Missense mutations in the human SDHB gene ... - The FASEB Journal

The FASEB Journal article fj.12-210146. Published online July 26, 2012.

The FASEB Journal • Research Communication

Missense mutations in the human SDHB gene increase protein degradation without altering intrinsic enzymatic function Chunzhang Yang,* Joey C. Matro,† Kristin M. Huntoon,* Donald Y. Ye,* Thanh T. Huynh,† Stephanie M. J. Fliedner,† Jan Breza,‡ Zhengping Zhuang,*,1 and Karel Pacak†,1 *Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, and †Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA; and ‡ Department of Urology, School of Medicine, Comenius University, Bratislava, Slovakia Mutations of succinate dehydrogenase subunit B (SDHB) play a crucial role in the pathogenesis of the most aggressive and metastatic pheochromocytomas (PHEOs) and paragangliomas (PGLs). Although a variety of missense mutations in the coding sequence of the SDHB gene have been found in PHEOs and PGLs, it has been unclear whether these mutations impair mRNA expression, protein stability, subcellular localization, or intrinsic protein function. RT-PCR and Western blot analysis of SDHB mRNA and protein expression from SDHB-related PHEOs and PGLs demonstrated intact mRNA expression but significantly reduced protein expression compared to non-SDHB PHEOs and PGLs. A pulse-chase assay of common SDHB missense mutations in transfected HeLa cell lines demonstrated that the loss of SDHB function was due to a reduction in mutant protein half-life, whereas colocalization of SDHB with mitochondria and immunoprecipitation with SDHA demonstrated intact subcellular localization and complex formation. The half-life of the SDHB protein increased after treatment with histone deacetylase inhibitors (HDACis), implicating the protein quality control machinery in the degradation of mutant SDHB protein. These findings provide the first direct mechanism of functional loss resulting from SDHB mutations and suggest that reducing protein degradation with HDACis may serve as a novel therapeutic paradigm for preventing the development of SDHB-related tumors.—Yang, C., Matro, J. C., Huntoon, K. M., Ye, D. Y., Huynh, T. T., Fliedner, S. M. J., Breza, J., Zhuang, Z., Pacak, K.

ABSTRACT

Abbreviations: CHX, cycloheximide; HDACi, histone deacetylase inhibitor; HIF, hypoxia-inducible factor; MEN2, multiple endocrine neoplasia type 2; NF1, neurofibromatosis type 1; PGL, paraganglioma; PHD, prolyl hydroxylase; PHEO, pheochromocytoma; ROS, reactive oxygen species; SAHA, suberoylanilide hydroxamic acid; SDH, succinate dehydrogenase; SDHA, succinate dehydrogenase subunit A; SDHB, succinate dehydrogenase subunit B; SDHC, succinate dehydrogenase subunit C; SDHD, succinate dehydrogenase subunit D; VHL, von Hippel-Lindau 0892-6638/12/0026-0001 © FASEB

Missense mutations in the human SDHB gene increase protein degradation without altering intrinsic enzymatic function. FASEB J. 26, 000 – 000 (2012). www.fasebj.org Key Words: pheochromocytoma 䡠 paraganglioma 䡠 succinate dehydrogenase subunit B 䡠 histone deacetylase inhibitor Pheochromocytomas (PHEOs) are rare but lifethreatening catecholamine-producing neuroendocrine tumors that arise from chromaffin cells in the adrenal medulla; tumors arising from the peripheral sympathetic or parasympathetic nervous system are called paragangliomas (PGLs) (1). Symptoms and signs of these tumors are a direct result of either tumor mass effect or hypersecretion of catecholamines (e.g., sustained or paroxysmal elevations in blood pressure, tachyarrhythmia, headaches, profuse sweating, pallor, and anxiety; ref. 2). In addition, ⬃10 –30% of PHEOs and PGLs give rise to metastases, for which there are currently limited and suboptimal chemotherapeutic options (3). PHEOs and PGLs mainly occur as sporadic tumors, but some can be associated with well-known inherited neuroendocrine disorders, including multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau (VHL) disease, and neurofibromatosis type 1 (NF1) (4 –7). Recent studies have suggested that changes in an energy metabolism-related gene, succinate dehydrogenase (SDH), are major contributors to the pathogenesis of these mainly aggressive and metastatic tumors (4, 8, 9). 1 Correspondence: K.P., Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 10, CRC, Rm. 1E-3140, 10 Center Dr., MSC-1109, Bethesda, MD 20892-1109, USA. E-mail: [email protected]; Z.Z., Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 10, Rm. 3D20, Bethesda, MD 20892-1414, USA. E-mail: [email protected] doi: 10.1096/fj.12-210146

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SDH is the mitochondrial protein complex II that is vital for mitochondrial electron transport, as well as Krebs cycle function. It catalyzes the oxidation of succinate to fumarate and transfers electrons to ubiquinone through the coordination of its four subunits (SDHA, SDHB, SDHC, and SDHD; refs. 10 –12). Genetic defects in SDH coding sequences largely affect its physiological functions and predispose carriers to the development of PHEOs and PGLs, as well as renal cell carcinoma (4, 13–16). The pathogenesis of these tumors reflects the abnormal mitochondrial electron transport that results in a state of normoxic pseudohypoxia. Loss of SDH activity prevents the conversion of succinate to fumarate, which leads to mitochondrial and cytoplasmic accumulation of succinate. Previous studies have shown succinate to inhibit prolyl hydroxylases (PHDs), which are responsible for the initial hydroxylation and subsequent degradation of hypoxiainducible factor ␣ (HIF-␣; refs. 17–20). Stabilization of HIF-1␣ and HIF-2␣ leads to the overexpression of a large subset of gene factors that increase cellular survival and proliferation and contribute substantially to the tumorigenesis of PHEOs and PGLs (21–23). The disruption of mitochondrial electron transfer may also lead to the excess generation of reactive oxygen species (ROS) that have been shown to independently induce a state of pseudohypoxia by inhibiting PHDs (24 –27). In addition, oxidative stress alters the expression of other tumor suppressor genes or oncogenes through mutagenesis and initiates tumorigenesis. Germline mutations in the SDHB gene correspond to an autosomal dominant form of PHEO or PGL. In addition, ⬎50% of SDHB-associated PHEOs and PGLs carry missense mutations in the coding sequence of the gene (28, 29). These mutations commonly cause a single amino acid substitution in the protein product, which is sufficient for tumor development after the loss of heterozygosity of the second wild-type allele. However, the precise mechanism of how gene mutations in SDHB lead to its functional loss is currently unknown. The aim of the present study was to gain insight into the mechanism underlying the functional loss of SDHB that results from the variety of missense mutations. We conducted a series of examinations to assess multiple processes from gene transcription to protein translation, modification, and function. Discovering a mechanism for functional loss of SDHB would be a key advancement in designing novel therapeutic options for these very aggressive and often lethal tumors.

tissue was removed, dimensions of tumors were recorded, and samples were dissected away from surrounding tissue, then divided into smaller 10- to 50-mg pieces, frozen on dry ice or optimal cutting temperature (OCT; Sakura Finetek, Torrance, CA, USA) blocks and stored at ⫺80°C. The normal adrenal tissues were collected during radical nephrectomy or from autopsy materials. Whole adrenal glands were snapfrozen on removal and stored at ⫺80°C (30). Tissue information has been summarized in Supplemental Table S1. Tissue microdissection was performed as described previously (31, 32). Reverse transcription and real-time PCR RNA was extracted from frozen tissue samples of PHEOs and PGLs with homogenization in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) followed by RNeasy Maxi (Qiagen, Valencia, CA, USA), according to the manufacturer’s recommendations. Total RNA (1 ␮g) was reverse transcribed to cDNA using random hexamers. Quantitative PCR (TaqMan PCR), using a 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA), was used for quantification of mRNA for SDHB. The primers and TaqMan probe premade set were ordered through Applied Biosystems. 18S ribosomal RNA was used as a housekeeping gene. Reaction tubes contained 25 ng of cDNA product as a template, 1⫻ TaqMan Universal PCR Master Mix, 1⫻ of premade sets of primers and TaqMan probes for the SDHB gene or 18S ribosomal RNA, adjusted to a final volume of 50 ␮l with H2O. PCR consisted of 45 cycles at the following temperature parameters: 15 s at 95°C, 1 min at 60°C. SDHB mRNA expression was evaluated using the ⌬⌬Ct method. Western blot analysis Western blot analysis was performed as described previously, with minor modifications (33). Microdissected tumor tissue and cell pellets were extracted for protein using RIPA lysis buffer supplemented with Halt proteinase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Protein was vortexed at 4°C for 20 min and centrifuged at 12,000 g for 10 min at 4°C. Supernatant was collected, and protein quantity was identified through a Bio-Rad (Hercules, CA, USA) protein assay kit. Equal amounts of proteins were separated on a NuPAGE 4 –12% Bis-Tris gel (Invitrogen) and transferred to PVDF membranes (Invitrogen). Membranes were blocked in 5% dried skim milk in PBST and blotted with primary antibody. Protein expression was identified through a chemiluminescence kit (Thermo Scientific, Waltham, MA, USA). The following antibodies were used: SDHA (1:1000; Cell Signaling Technology, Beverly, MA, USA), SDHB (1:1000; Sigma-Aldrich, St. Louis, MO, USA), Flag (1:2000; Origene, Rockville, MD, USA), ubiquitin (1:1000; Abcam, Cambridge, MA, USA), HA (1:2000; Origene, Rockville, MD, USA), Hsp90 (1:1000; Cell Signaling Technology), and ␤-actin (1: 1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

MATERIALS AND METHODS Immunoprecipitation SDHB tumor samples and tissue dissection Tumor tissue samples and normal human adrenal glands were obtained from patients recruited to the U.S. National Institute of Child Health and Human Development and the National Institutes of Health (NICHD/NIH) under studies approved by the Intramural Review Board of NICHD/NIH, with informed consent obtained from all patients. Extraneous 2

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Immunoprecipitation was performed as described previously (34). Protein was extracted from cell cultures using IP lysis buffer with Halt proteinase inhibitor cocktail (Thermo Scientific). Total protein (400 ␮g) was precipitated with Flag antibody (1:200; Origene) using a DynaBeads Protein G immunoprecipitation kit (Invitrogen). Proteins were precipitated overnight at 4°C and eluted for Western blot analysis.

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Immunofluorescence Cells were preloaded with Mitotracker Red for 20 min before fixation. Cells were then washed 3 times in PBS and fixed in Histochoice for 15 min. SDHB mutants were labeled with anti-Flag antibody (1:200; Origene). Cell nuclei were counterstained with Hoechst 33342 (Invitrogen). The specimens were visualized using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY, USA). Immunohistochemistry staining Immunohistochemistry staining was performed using commercially available SDHB antibody (Sigma-Aldrich) on formalin-fixed paraffin-embedded tissue mounted on positively charged slides. The primary antibody was used at a dilution of 1:500 after heat-induced antigen retrieval using 1 mM EDTA. Samples were then labeled and visualized using a DAB staining kit (Envision⫹Kit; Dako, Carpinteria, CA, USA). Cell culture and transfection HeLa cells were maintained in DMEM containing 10% FBS (Invitrogen). Cells were transfected with SDHB vectors by FuGene 6 transfection reagent (Roche, Indianapolis, IN, USA). The medium was changed 4 h after transfection, and cells were maintained for 48 h before cycloheximide (CHX; 20 ␮g/ml, Sigma-Aldrich) treatment. DNA cloning and site-directed mutagenesis The ubiquitin-HA vector was described previously (35). The wild-type human SDHB gene was cloned into a pCMV6-Entry vector (Origene). A C-terminal Flag tag was used in all SDHB recombinants for immunodetection. Hot spot missense mutations in SDHB-related PHEOs and PGLs were based on previous findings (36 –38). The mutant SDHB recombinant vector was established using a standardized Quikchange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). Briefly, the pCMV6-SDHB vector was used as a template for mutagenesis. Plasmid (500 ng) was incubated with mutagenesis primer and enzyme mix, according to the manufacturer’s protocol. For each mutant, the sequence of the SDHB gene was verified by sequencing the entire coding region of the gene. Vectors were purified through the PureLink HiPure Plasmid Maxiprep kit (Invitrogen) for transfection and cell-free protein expression. Pulse-chase assay Radioactive pulse-chase assay was performed as described previously (39). A total of 106 HeLa cells were transfected with SDHB vectors 12 h before labeling. Cells were starved in methionine-free DMEM with 10% FBS for 5 min, followed by 15 min incubation in the same medium supplemented with 0.2 mCi/ml [35S]-methionine (⬎1000 Ci/mmol specific activity; Perkin Elmer, Waltham, MA, USA). Cells were then washed and chased in DMEM with 10% FBS supplemented with 3 mg/ml methionine. At the end of each time period, proteins were extracted from the cells using RIPA lysis buffer with Halt proteinase inhibitor cocktail. Mutant SDHB proteins were precipitated from 200 ␮g total lysate with Flag antibody. Proteins were eluted and separated on a 4 –12% Bis-Tris gel or measured by liquid scintillation counting. Gels were fixed and incubated with EN3HANCE (Perkin Elmer) for 30 min and dried for autoradiography. For the CHX assay, HeLa cells were transfected with SDHB SDHB MISSENSE MUTATIONS INCREASE PROTEIN DEGRADATION

expression vectors 2 d before protein stability measurement. Cells were exposed to CHX (20 ␮g/ml) for the period as indicated. Total protein was extracted with RIPA lysis buffer supplemented with protease inhibitors, and mutant SDHB residues were examined through Western blot and Flag-tag immunoblot. In vitro synthesis of mutant SDHB proteins Mutated SDHB sequences were cloned into the MCS region of a pCMV6-Entry vector, which could be driven by a T7 promoter in the upstream sequence. In vitro translations were performed using a TNT T7 Quick Coupled Transcription/ Translation System (Promega, Madison, WI, USA). For each mutant, 2 ␮g of plasmid DNA was incubated with 40 ␮l TNT Quick Master Mix and 20 ␮Ci [35S]-methionine (⬎1000 Ci/mmol specific activity; Perkin Elmer) at 30°C for 2 h. Synthesized SDHB proteins were loaded on 4 –12% Bis-Tris gels and analyzed through autoradiography. In vitro mitochondria binding and insertion assay for mutant SDHB proteins The mitochondria binding and insertion assay was performed on the basis of the protocol developed in our lab, as described previously (40, 41). Mitochondria were isolated from HeLa cells using the Qproteome Mitochondria Isolation Kit (Qiagen). Equal amounts of radioactively labeled SDHB protein and isolated mitochondria were incubated with 4 mM ATP, 10 mM creatine phosphate, 1 U/ml creatine phosphokinase, 10 mM methionine, 10 mM MgCl2, 40 mM KCl, 0.4 mM dithiothreitol, 0.3 M sucrose, and 25 mM HEPES buffered to a pH of 7.5 with KOH. The reaction mixture was kept at 37°C for 30 min and then placed on ice. Mitochondria was washed and collected to evaluate mitochondria binding. For measurement of mitochondria insertion, the reaction mixture was treated with 10 ␮g proteinase K for 15 min on ice. Treatment was terminated by the addition of 2 mM PMSF. Mitochondria were washed and lysed in 50 ␮l RIPA lysis buffer with Halt proteinase inhibitor cocktail. SDHB residues were analyzed by 4 –12% Bis-Tris gels and autoradiography.

RESULTS Quantitative loss of SDHB protein expression in PHEOs and PGLs To assess the level of SDHB expression in these tumors, we investigated SDHB protein expression through immunohistochemistry staining (Fig. 1A). We identified reduced SDHB protein expression in SDHB-associated tumor tissues. A significant reduction of SDHB protein expression was identified for a tumor sample that contained a missense mutation (⌬SDHB-R11H), as well as for a tumor sample with a nonsense mutation (⌬SDHB-T115X) (Fig. 1A). In contrast, robust SDHB protein expression was found in the cytoplasm of adjacent normal adrenal cells. Quantification of SDHB revealed a 90.0 and 72.5% reduction in the SDHB protein in tumors with nonsense and missense mutations, respectively. Much of the remaining SDHB protein, particularly in tumor samples associated with nonsense mutations, likely derived from normal tissue mingled with tumor cells. To confirm this protein 3

Figure 1. SDHB protein expression and enzymatic activity are altered in missense/nonsense mutation-associated tumor tissue. A) Immunohistochemical staining for SDHB in tumor tissue with missense (⌬SDHB-R11H) and nonsense (⌬SDHB-T115X) mutations. B) Western blot of the SDHB protein in microdissected PHEOs and PGLs associated with mutations in the SDHB, MEN1, or VHL genes. C) Quantification of the SDHB protein in microdissected tumor tissue (n⫽3 for each type of tumor). D) Real-time-PCR quantification of SDHB mRNA transcription in microdissected tumors (n⫽4; 3 technical replicates for each sample). Normal adrenal tissue (NAM) was used as a positive control. E) SDHB enzyme activity of tumor samples with SDHB gene nonsense or missense mutations (n⫽6; 3 technical replicates for each sample). NAM was used as a positive control.

quantity change, we next measured the SDHB protein in microdissected tumor samples with SDHB, MEN2, and VHL mutations using Western blot analysis (Fig. 1B). Consistent with the immunohistochemistry analysis, substantially lower expression of the SDHB protein was present, with approximately only 30% in SDHB PHEO/PGL samples (n⫽3) as compared with normally expressed SDHB protein in VHL (P⬍0.05, n⫽3) or MEN2 (P⬍0.01, n⫽3) PHEOs (Fig. 1C). To determine the mechanism of protein loss, we measured SDHB mRNA transcription in dissected tumor specimens through real-time PCR (Fig. 1D). We identified only a slight reduction in mRNA expression in tumors with missense mutations as compared with normal adrenal medullary tissue (P⬎0.05), suggesting mRNA transcription efficiency is intact in tumor cells. In addition, we tested the gene integrity of SDHB through radioactively labeled short tandem repeat PCR in tumor specimens. This analysis confirmed that there was a loss of heterozygosity in the SDHB gene in tumor specimens, but both alleles remained intact in normal tissue (Supplemental Table S1). These results indicate that mutant mRNA was intact at the transcriptional level in the absence of the wild-type SDHB gene. SDHB is an essential subunit of mitochondrial complex II on the inner membrane of the mitochondria. To evaluate SDHB enzyme activity, we measured mito4

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chondrial complex II activity in SDHB-associated tumor specimens compared to normal human adrenal medullary tissue (Fig. 1E). Consistent with protein loss, we identified a parallel reduction in the enzymatic activity of complex II in tumors with SDHB nonsense and missense mutations. The complex II activity in all specimens tested was consistent with a quantitative loss of SDHB protein product in these tumors. Tumors with nonsense mutations showed a loss of 93.4% of normal enzymatic activity, while tumors with missense mutations showed a loss of 86.5% of normal enzymatic activity. Impaired protein stability causes quantitative loss of the SDHB protein The intact mRNA expression and quantitative loss of the SDHB protein product in tumors suggested that post-translational changes occur in mutant SDHB proteins that could lead to their premature quantitative loss. We hypothesized that increased protein misfolding and degradation would underlie the net loss of the functional SDHB subunit in these tumors. To test this hypothesis, we compared the protein stability of the wild-type and mutant SDHB protein. Hot spot missense mutations in the SDHB gene were selected on the basis of previous findings (36 –39) and constructed in


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pCMV6-Entry mammalian expression vectors. The mutant SDHB vectors were transfected into HeLa cells, which were later treated with CHX to inhibit protein synthesis. SDHB protein residue was measured by Western blot analysis (Fig. 2A). We successfully identified expression of both wild-type and mutant SDHB protein at the beginning of the treatment. The wild-type SDHB protein was stable after synthesis and was observed at ⬎80% after 4 h of CHX treatment. The protein half-life of the wild-type SDHB was 10.43 h. We also observed a rapid and significant loss of mutant SDHB proteins across all mutations measured in the same assay. The protein residue was found to be reduced to ⬍45% among all mutants. One-phase decay nonlinear regression showed that A43P, R46Q, L65P, and W200C mutant SDHB protein half-lives were reduced to 0.58, 0.74, 0.43, and 1.54 h, respectively (Fig. 2B). We confirmed this protein stability change through a [35S]-methionine-mediated pulse-chase assay. Wild-type and ⌬SDHB-R46Q vectors were delivered into HeLa cells, and the protein was pulse labeled with [35S]methionine. The SDHB protein was precipitated and analyzed through autoradiography (Fig. 2C). Consistent with effective protein translation, the pulse-chase analysis revealed that both wild-type and mutant SDHB were synthesized in their full length immediately after labeling and that no notable differences in expression levels were observed after initial translation. We identified a marked reduction in the [35S]-labeled SDHB

protein in the R46Q mutant over the course of 12 h, which is in agreement with the data from the CHX treatment. Autoradiography and [35S] scintillation demonstrated that there was a minimal reduction in wild-type SDHB expression 4 h after labeling and ⬎75% of the labeled protein remained intact. After 12 h of pulse labeling, ⬃50% of the wild-type protein was degraded, indicating an efficient turnover of the SDHB protein under normal conditions. On the contrary, the R46Q mutant protein exhibited accelerated degradation. Radioactively labeled protein was lost extensively within 6 h of the chase (Fig. 2D). Similar protein losses were observed in other tumorigenic missense mutants (A43P, C196Y, and W200C; data not shown). To understand the degradation dynamic of the mutant SDHB protein, the protein half-life was calculated through liquid scintillation counting of the radioactively labeled SDHB protein (Fig. 2D). Consistent with the findings above, the wild-type SDHB protein was found to be stable, and the half-life was 11.23 h, whereas a significant loss of protein stability was found in the R46Q mutant. The [35S]-labeled SDHB protein decreased to ⬍50% within 3 h of labeling and further reduced below 5% at 12 h after labeling. Consistent with CHX treatment, the half-life of the mutant SDHB protein was reduced to 1.73 h. To confirm a rapid degradation of the mutant SDHB protein, we investigated the ubiquitination status of the SDHB protein with hot spot missense mutations (Fig.

Figure 2. Missense mutations in the SDHB gene decrease protein stability. A) Western blot demonstrating loss of protein stability due to hot spot SDHB missense mutations through CHX treatment. B) Hot spot missense mutations of the SDHB protein decrease protein half-life. C) [35S]-methionine pulse-chase assay measuring radiolabeled SDHB 0 –12 h in wild-type and R46Q mutant SDHB. D) Liquid scintillation measurement of wild-type and R46Q mutant SDHB 0 –12 h after radiolabeling. E) Immunoprecipitation assay of mutant SDHB protein ubiquitin binding. F) Quantification of mutant SDHB ubiquitination relative to wild-type SDHB. SDHB MISSENSE MUTATIONS INCREASE PROTEIN DEGRADATION

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2E). The wild-type SDHB protein was found to be ubiquitinated, indicating a rapid turnover and steady degradation in physiological conditions. However, elevated ubiquitin binding by an average of 57.4% was found across all missense mutants tested, suggesting the mutant proteins undergo an ubiquitin-proteasome derived degradation pathway (Fig. 2F). Mutant SDHB proteins maintain intrinsic functions Missense mutations may alter the 3-dimensional protein structure, leading to both protein misfolding and degradation, as well as to changes to its intrinsic biological function. Since the quantitative level of protein loss was in agreement with functional loss of complex II activity, we hypothesized that missense mutations of SDHB would have minimal effects on its intrinsic targeting and transport into the mitochondria, complex formation, and biological function. To test these possibilities, we investigated mitochondrial binding, insertion, and mitochondrial complex II assembly for tumorigenic missense mutants. We first investigated mutant SDHB localization through immunofluorescent staining (Fig. 3A). SDHB mitochondrial localization was measured on the basis of colocalization with Mitotracker. Wild-type SDHB was found to colocalize with Mitotracker, suggesting correct mitochondrial localization and assembly. Interestingly,

mutant SDHB proteins were also found to colocalize with Mitotracker, suggesting that mutants of SDHB can also be assembled in the mitochondria. A small amount of mutant SDHB protein was found trapped in the cytoplasm, which results from a “leaky” distribution outside the mitochondria. To understand whether complex II assembly efficiency is affected by missense SDHB mutations, we performed a mitochondrial binding and insertion assay, as described previously (40, 41). Mutant SDHB protein was synthesized in a cell-free system supplemented with [35S]-methionine. Radioactive SDHB proteins were then incubated with isolated HeLa mitochondria to investigate protein binding. Mitochondrial insertion was further tested through proteinase K incubation. The wild-type SDHB protein appeared to undergo both successful mitochondrial binding and insertion. On the contrary, green fluorescent protein (GFP), a cytoplasmic protein, showed little mitochondrial binding and undetectable insertion. We tested seven SDHB missense mutants in the same assay and found almost every mutant protein exhibited both successful binding and insertion (Fig. 3B). Quantification on autoradiography and [35S] scintillation demonstrated slightly increased mitochondrial binding and minimal changes in insertion in the mutant protein, which is in agreement with the immunofluorescent staining data.

Figure 3. Normal mitochondrial localization of missense SDHB mutant proteins. A) Immunofluorescent detection of SDHB mitochondrial localization. Cells were labeled with anti-Flag (green), Mitotracker (red), and Hoechst33342 (blue). B) Autoradiography of [35S]-methionine labeled SDHB binding and insertion into isolated mitochondria. Liquid scintillation quantification of binding and insertion of mutant SDHB proteins relative to wild-type SDHB. C) Immunoprecipitation assay of SDHA binding to SDHB mutants. D) Quantification of SDHA binding to mutant SDHB relative to wild type SDHB. 6

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SDHB forms mitochondrial complex II through assembly with the SDHA, SDHC, and SDHD subunits. SDHB is physically associated with the SDHA subunit on the hydrophilic catalytic end of complex II (42). We further evaluated mitochondrial complex II assembly by testing SDHA binding to mutant SDHB proteins. Wild-type and mutant SDHB proteins were immunoprecipitated and tested for SDHA binding through Western blot analysis (Fig. 3C). The binding of SDHA was detected on both wild-type and mutant SDHB proteins. The binding efficiency of SDHA was similar between wild-type and mutant proteins, indicating that mitochondria II complex assembly of SDHB in missense mutants was intact (Fig. 3D). Increase of functional SDHB subunit through proteostasis modulators The missense SDHB proteins tested in these studies not only remained intact but appeared to be functional as well. Therefore, these same mutated SDHB proteins are likely quantitatively insufficient in PHEOs and PGLs due to rapid degradation by ubiquitin-proteasome degradation. A potential treatment strategy would be to target the mediators that lead to the rapid degradation

of mutant SDHB proteins. This may increase the functional SDHB subunit in mitochondria, recovering adequate succinate metabolism to reverse the state of pseudohypoxia. Specifically, we applied small-molecule compounds known to modulate protein stability and proteostasis, including histone deacetylase inhibitors (HDACis), celastrol, and quercetin, and investigated their effect on protein half-lives. We tested SDHB stability changes through a [35S]-methionine pulsechase assay. The R46Q and W200C mutants were transiently labeled with [35S]-methionine. Cells were chased for 4 h under various treatments. Protein residues were measured through autoradiography and liquid scintillation (Fig. 4A, B). Interestingly, we observed SDHB protein stabilization in both mutants under the treatment. In particular, the W200C mutant was found to be more responsive to the treatment, with increases approaching 5-fold, according to the liquid scintillation. To confirm the effect of the drug on mutant protein stability, we measured ⌬SDHB-L65P protein half-life through CHX treatment (Fig. 4C, D). Consistent with the data shown above, the L65P mutant protein has an extremely short half-life, and the protein residue was reduced to ⬍50% within 1 h. Two HDACis, LB-201 and suberoylanilide hydroxamic acid (SAHA), were tested

Figure 4. Effect of proteostasis modulators on SDHB protein stability. A) Autoradiography of [35S]-methionine-labeled mutant SDHB 4 h after administration of proteostasis modulators LB-201, LB-202, SAHA, celastrol, and quercetin. B) Liquid scintillation quantification of [35S]-methionine-labeled SDHB 4 h after administration of proteostasis modulators. C) Western blot of ⌬SDHB-L65P 0 –2 h after administration of CHX following treatment with LB-201, SAHA, and celastrol. D) Relative quantity of ⌬SDHB-L65P 0 –2 h after administration of CHX following treatment with LB-201, SAHA, and celastrol. E) Western blot of ubiquitin and Hsp90 binding to immunoprecipitated ⌬SDHB-R49Q following treatment with LB-201, LB-205, and SAHA. SDHB MISSENSE MUTATIONS INCREASE PROTEIN DEGRADATION

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and were found to slow the degradation of mutant protein in the same assay. Celastrol exhibited a stronger protein stabilization effect; more than 50% of the SDHB protein remained intact after a 2-h CHX treatment. Half-life calculations showed that LB-201, SAHA, and celastrol lengthened the L65P protein half-life to 0.67, 2.36, and 2.67 h, respectively. The effect of HDACis was confirmed through investigation of the ubiquitination of mutant SDHB protein through immunoprecipitation (Fig. 4E). We found that LB-201, LB-205, and SAHA decreased mutant SDHB protein ubiquitination. Hsp90 binding, which targets misfolded proteins to the ubiquitin-proteasome degradation pathway, was also decreased in the same assay.

DISCUSSION Classically, the mechanism for the genetic pathogenesis of solid inherited neoplastic syndromes has fallen under the generic umbrella of the Knudson 2-hit paradigm (43– 45). However, in many cases of cancer, the precise mechanism of how missense mutations contribute to the loss of the functional gene-specific protein and lead to tumor formation remains unclear. PHEOs and PGLs can often be attributed to well-known defects in the VHL tumor suppressor pathway and stabilization of HIF-␣ coupled with changes in expression of many genes that lead to unopposed proliferation and tumor development (46, 47). However, PHEOs and PGLs due to SDHB mutations are less well understood. Various missense mutations in the SDHB gene have been described in these tumors, but little is understood about how these mutations lead to the loss of SDHB function, as well as the fate of the resulting mutant proteins. Many defects along the pathway from gene to protein could play a role in the functional loss of the mutated SDHB protein in these tumors. Reduced transcription, impaired mRNA stability, decreased translation, increased protein degradation, impaired mitochondrial localization and transport, and loss of intrinsic function all prevent proper formation and function of mitochondrial complex II and inhibit the conversion of succinate to fumarate. However, in our investigations of these various potential mechanisms, we demonstrate that accelerated protein degradation gives rise to the quantitative loss of mutant SDHB protein and results in the functional insufficiency of protein function (Fig. 2). Moreover, the use of proteostasis regulators like HDACi can be used to prevent the degradation of mutant SDHB proteins and potentially reduce the proliferative signals of pseudohypoxia that lead to tumorigenesis. A recent study observed an absence of the SDHB protein in PHEOs and PGLs harboring an SDHB missense mutation (48, 49). We confirmed this and demonstrated a significant decrease in the SDHB protein by both immunohistochemistry and by Western blot analysis of cells isolated from biopsy specimens from SDHB-related tumors compared to VHL- and 8

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MEN2-related PHEOs and PGLs (Fig. 1A–C). Together, our findings support that this quantitative absence of proteins is the hallmark feature of these tumors and central to the functional mechanism of SDHB mutations in related PHEOs and PGLs. However, these tumor specimens only demonstrate slight changes in the yield of corresponding extracted mRNA, which is insufficient to account for the amount of protein losses observed. Likewise, mitochondrial complex II activity in SDHB-related tumor samples was reduced in a manner parallel to a quantitative loss of SDHB protein product, suggesting that the pathogenesis of PHEOs and PGLs in SDHB mutations are due to changes at the protein level and not changes at the RNA level (Fig. 1D). Several processes play a role in protein synthesis, degradation, and function, and these processes are not mutually exclusive in reducing enzymatic activity to the levels observed in SDHB-related tumors. To elucidate which mechanisms are responsible for the findings from SDHB tumor samples, we examined protein synthesis, protein stability, targeting to the mitochondria, import into the mitochondria, and intrinsic enzyme activity. On the basis of previous studies related to neurofibromatosis type 2 and Gaucher’s disease (34, 39), we believe that quantitative loss of SDHB is due to a defect in protein stability and an increase in ubiquitin-mediated proteasomal degradation. Post-translational modifications are essential mechanisms that cells utilize to sort out premature target proteins for degradation as part of the endogenous protein quality control system (50, 51). Mutations of single amino acid substitutions lead to post-translational ubiquitination and premature degradation of merlin and glucocerebrosidase, suggesting that the protein quality control system is responsible for regulating mutant proteins by accelerating degradation (34, 39). Accelerated degradation for mutant SDHB proteins can occur as a result of protein misfolding during maturation, a mechanism extensively studied in VHL mutations (52–54). We observed similar misfolding reactions through changes in Hsp90 binding to mutant SDHB proteins, which has been demonstrated to be essential for the establishment of early degradation complexes (55). Further investigation will be necessary to understand the dimensional changes caused by missense mutations and explain the targeting of SDHB to the ubiquitin-proteasome degradation pathway. Missense mutations may affect not only protein instability but also changes in intrinsic protein targeting, transport, and function in the target organelle. Although not demonstrated for every potential variant of SDHB mutations, the hot spot SDHB mutations that we analyzed maintained the proper conformation for transport into the mitochondria and formed an active mitochondrial complex II, as supported by immunofluorescence and biochemical assays (Figs. 1E and 3). Furthermore, SDHB appears to maintain proper function once imported into the mitochondria, as these mutant SDHB proteins maintained their ability to associate with SDHA, another subunit in mitochondrial


YANG ET AL.

complex II. The physical interaction between mutated SDHB proteins and SDHA confirms that although these mutants may have increased susceptibility for degradation, they still retain their intrinsic capacity to target and insert into the proper mitochondrial compartment and form complex II associations (Fig. 3C, D). Therefore, the loss of mitochondrial complex II enzymatic activity occurs more likely due to a quantitative loss of the mutated SDHB protein rather than an intrinsic loss of enzymatic activity. The demonstration that PHEOs and PGLs arise from a quantitative loss of SDHB rather than a qualitative defect in its localization and complex formation may explain some aspects of its tumorigenic risk. Interestingly, Ricketts et al. (15) describes an increased risk for the development of PHEOs and PGLs in patients with missense mutations of SDHB compared to patients with nonsense mutations of SDHB. This finding seems counterintuitive, given the proposed mechanism of pseudohypoxia that leads to tumorigenesis in patients with SDHB mutations. We found that patients with nonsense mutations had a greater reduction in SDHB protein expression in their tumors than patients with missense mutations (Fig. 1E). These patients should also have less functional activity of mitochondrial complex II, greater accumulation of succinate in their cytoplasm, and increased stabilization of HIF-␣. However, because loss of heterozygosity appears to be a necessary precursor to tumorigenesis in SDHB-related PHEOs and PGLs, and a quantitative loss of protein due to degradation occurs rather than a change in the intrinsic function of missense mutations of SDHB, the increased risk of tumorigenesis in missense mutations is consistent with other solid tumor neoplasia syndromes, such as VHL (56, 57). Complete loss of both copies of the gene is more likely to cause severe dysfunction of the target cell that is developmentally lethal and not conducive to tumor formation. However, partial quantitative loss of a missense mutation followed by a deletion of the second allele leads to an insufficiency of protein function that results in an imbalance of cell growth toward proliferation that underlies tumorigenesis. By understanding the underlying mechanism that leads to tumorigenesis, this study also provides significant clinical implications for both diagnosis and future treatment options for tumors associated with SDHB mutations. The current diagnosis of tumors with SDHB mutations largely relies on the detection of a loss of heterozygosity (28, 58), which applies comparisons of single nucleotide polymorphisms of different alleles. This requires a large amount of tumor sample in order to obtain consistent results, leading to significant variability in predictive value. However, this study suggests an alternative diagnostic approach via direct quantification of the SDHB protein or measurements of complex II activity from relatively small amounts of sample. Because the pathogenesis of these tumors occurs due to changes in protein quantity, a technique that relies on protein content and not on genetic material could SDHB MISSENSE MUTATIONS INCREASE PROTEIN DEGRADATION

provide more consistent diagnosis and prognosis of these aggressive and lethal tumors. This understanding also provides a mechanism of reversing much of the risk associated with SDHB mutations. In this study, we demonstrate the potential of proteostasis regulators, such as HDACis, to increase the quantity of functional SDHB (Fig. 4). The manipulation of the protein quality control system via HDACis and other proteostasis regulators results in increased stability of the protein, increasing the total amount of SDHB protein in the mitochondria. This may lead to the formation of sufficient functional mitochondrial complex II to rescue cellular enzymatic activity in order to scavenge succinate, reduce the formation of ROS, and destabilize HIF-␣. In this way, HDACis may limit, if not prevent, the perturbations that lead to the pathogenesis of PHEO and PGL. The use of such specific molecular mediators targeting the ubiquitin-proteasome degradation pathway may provide an effective treatment paradigm for SDHB-related neoplasias and other similar human diseases of accelerated protein degradation. This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the National Institute of Neurological Disorders and Stroke at the U.S. National Institutes of Health.

REFERENCES 1. 2. 3.

4.

5. 6.

7. 8.

9.

DeLellis, R. A., Lloyd, R. V., Heitz, P. U., and Eng, C. (2004) Tumors of Endocrine Organs, IARC Press, Lyon, France Lenders, J. W., Eisenhofer, G., Mannelli, M., and Pacak, K. (2005) Phaeochromocytoma. Lancet 366, 665–675 Huang, H., Abraham, J., Hung, E., Averbuch, S., Merino, M., Steinberg, S. M., Pacak, K., and Fojo, T. (2008) Treatment of malignant pheochromocytoma/paraganglioma with cyclophosphamide, vincristine, and dacarbazine: recommendation from a 22-year follow-up of 18 patients. Cancer 113, 2020 –2028 Neumann, H. P., Bausch, B., McWhinney, S. R., Bender, B. U., Gimm, O., Franke, G., Schipper, J., Klisch, J., Altehoefer, C., Zerres, K., Januszewicz, A., Eng, C., Smith, W. M., Munk, R., Manz, T., Glaesker, S., Apel, T. W., Treier, M., Reineke, M., Walz, M. K., Hoang-Vu, C., Brauckhoff, M., Klein-Franke, A., Klose, P., Schmidt, H., Maier-Woelfle, M., Peczkowska, M., and Szmigielski, C. (2002) Germ-line mutations in nonsyndromic pheochromocytoma. N. Engl. J. Med. 346, 1459 –1466 Kaelin, W. G. (2007) Von Hippel-Lindau disease. Annu. Rev. Pathol. 2, 145–173 Gutmann, D. H., Aylsworth, A., Carey, J. C., Korf, B., Marks, J., Pyeritz, R. E., Rubenstein, A., and Viskochil, D. (1997) The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2. JAMA 278, 51–57 Eng, C. (1999) RET proto-oncogene in the development of human cancer. J. Clin. Oncol. 17, 380 –393 Baysal, B. E., Ferrell, R. E., Willett-Brozick, J. E., Lawrence, E. C., Myssiorek, D., Bosch, A., van der Mey, A., Taschner, P. E., Rubinstein, W. S., Myers, E. N., Richard, C. W., 3rd, Cornelisse, C. J., Devilee, P., and Devlin, B. (2000) Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287, 848 –851 Amar, L., Bertherat, J., Baudin, E., Ajzenberg, C., Bressac-de Paillerets, B., Chabre, O., Chamontin, B., Delemer, B., Giraud, S., Murat, A., Niccoli-Sire, P., Richard, S., Rohmer, V., Sadoul, J. L., Strompf, L., Schlumberger, M., Bertagna, X., Plouin, P. F., Jeunemaitre, X., and Gimenez-Roqueplo, A. P. (2005) Genetic

9

10. 11. 12. 13. 14.

15.

16.

17.

18.

19.

20.

21.

22. 23.

24.

25.

10

testing in pheochromocytoma or functional paraganglioma. J. Clin. Oncol. 23, 8812–8818 Gottlieb, E., and Tomlinson, I. P. (2005) Mitochondrial tumour suppressors: a genetic and biochemical update. Nat. Rev. Cancer 5, 857–866 Eng, C., Kiuru, M., Fernandez, M. J., and Aaltonen, L. A. (2003) A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat. Rev. Cancer 3, 193–202 Baysal, B. E. (2003) On the association of succinate dehydrogenase mutations with hereditary paraganglioma. Trends Endocrinol. Metab. 14, 453–459 Hensen, E. F., and Bayley, J. P. (2011) Recent advances in the genetics of SDH-related paraganglioma and pheochromocytoma. Fam. Cancer 10, 355–363 Ricketts, C., Woodward, E. R., Killick, P., Morris, M. R., Astuti, D., Latif, F., and Maher, E. R. (2008) Germline SDHB mutations and familial renal cell carcinoma. J. Natl. Cancer Inst. 100, 1260 –1262 Ricketts, C. J., Forman, J. R., Rattenberry, E., Bradshaw, N., Lalloo, F., Izatt, L., Cole, T. R., Armstrong, R., Kumar, V. K., Morrison, P. J., Atkinson, A. B., Douglas, F., Ball, S. G., Cook, J., Srirangalingam, U., Killick, P., Kirby, G., Aylwin, S., Woodward, E. R., Evans, D. G., Hodgson, S. V., Murday, V., Chew, S. L., Connell, J. M., Blundell, T. L., Macdonald, F., and Maher, E. R. (2010) Tumor risks and genotype-phenotype-proteotype analysis in 358 patients with germline mutations in SDHB and SDHD. Hum. Mutat. 31, 41–51 Gill, A. J., Pachter, N. S., Clarkson, A., Tucker, K. M., Winship, I. M., Benn, D. E., Robinson, B. G., and Clifton-Bligh, R. J. (2011) Renal tumors and hereditary pheochromocytoma-paraganglioma syndrome type 4. N. Engl. J. Med. 364, 885–886 Guzy, R. D., Sharma, B., Bell, E., Chandel, N. S., and Schumacker, P. T. (2008) Loss of the SdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Mol. Cell. Biol. 28, 718 –731 Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson, D. G., Mansfield, K. D., Pan, Y., Simon, M. C., Thompson, C. B., and Gottlieb, E. (2005) Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell 7, 77–85 Astuti, D., Ricketts, C. J., Chowdhury, R., McDonough, M. A., Gentle, D., Kirby, G., Schlisio, S., Kenchappa, R. S., Carter, B. D., Kaelin, W. G., Jr., Ratcliffe, P. J., Schofield, C. J., Latif, F., and Maher, E. R. (2011) Mutation analysis of HIF prolyl hydroxylases (PHD/EGLN) in individuals with features of phaeochromocytoma and renal cell carcinoma susceptibility. Endocr. Relat. Cancer 18, 73–83 Lee, S., Nakamura, E., Yang, H., Wei, W., Linggi, M. S., Sajan, M. P., Farese, R. V., Freeman, R. S., Carter, B. D., Kaelin, W. G., Jr., and Schlisio, S. (2005) Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: developmental culling and cancer. Cancer Cell 8, 155–167 Eisenhofer, G., Bornstein, S. R., Brouwers, F. M., Cheung, N. K., Dahia, P. L., de Krijger, R. R., Giordano, T. J., Greene, L. A., Goldstein, D. S., Lehnert, H., Manger, W. M., Maris, J. M., Neumann, H. P., Pacak, K., Shulkin, B. L., Smith, D. I., Tischler, A. S., and Young, W. F., Jr. (2004) Malignant pheochromocytoma: current status and initiatives for future progress. Endocr. Relat. Cancer 11, 423–436 Favier, J., and Gimenez-Roqueplo, A. P. (2010) Pheochromocytomas: the (pseudo)-hypoxia hypothesis. Best Pract. Res. Clin. Endocrinol. Metab. 24, 957–968 Dahia, P. L., Ross, K. N., Wright, M. E., Hayashida, C. Y., Santagata, S., Barontini, M., Kung, A. L., Sanso, G., Powers, J. F., Tischler, A. S., Hodin, R., Heitritter, S., Moore, F., Dluhy, R., Sosa, J. A., Ocal, I. T., Benn, D. E., Marsh, D. J., Robinson, B. G., Schneider, K., Garber, J., Arum, S. M., Korbonits, M., Grossman, A., Pigny, P., Toledo, S. P., Nose, V., Li, C., and Stiles, C. D. (2005) A HIF1␣ regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas. PLoS Genet. 1, 72–80 Gerald, D., Berra, E., Frapart, Y. M., Chan, D. A., Giaccia, A. J., Mansuy, D., Pouyssegur, J., Yaniv, M., and Mechta-Grigoriou, F. (2004) JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118, 781–794 Gao, P., Zhang, H., Dinavahi, R., Li, F., Xiang, Y., Raman, V., Bhujwalla, Z. M., Felsher, D. W., Cheng, L., Pevsner, J., Lee,

Vol. 26

November 2012

26.

27. 28.

29.

30.

31.

32.

33.

34.

35. 36. 37.

38. 39.

40. 41. 42.

43.

L. A., Semenza, G. L., and Dang, C. V. (2007) HIF-dependent antitumorigenic effect of antioxidants in vivo. Cancer Cell 12, 230 –238 Slane, B. G., Aykin-Burns, N., Smith, B. J., Kalen, A. L., Goswami, P. C., Domann, F. E., and Spitz, D. R. (2006) Mutation of succinate dehydrogenase subunit C results in increased O2⫺, oxidative stress, and genomic instability. Cancer Res. 66, 7615– 7620 Tormos, K. V., and Chandel, N. S. (2010) Inter-connection between mitochondria and HIFs. J. Cell. Mol. Med. 14, 795–804 Gimenez-Roqueplo, A. P., Favier, J., Rustin, P., Rieubland, C., Crespin, M., Nau, V., Khau Van Kien, P., Corvol, P., Plouin, P. F., and Jeunemaitre, X. (2003) Mutations in the SDHB gene are associated with extra-adrenal and/or malignant phaeochromocytomas. Cancer Res. 63, 5615–5621 Neumann, H. P., Pawlu, C., Peczkowska, M., Bausch, B., McWhinney, S. R., Muresan, M., Buchta, M., Franke, G., Klisch, J., Bley, T. A., Hoegerle, S., Boedeker, C. C., Opocher, G., Schipper, J., Januszewicz, A., and Eng, C. (2004) Distinct clinical features of paraganglioma syndromes associated with SDHB and SDHD gene mutations. JAMA 292, 943–951 Fliedner, S. M., Breza, J., Kvetnansky, R., Powers, J. F., Tischler, A. S., Wesley, R., Merino, M., Lehnert, H., and Pacak, K. (2010) Tyrosine hydroxylase, chromogranin A, and steroidogenic acute regulator as markers for successful separation of human adrenal medulla. Cell Tissue Res. 340, 607–612 Furuta, M., Weil, R. J., Vortmeyer, A. O., Huang, S., Lei, J., Huang, T. N., Lee, Y. S., Bhowmick, D. A., Lubensky, I. A., Oldfield, E. H., and Zhuang, Z. (2004) Protein patterns and proteins that identify subtypes of glioblastoma multiforme. Oncogene 23, 6806 –6814 Zhuang, Z., Bertheau, P., Emmert-Buck, M. R., Liotta, L. A., Gnarra, J., Linehan, W. M., and Lubensky, I. A. (1995) A microdissection technique for archival DNA analysis of specific cell populations in lesions ⬍1 mm in size. Am. J. Pathol. 146, 620 –625 Zhuang, Z., Qi, M., Li, J., Okamoto, H., Xu, D. S., Iyer, R. R., Lu, J., Yang, C., Weil, R. J., Vortmeyer, A., and Lonser, R. R. (2001) Proteomic identification of glutamine synthetase as a differential marker for oligodendrogliomas and astrocytomas. J. Neurosurg. 115, 789 –795 Lu, J., Chiang, J., Iyer, R. R., Thompson, E., Kaneski, C. R., Xu, D. S., Yang, C., Chen, M., Hodes, R. J., Lonser, R. R., Brady, R. O., and Zhuang, Z. (2010) Decreased glucocerebrosidase activity in Gaucher disease parallels quantitative enzyme loss due to abnormal interaction with TCP1 and c-Cbl. Proc. Natl. Acad. Sci. U. S. A. 107, 21665–21670 Kamitani, T., Kito, K., Nguyen, H. P., and Yeh, E. T. (1997) Characterization of NEDD8, a developmentally down-regulated ubiquitin-like protein. J. Biol. Chem. 272, 28557–28562 Van Nederveen, F. H., Dinjens, W. N., Korpershoek, E., and De Krijger, R. R. (2006) The occurrence of SDHB gene mutations in pheochromocytoma. Ann. N. Y. Acad. Sci. 1073, 177–182 Henderson, A., Douglas, F., Perros, P., Morgan, C., and Maher, E. R. (2009) SDHB-associated renal oncocytoma suggests a broadening of the renal phenotype in hereditary paragangliomatosis. Fam. Cancer 8, 257–260 Pawlu, C., Bausch, B., and Neumann, H. P. (2005) Mutations of the SDHB and SDHD genes. Fam. Cancer 4, 49 –54 Yang, C., Asthagiri, A. R., Iyer, R. R., Lu, J., Xu, D. S., Ksendzovsky, A., Brady, R. O., Zhuang, Z., and Lonser, R. R. (2011) Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function. Proc. Natl. Acad. Sci. U. S. A. 108, 4980 –4985 Zhuang, Z., Hogan, M., and McCauley, R. (1988) The in vitro insertion of monoamine oxidase B into mitochondrial outer membranes. FEBS Lett. 238, 185–190 Zhuang, Z. P., Marks, B., and McCauley, R. B. (1992) The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria. J. Biol. Chem. 267, 591–596 Yankovskaya, V., Horsefield, R., Tornroth, S., Luna-Chavez, C., Miyoshi, H., Leger, C., Byrne, B., Cecchini, G., and Iwata, S. (2003) Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 299, 700 –704 Knudson, A. G., Jr. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad. Sci. U. S. A. 68, 820 –823


YANG ET AL.

44.

45. 46.

47.

48.

49.

50. 51.

Trotman, L. C., Niki, M., Dotan, Z. A., Koutcher, J. A., Di Cristofano, A., Xiao, A., Khoo, A. S., Roy-Burman, P., Greenberg, N. M., Van Dyke, T., Cordon-Cardo, C., and Pandolfi, P. P. (2003) Pten dose dictates cancer progression in the prostate. PLoS Biol. 1, E59 Berger, A. H., Knudson, A. G., and Pandolfi, P. P. (2011) A continuum model for tumour suppression. Nature 476, 163–169 Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) HIF␣ targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464 –468 Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe, P. J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271–275 Astuti, D., Douglas, F., Lennard, T. W., Aligianis, I. A., Woodward, E. R., Evans, D. G., Eng, C., Latif, F., and Maher, E. R. (2001) Germline SDHD mutation in familial phaeochromocytoma. Lancet 357, 1181–1182 Van Nederveen, F. H., Gaal, J., Favier, J., Korpershoek, E., Oldenburg, R. A., de Bruyn, E. M., Sleddens, H. F., Derkx, P., Riviere, J., Dannenberg, H., Petri, B. J., Komminoth, P., Pacak, K., Hop, W. C., Pollard, P. J., Mannelli, M., Bayley, J. P., Perren, A., Niemann, S., Verhofstad, A. A., de Bruine, A. P., Maher, E. R., Tissier, F., Meatchi, T., Badoual, C., Bertherat, J., Amar, L., Alataki, D., Van Marck, E., Ferrau, F., Francois, J., de Herder, W. W., Peeters, M. P., van Linge, A., Lenders, J. W., GimenezRoqueplo, A. P., de Krijger, R. R., and Dinjens, W. N. (2009) An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis. Lancet Oncol. 10, 764 –771 Bukau, B., Weissman, J., and Horwich, A. (2006) Molecular chaperones and protein quality control. Cell 125, 443–451 McClellan, A. J., Tam, S., Kaganovich, D., and Frydman, J. (2005) Protein quality control: chaperones culling corrupt conformations. Nat. Cell Biol. 7, 736 –741

SDHB MISSENSE MUTATIONS INCREASE PROTEIN DEGRADATION

52.

53.

54.

55.

56.

57.

58.

Feldman, D. E., Spiess, C., Howard, D. E., and Frydman, J. (2003) Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol. Cell. 12, 1213–1224 McClellan, A. J., Scott, M. D., and Frydman, J. (2005) Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell 121, 739 –748 Melville, M. W., McClellan, A. J., Meyer, A. S., Darveau, A., and Frydman, J. (2003) The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex. Mol. Cell. Biol. 23, 3141–3151 Dickey, C. A., Kamal, A., Lundgren, K., Klosak, N., Bailey, R. M., Dunmore, J., Ash, P., Shoraka, S., Zlatkovic, J., Eckman, C. B., Patterson, C., Dickson, D. W., Nahman, N. S., Jr., Hutton, M., Burrows, F., and Petrucelli, L. (2007) The high-affinity HSP90CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J. Clin. Invest. 117, 648 –658 Chen, F., Kishida, T., Duh, F. M., Renbaum, P., Orcutt, M. L., Schmidt, L., and Zbar, B. (1995) Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor suppressor gene. Cancer Res. 55, 4804 –4807 Chen, F., Kishida, T., Yao, M., Hustad, T., Glavac, D., Dean, M., Gnarra, J. R., Orcutt, M. L., Duh, F. M., Glenn, G., Green, J., Hsia, Y. E., Lamiell, J., Li, H., Wei, M. H., Schmidt, L., Tory, K., Kuzmin, I., Stackhouse, T., Latif, F., Linehan, W. M., Lerman, M., and Zbar, B. (1995) Germline mutations in the von HippelLindau disease tumor suppressor gene: correlations with phenotype. Hum. Mutat. 5, 66 –75 Benn, D. E., Croxson, M. S., Tucker, K., Bambach, C. P., Richardson, A. L., Delbridge, L., Pullan, P. T., Hammond, J., Marsh, D. J., and Robinson, B. G. (2003) Novel succinate dehydrogenase subunit B (SDHB) mutations in familial phaeochromocytomas and paragangliomas, but an absence of somatic SDHB mutations in sporadic phaeochromocytomas. Oncogene 22, 1358 –1364 Received for publication April 16, 2012. Accepted for publication July 17, 2012.

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