. Al., (2002), Inhibition of Fe—S cluster biosynthesis decreases mitochondrial iron export: evidence that Yfh1p affects F—S cluster synthesis, Proc Natl Acad Sci U S A 99, 12321-12326, incorporated herein by reference) and normalized to total mitochondrial protein.
Yeast strains were grown to mid-log phase in SRaff (2% raffinose) liquid media and 2 OD of each culture was resuspended in 3% glycerol solution after washing with sterile water. The rate of oxygen consumption was measured using a 5300A Biological Oxygen Monitor (Yellow Springs Instrument Co.). The linear region of data was used for calculation.
The nickel chromatography and anti-HA affinity chromatography two-step purification was performed as described (Honey et al., 2001), except that purified mitochondria and commercial anti-HA conjugated agarose beads (Sigma) were used for purification and elution was achieved by 200 ng/ml HA peptide. The final eluates were precipitated in 10% TCA and 200ug/m1 sodium deoxycholate, resolved using 15% SDS-PAGE and visualized by SilverSNAP Stain (Pierce) or immunoblot with indicated antibodies. Validated samples were then subjected to LC-MS-MS for protein identification.
At 30 h post-transfection, HEK293 cells grown on 10 cm plates were harvested by trypsinization, washed with PBS and frozen at −80° C. Cells were then resuspended in 500 ul binding buffer (10 mM Tris-HCl pH7.4, 150 mM NaCl, 0.1% NP-40, 0.5 mM EDTA and 10% glycerol), passed through an insulin syringe 10 times to shear DNA, and centrifuged at 14,000 rpm for 10 min at 4° C. to remove cell debris. Cleared lysates were incubated with equilibrated anti-Myc conjugated agarose beads (Sigma) at 4° C. for 2 h. After extensive washing, bound proteins were eluted in 1×SDS sample buffer for immunoblot.
Yeast proteins were extracted by a NaOH-based method as described (Kushnirov, V.V. (2000), Rapid and reliable protein extraction from yeast, Yeast 16, 857-860, incorporated herein by reference) or by directly dissolving purified mitochondria in 1×SDS sample buffer. Tissue lysates were prepared by homogenizing approximately 30 mg frozen human PGL tumor or mouse tissue in cell lysis buffer (Cell Signaling Technology) with a Tissue-Tearer rotor after pulverization by Bessman tissue pulverizer. HEK293 and
HepG2 cell lysates were prepared by sonication on ice of harvested cells in above cell lysis buffer. After centrifugation at 14,000 rpm for 20 min at 4° C., cleared lysates were collected to measure protein concentration. Protein samples were then separated by SDS-PAGE and transferred to nitrocellulose for blocking and incubation with primary antibody and secondary antibody conjugated with infared dyes for detection by the Odyssey system (LI-COR). Quantitation of protein bands was performed using the built-in Odyssey software. The sources of primary antibodies are indicated: anti-HA (Roche); anti-Myc (Covance); anti-Porin and anti-PGK (Invitrogen); anti-SDHA (Mitosciences); anti-GAPDH (Chemicon); anti-Mge1 and anti-Fzo1 (Janet Shaw, University of Utah); anti-Aco1 (Jerry Kaplan, University of Utah); anti-Sdh1 and anti-Sdh2 (Bernard Lemire); anti-Tim10 (Carolyn Outten).
The flavination assay is adapted from a UV-transilluminator method described previously (Bafunno et al., (2004), Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria: involvement of the Flxlp carrier in FAD export, J Biol Chem 279, 95-102, incorporated herein by reference). After SDS-PAGE separation of mitochondrial proteins or whole cell extracts, the protein gel was incubated for 20 min in 10% acetic acid to adjust pH. 526 nm fluorescence emission by covalently bound FAD upon excitation at 488 nm was measured by the Typhoon imager (GE Healthcare). Quantitation of fluorescence was done by densitometry using AlphaEaseFC™ software.
BN-PAGE was performed essentially as described (Wittig et al., (2006), Blue native PAGE, Nat Protoc 1, 418-428; Pierrel et al., (2007), Coal links the Mss51 post-translational function to Coxl cofactor insertion in cytochrome c oxidase assembly, Embo J 26, 4335-4346; both of which are incorporated herein by reference), with slight modification. In-gel activity assay of respiratory complexes after BN-PAGE was performed as previously described (Zerbetto et al., (1997), Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels, Electrophoresis 18, 2059-2064; Jung et al., (2000), Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis, Anal Biochem 286, 214-223; both of which are incorporated herein by reference). Coomassie stain and immunoblot were performed according to standard protocols for SDS-PAGE, except that 0.1% SDS is supplemented in the transfer buffer to facilitate protein transfer.
The sdh5Δ mutant strain transformed with both pSdh5-GFP and pMito-RFP plasmids was grown to mid-log phase medium containing 2% glucose or 2% raffinose and imaged using a Zeiss Axioplan 2 Imaging microscope as described (Kondo-Okamoto et. Al., (2003), Mmmlp spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation, J Biol Chem 278, 48997-49005. Epub 42003 Sep 48912, incorporated herein by reference). Human HEK293 cells were transfected with pDsRed2-Mito (Mito-RFP) and pcDNA3-hSDH5-GFP or pcDNA3-hSDH5(G78R)-GFP in 35mm plates. At 24 h post transfection, cells were photographed using the Olympus IX81 microscope at the University of Utah Fluorescence Microscopy Core Facility.
BL21(DE3) CodonPlus-RIL (Stratagene) E. coli strain was transformed with pRSFDuet-His-Sdh1 alone or together with pCDFDuet-Sdh2-Myc or pETDuet-Sdh5-HA. 0.2 mM final concentration of IPTG was added to 1 liter culture of each strain at OD=0.8 to induce protein expression for 6 h at 37° C. The cells were harvested and His-tagged proteins were purified using Ni-NTA agarose beads (Qiagen) according to the manufacturer's protocol.
Primers for amplification of exons and exon-intron boundaries of the Cllorf79 gene (Ensembl gene number ENSG00000167985; http://www.ensembl.org/index.html) were designed using ExonPrimer (http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html) and the reference sequence NT—033903. Primer sequences are provided in Table 2. (SEQ ID 15-24). For amplification, 50 ng of genomic DNA was used as the starting material and primer annealing was performed at 58° C. The MgCl2 concentration in the reaction mix was 2 mM and for amplification of exon 1, 10% DMSO was added.
PCR fragments were purified using NucleoFast 96 PCR plates (Clontech). Sequence analysis was performed with the ABI PRISM Big Dye Terminator Cycle Sequencing V2.0 Ready Reaction kit and the ABI PRISM 3730 DNA analyzer (Applied Biosystems). As a reference sequence NM017841 was used.
For the presence of the mutation in control individuals, exon 2 was amplified as described for sequencing. The fragments were purified with NucleoFast 96 PCR plates (Clontech) and digested with BseRl (New England Biolabs) according to manufacturer's protocols. Restriction fragments were analyzed on 2% agarose gels.
The sdh5Δ strain containing both a plasmid expressing Sdh5-GFP under the native SDH5 promoter and a plasmid expressing mito-RFP (a fusion of the N. crassa F0-ATPase Su9 presequence to RFP) was grown in SD-Ura-Leu and harvested at mid-log phase for fluorescence microscopy. A representative population of cells is shown in
Wild-type and sdh5Δ mutant strains were transformed with empty vector (EV) or a plasmid expressing Sdh5-HA under control of the native SDH5 promoter. Serial 5-fold dilutions of saturated SD-Ura liquid culture of each strain were spotted on S-URA solid medium with either 2% glucose or 3% glycerol as the carbon source and grown at 30° C. for 2 days (glucose) or 3 days (glycerol). (See
Mitochondria were purified from 6 liter cultures of the wild-type strain or the sdh5Δ strain containing a plasmid expressing C-terminally His and HA-tagged Sdh5 under the native SDH5 promoter. Lysate from purified mitochondria was subjected to nickel chromatography and anti-HA affinity chromatography, finally eluting with 200 ng/ml HA peptide. As is shown in
Purified mitochondria from wild-type and sdh5Δ strains grown in YPA-Raff media were assayed for succinate dehydrogenase and malate dehydrogenase activity. Activity measurements were normalized to the total protein amount and expressed as a percentage relative to the wild-type strain. Data represent the average±standard deviation of 3 replicates per strain. (See
Of course, it is to be understood that the above-described arrangements are only illustrative of the application of the principles of the present invention. Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements. Thus, while the present invention has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiments of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, variations in size, materials, shape, form, function and manner of operation, assembly and use may be made without departing from the principles and concepts set forth herein.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/270,259, filed on Jul. 6, 2009, which is incorporated herein by reference in its entirety.
This invention was made with government support under NIH grants DK071962 and GM087346. The United States government has certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61270259 | Jul 2009 | US |
