Regulation of 2-Oxoglutarate (α-Ketoglutarate) Dehydrogenase Stability by the RING Finger Ubiquitin Ligase Siah*

The 2-oxoglutarate dehydrogenase complex (OGHDC) (also known as the α-ketoglutarate dehydrogenase complex) is a rate-limiting enzyme in the mitochondrial Krebs cycle. Here we report that the RING finger ubiquitin-protein isopeptide ligase Siah2 binds to and targets OGDHC-E2 for ubiquitination-dependent degradation. OGDHC-E2 expression and activity are elevated in Siah2-/- cells compared with Siah2+/+ cells. Deletion of the mitochondrial targeting sequence of OGDHC-E2 results in its cytoplasmic localization and rapid proteasome-dependent degradation in Siah2+/+ but not in Siah2-/- cells. Significantly, because of its overexpression or disruption of the mitochondrial membrane potential, the release of OGDHC-E2 from mitochondria to the cytoplasm also results in its concomitant degradation. The role of the Siah family of ligases in the regulation of OGDHC-E2 stability is expected to take place under pathological conditions in which the levels of OGDHC-E2 are altered.

Siah was first identified in Drosophila melanogaster as seven in absentia (sina) (1,2). Three murine (Siah1a, Siah1b, and Siah2) and two human (SIAH1 and SIAH2) homologues share significant homology (1,2). The protein products of sina and Siah1a, Siah1b, and Siah2 are RING finger proteins with E3 1 ligase activity (1,2) and have been implicated in the ubiquitination and proteasome-dependent degradation of Bag-1, DCC (deleted in colon cancer), N-CoR, c-Myb, Kid, OBF1, ␤-catenin, Numb, and ␣-synuclein, while also limiting their own availability through efficient self-ubiquitination and degradation (3)(4)(5)(6)(7)(8)(9)(10). We have demonstrated that Siah2 targets TRAF2 for proteasome-dependent degradation under stress conditions, which is required for the cell to commit to undergoing apoptosis (11). The C terminus of Siah, which is structurally very similar to the TRAF-C domain of the TRAF family of proteins, serves as the substrate binding domain for Siah substrates (12). Common to both sina and Siah proteins is their association with phyllopod and SIP, respectively, adaptor proteins that mediate efficient targeting of Siah substrates (13,14). A search for novel Siah2 substrates led to the identification of prolyl hydroxylases PHD1 and PHD3 as Siah2-binding proteins. Siah1a and Siah2 regulate the abundance of both PHD1 and PHD3 by targeting their proteasome-dependent degradation more under hypoxic than normoxic conditions, thereby constituting a novel layer in the regulation of hypoxia-inducible factor (HIF1␣) stability, as in the regulation of the cellular hypoxia response (15). The same analysis also identified an E2 subunit of the 2-oxoglutaric acid dehydrogenase complex (OGDHC) (also known as the ␣-ketoglutarate dehydrogenase complex), which serves as a rate-limiting enzyme in the Krebs cycle as a Siah2-associated protein. Chronic hypoxia and oxidative stress decrease OGDHC activity, especially in brain regions (olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum) (16,17). A decrease in OGDHC activity is also noted in a number of neurodegenerative diseases, including thiamine deficiency, and Alzheimer and Parkinson diseases (18).
Primary biliary cirrhosis (PBC) is an organ-specific autoimmune disease that affects predominantly women and is characterized by the progressive destruction of small intrahepatic bile ducts with portal inflammation and ultimately fibrosis (19). The serological hallmark of PBC is the presence of antimitochondrial antibodies directed against E2 components of the 2-oxoacid dehydrogenase complex family (2-OADHC-E2), including the E2 component of the OGDHC (OGDHC-E2), the pyruvate dehydrogenase complex (PDHC-E2), and the branched chain 2-oxoacid dehydrogenase complex (BCOADHC-E2) (19). The up-regulation of 2-OADHC-E2 is often seen in biliary epithelial cells (BEC) in the natural history of PBC, as well as in BEC in allografts of patients with recurrent PBC following liver transplantation (20 -23), suggesting that these antigens have a causative role in the pathogenesis and/or progression of PBC.
2-OADHC-E2 proteins are localized normally within the mitochondrial matrix. Enhanced synthesis, impaired degradation, or abnormal targeting of these E2s to the surface and apical regions of BEC may induce the production of anti-2-OADHC-E2 antibodies, which in turn induces an autoimmune response resulting in the selective destruction of BEC in PBC patients (19). In situ hybridization of PDHC-E2, OGDHC-E2, and BCOADHC-E2 mRNA, however, does not reveal differences in the number of transcripts present in PBC compared with other liver diseases (24), suggesting that the elevated expression of E2 is the result of post-translational events.
In the present study, we demonstrate that both Siah1 and Siah2 bind specifically to OGDHC-E2 and target the precursor form of OGDHC-E2 or the OGDHC-E2 that is released from mitochondria for ubiquitination-dependent degradation. Our data highlight a newly recognized function for the Siah family of ligases, which act through the regulation of OGDHC-E2 stability under certain pathological conditions.

MATERIALS AND METHODS
Cell Culture and Reagents-HeLa and 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with calf serum (10%) and antibiotics. Wild-type and Siah2 Ϫ/Ϫ MEFs were maintained in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%), 0.02 mM ␤-mercaptoethanol, sodium pyruvate, and antibiotics. Antibodies (Ab) and reagents were purchased as follows: anti-␣-tubulin Ab, anti-FLAG Ab, carbonyl cyanide p-chlorophenylhydrazone (CCCP), and actinomycin D were from Sigma; anti-HA Ab was from Zymed Laboratories; anti-cytochrome c and anti-cytochrome oxidase subunit I (COX I) Ab were from Molecular Probes; mTNF␣ was from Roche Applied Science; and hTNF␣ was from R&D Systems. The generation of anti-OGDHC-E2 (6G10) and PDHC-E2 (4C8) monoclonal Ab was described previously (22).
Plasmids and Transfection-The FLAG-OGDHC-E2 expression vector was generated by PCR amplification of OGDHC-E2 cDNA from the HeLa cell cDNA library followed by in-frame cloning downstream of the FLAG tag in pCDNA3-FLAG. ⌬M-OGDHC-E2-HA was generated by cloning OGDHC-E2 cDNA, from which an N-terminal mitochondrial targeting sequence (MLSRSRCVSRAFSRSLSAFQKGNCP-LGRRSLPGVSLCQGPGYPNSRKVVINNSVFSVRFFRT) was deleted into pCDNA3 by PCR amplification using a C-terminal HA-tagged primer. GST-OGDHC-E2 was generated by cloning OGDHC-E2 cDNA in-frame into pGEX-4T-2. All plasmids were confirmed by DNA se-quencing. FLAG-Siah2 and GST-Siah2 plasmids were described previously (11). GST-OGDHC-E2 and GST-Siah2 were expressed in Escherichia coli BL21 and purified using immobilized glutathione beads (Pierce) as described previously (11). 293T cells were transfected using the calcium phosphate precipitation method. HeLa and MEF cells were transfected with Lipofectamine PLUS reagent (Invitrogen).
Identification of OGDC-E2 as a Siah-binding Protein-A FLAG-Siah2-Rm or an empty pCDNA3 (as a control) plasmid was transfected into 293T cells (40 plates). 36 h after transfection, cells were washed and harvested, and cell pellets were lysed in TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0% Nonidet P-40, 10% glycerol, 1.0 mM EGTA, 1 mM EDTA, 1.0 mM dithiothreitol, 1.0 mM NaVO 3 , 5 mM ␤-glycerol phosphate, 0.5 mM phenylmethylsulfonyl fluoride, and 1ϫ protease inhibitor mixture) for 30 min on ice. The lysates were cleared by ultracentrifugation at 37,000 ϫ g for 45 min at 4°C, and FLAG-Siah2-Rm was immunoprecipitated with anti-FLAG M2 beads. After three washes with the same TNE lysis buffer, Siah2-bound proteins were eluted with 0.5 mg/ml FLAG-peptides, precipitated with 15% tricarboxylic acid, and separated on two-dimensional PAGE as described previously (25). The specific Siah2-binding proteins (compared with the control sample) were identified via silver staining and subjected to mass spectrometry analysis as described previously (15,26).
Immunoprecipitation and Western Blotting-Cells were lysed in TNE buffer for 30 min at 4°C. After centrifugation at 12,000 ϫ g for 20 min at 4°C, 1.0 mg of protein was incubated overnight with 1 g of anti-FLAG or anti-OGDHC-E2 Ab and then further incubated for 2 h with 20 l of protein G beads (Invitrogen). The beads were washed three times with the same TNE lysis buffer, resuspended in loading buffer, boiled, centrifuged, resolved by SDS-PAGE, and analyzed by Western blot as described previously (11,27). Isolation of Mitochondria-Cells were suspended in 1.0 ml of ice-cold homogenization buffer (20 mM HEPES-KOH, pH 7.5, 1.0 mM EDTA, 0.25 M sucrose, 1.0 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1ϫ mixture of protease inhibitors) and disrupted in a 2.0-ml Teflon Dounce homogenizer with 30ϫ up-and-down strokes with a tightly fitting pestle. The cellular lysates were centrifuged at 750 ϫ g for 10 min in a swing-out rotor, and then the supernatants were centrifuged at 8000 ϫ g for 10 min to pellet the mitochondria. Postmitochondria supernatants were further centrifuged at 14,000 ϫ g for 30 min, and the supernatants were used as the cytoplasm protein fraction. The mitochondria were lysed in TNE buffer for further analysis.
In Vitro Binding Assay-Bacterially expressed and purified GST, GST-OGDHC-E2, or GST-Siah2 bound to glutathione beads was incubated first with 1% bovine serum albumin in PBS for 1 h followed by incubation with 35 S-labeled Siah2 or OGDHC-E2, which was in vitro translated using the TNT-coupled rabbit reticulocyte lysate system (Promega). Bead-bound materials were washed four times with PBS containing 0.25% Nonidet P-40, 0.1% ␤-mercaptoethanol, and 2 mM EDTA before being subjected to separation by SDS-PAGE followed by autoradiography.
In Vivo Ubiquitination Assay-FLAG-OGDHC-E2 (1.0 g) and/or HA-ubiquitin (2.0 g) was transfected into HeLa cells. Thirty-six hours later, cells were treated with or without MG132 for 4 h, cell pellets were lysed, and FLAG-OGDHC-E2 was immunoprecipitated as described previously (27). Bead-bound proteins were then eluted in SDS sample buffer and subjected to immunoblot analysis with anti-HA Ab. The same membrane was stripped and reprobed with anti-FLAG Ab.
OGDHC Activity Assay-The activity of OGDHC was assayed by measuring the reduction of NAD ϩ at 340 nm (16). The assay mixture contained 50 mM HEPES, pH 7.4, 0.1% Triton X-100, 0.5 mg/ml bovine serum albumin, 0.2 mM thiamine pyrophosphate chloride, 2 mM NAD ϩ , 1 mM MgCl 2 , 0.3 mM dithiothreitol, 5 mM ␣-ketoglutarate, 0.1 mM CoA, and 25 g of mitochondrial proteins. The reaction was initiated by the addition of CoA, and the initial rate was measured. The OGDHC activities obtained from wild-type MEF or 293T cells that were transfected with empty pCDNA3 were set as 100%, and the rest were calculated relative to the value of control.
Immunofluorescence Microscopy-OGDHC-E2-HA-transfected cells grown on glass coverslips were fixed with 3% paraformaldehyde and 2% sucrose in PBS for 20 min at room temperature, washed twice with PBS, and then incubated for 1 min in permeabilization buffer (0.3% Triton X-100, 3 mM MgCl 2 , and 6.8% sucrose). After blocking the cells with 3% bovine serum albumin/PBS for 30 min, cells were incubated with 2.0 g/ml anti-HA Ab for 1 h, washed, and further incubated with fluorescein isothiocyanate-labeled secondary Ab (1:1000 dilutions) for 1 h. After they were stained with 4,6-diamidino-2-phenylindole, coverslips were inverted and mounted on slides with Vectashield and affixed with nail polish. Fluorescence was monitored using a Leica TCS-SP (UV) confocal microscope.

Identification of OGDHC-E2 as a Siah-binding Protein-To
identify novel Siah2-binding proteins that might serve as adaptors or substrates, we expressed FLAG-tagged and RING domain-mutated Siah2 (FLAG-Siah2-Rm) in 293T cells followed by immunoprecipitation with an anti-FLAG antibody and separation by two-dimensional PAGE. The selection of Siah2bound proteins was based on a comparison with control immunoprecipitation, subject to the same analysis (Fig. 1A). Mass spectrometry analysis identified three of six positive spots as components of OGDHC, as follows: E1 (2-oxoglutarate dehydrogenase (gene name OGDH)), E2 (dihydrolipoamide S-succinyltransferase (gene name DLST)), and E3 (dihydrolipoamide dehydrogenase (gene name DLD)). OGDHC is a rate-limiting enzyme complex in the tricarboxylic acid (Krebs) cycle, localized within the mitochondrial matrix, which converts ␣-ketoglutarate to high energy succinyl-CoA and CO 2 and reduces NAD ϩ to NADH (18). An in vitro GST fusion protein pull-down assay confirmed that both Siah1 and Siah2 interact efficiently with OGDHC-E2 (Fig. 1B). OGDHC-E3 is a common subunit for all 2-OADHC family members, and Siah2 binding to OGDHC-E1 was very weak (Fig. 1A). We thus elucidated the role of Siah in the regulation of OGDHC-E2 stability and activity.
Siah2 Targets Exogenously Expressed OGDHC-E2 for Ubiquitination and Degradation-Siah2 is a potent E3 ligase that targets most but not all of its associated proteins for proteasomedependent degradation (11,15). To determine whether Siah2 targets OGDHC-E2 for ubiquitination and degradation, we gen-erated two different OGDHC-E2 expression vectors, FLAG-OGDHC-E2, in which the FLAG tag was added to the N terminus of OGDHC-E2 cDNA, and OGDHC-E2-HA, in which the HA tag was added to the C terminus of OGDHC-E2. Expression of these two plasmids in 293T cells followed by Western blot analysis revealed that FLAG-OGDHC-E2 migrates as a 62-kDa protein, whereas OGDHC-E2-HA migrates as a 52-kDa protein ( Fig. 2A). Common to mitochondrial proteins synthesized in the cytoplasm and imported to the mitochondria and also seen in OGDHC-E2 is an N-terminal mitochondrial targeting sequence that is cleaved proteolytically on import into the mitochondria, generating the mature form of the protein. We failed to detect the precursor form of OGDHC-E2-HA by Western blot even after exogenous overexpression ( Fig. 2A). Because removal of mitochondrial targeting sequence takes place within the mitochondria, the lack of a precursor form of OGDHC-E2-HA indicates that OGDHC-E2 is imported into mitochondria immediately after translation. The attachment of the FLAG tag in front of the mitochondrial targeting sequence impaired the efficient import of OGDHC-E2 into mitochondria ( Fig. 2A). The expression of wild-type HA-Siah2 efficiently reduced the steady-state level of FLAG-OGDHC-E2, whereas the expression of RING mutant HA-Siah2 increased the level of FLAG-OGDHC-E2 (Fig. 2B). The Siah2-induced decrease in the FLAG-OGDHC-E2 protein level is proteasome-dependent, because it is inhibited by the proteasome inhibitor MG132 (Fig.  2B, lane 5). The expression of FLAG-Siah1 or FLAG-Siah2 also reduced the steady-state level of OGDHC-E2-HA (Fig. 2C). An in vivo ubiquitination assay also revealed that Siah2 induces FLAG-OGDHC-E2 ubiquitination in vivo, which is detected on treatment of the cells with the proteasome inhibitor (Fig. 2D,  lane 5). In addition, bacterially expressed and purified Siah2 also efficiently targeted in vitro translated [ 35 S]OGDHC-E2 for ubiq- uitination in vitro (Fig. 2E). Collectively, these data establish the role of Siah2 in mediating the ubiquitination and degradation of OGDHC-E2.
Siah2 Limits the Protein Level and Enzymatic Activity of OGDHC-E2 by Targeting Its Precursor Form for Degradation-To assess the role of Siah2 in bringing about endogenous OGDHC-E2 abundance, we exogenously expressed the wildtype and RING mutant Siah2 in 293T cells and analyzed the steady-state level of endogenous OGDHC-E2. Neither form of exogenously expressed Siah2 affected the steady-state levels of OGDHC-E2 significantly within 36 h after transfection (Fig.  3A). However, 72 h after transfection, wild-type Siah2, but not RING mutant Siah2, efficiently decreased the steady-state level of endogenous OGDHC-E2 in a dose-dependent manner (Fig. 3B). In line with this, OGDHC activities were also inhibited by exogenous overexpression of wild-type Siah2 but not by RING mutant Siah2 (Fig. 3C). Therefore, these data revealed that under normal growth conditions Siah2 targets the precursor form of OGDHC-E2 for ubiquitination-dependent degradation and that the OGDHC-E2 that is localized within the mi-tochondria is protected from Siah2-mediated degradation.
Expression and Activity of OGDHC-E2 Are Elevated in Siah2 Ϫ/Ϫ Cells-To further assess the role of Siah2 in the regulation of OGDHC-E2 stability, we used MEFs prepared from Siah2 ϩ/ϩ and Siah2 Ϫ/Ϫ mice. Interestingly, steady-state levels of endogenous OGDHC-E2 were found to be elevated in Siah2 Ϫ/Ϫ MEF cells compared with those in the Siah2 ϩ/ϩ MEFs (Fig. 3D). These data provide important genetically based support for the observation that Siah2 can target the degradation of a precursor form of OGDHC-E2. Along these lines, analysis of OGDHC-E2 activity also revealed an increase in the enzymatic activity of the protein that coincides with the increase seen in the steady-state level of the protein (Fig. 3E). Exposure to TNF␣ and actinomycin D, which causes apoptosis, a condition that is expected to release OGDHC-E2 from the mitochondria and make it available for Siah2 activity, did not reveal changes in the steady-state levels of OGDHC-E2 within the first 4 h (Fig. 3D), although such changes were observed at later time points (see below). Reconstitution experiments revealed that exogenous expression of wild-type but not RING  3F). However, pulse-chase analysis did not reveal significant differences in the half-life of OGDHC-E2 between Siah2 ϩ/ϩ and Siah2 Ϫ/Ϫ MEF cells (data not shown), suggesting that the difference seen in the steady-state level can be attributed to a higher amount of precursor protein that reaches the mitochondria, where it is protected from Siah2 E3 ligase activities. These data further confirm that Siah2 targets the precursor form of OGDHC-E2 for degradation.
OGDHC-E2 from Which the Mitochondrial Targeting Sequence Has Been Deleted Is Degraded Rapidly in Siah2 ϩ/ϩ but Not in Siah2 Ϫ/Ϫ Cells-Given that OGDHC-E2 is protected against Siah2 while residing in the mitochondria and in light of the differences observed in the steady state level of the protein between Siah2 Ϫ/Ϫ and Siah2 ϩ/ϩ MEFs, we further elucidated the ability of Siah2 to elicit degradation of the cytoplasmresident form of OGDHC-E2. To this end we generated an OGDHC-E2 expression vector in which an HA tag was added to the C terminus of the protein and the mitochondrial targeting sequence was deleted (⌬M-OGDHC-E2-HA). The expression of ⌬M-OGDHC-E2-HA in 293T cells revealed that it is substantially less stable compared with the wild-type form of the protein and that it is subject to efficient proteasome-dependent degradation (Fig. 4A). An analysis of ⌬M-OGDHC-E2-HA expression levels in Siah2 ϩ/ϩ and Siah2 Ϫ/Ϫ MEF cells revealed that it is more stable in Siah2 Ϫ/Ϫ than in Siah2 ϩ/ϩ MEF cells (Fig. 4B). These findings establish the role of Siah2 in targeting the degradation of OGDHC-E2 that lacks its mitochondrial targeting signal. Indeed, immunohistochemistry analysis revealed that ⌬M-OGDHC-E2-HA is located diffusely in the cytoplasm (Fig. 4C). Collectively, these observations substantiate the role of Siah2 in targeting the degradation of cytoplasmicresident forms of OGDHC-E2. Two possible forms that are expected to be targeted by Siah2 are the precursor form, prior to its entry into the mitochondria, and the mature form, on its leakage from the mitochondria.
Overexpression or Disruption of Mitochondrial Membrane Potential Causes OGDHC-E2 Release from Mitochondria and Its Degradation in Cytoplasm-The overexpression of OGDHC-E2-HA in 293T cells resulted in abundant expression within the mitochondria but also in leakage of the 52-kDa mature form of OGDHC-E2-HA from the mitochondria, which is seen more clearly in the presence of the proteasome inhibitor MG132 (Fig. 5A). This finding suggests that the leakage of mature OGDHC-E2-HA into the cytoplasm renders it unstable. To further substantiate this observation, we treated cells with a mitochondria-specific ionophore (CCCP) that disrupts the mitochondrial membrane potential (28). Exposure to CCCP (20 M for 4 h) resulted in the release of more OGDHC-E2-HA from mitochondria to the cytoplasm (Fig. 5A). Furthermore, the treatment of HeLa cells with CCCP (50 M for 8 h) induced the release of endogenous OGDHC-E2 from the mitochondria to the cytoplasm, which was detectable only in the presence of the proteasome inhibitor (Fig. 5B). Similarly, the treatment of MEF cells with CCCP (50 M) or mTNF␣/actinomycin D (10 ng/ml and 2 g/ml) for 8 h induced the release of endogenous OGDHC-E2 from mitochondria, which was detectable in Siah2 Ϫ/Ϫ but not in Siah2 ϩ/ϩ cells without MG132 treatment (Fig. 5C). More importantly, treatment of MEF cells with a high dose of mTNF␣ (50 ng/ml) alone was sufficient to induce the release of OGDHC-E2 from mitochondria, which was also detectable in Siah2 Ϫ/Ϫ MEF cells without treating the cells with proteasome inhibitor (Fig. 5D). These results suggest that Siah2 limits the availability of OGDHC-E2 that is released from the mitochondria on its overexpression or change in mitochondrial membrane potential. DISCUSSION The present study identified and characterized OGDHC-E2 as a newly recognized substrate for the RING finger E3 ligase Siah2. In vitro and in vivo data provided direct support for the association and ubiquitination-dependent degradation of OG-DHC-E2 by Siah. Importantly, Siah2 Ϫ/Ϫ cells exhibited elevated expression and activity of OGDHC-E2, providing genetic evidence for the regulation of OGDHC-E2 stability by Siah. Given the localization of OGDHC-E2 within the inner mitochondrial matrix, the ability of Siah2 to affect the stability of E2 was limited to situations in which E2 is situated within the cytoplasm.
Under physiological conditions, OGDHC-E2 is imported into the inner matrix of the double layer of the mitochondrial membrane immediately after translation in the cytoplasm (29). It is therefore plausible that OGDHC-E2 could be targeted by Siah2 for degradation prior to its entry into the mitochondria when it is present as a proenzyme. Our data support this possibility because we provide evidence of the ability of Siah to decrease the stability of OGDHC-E2 in which the N-terminal mitochondrial localization signal has been deleted. Thus, the disruption of the OGDHC-E2 import is expected to result in lower amounts of this enzyme within the mitochondria and concomitantly limits mitochondrial metabolism. Alternatively, an increase in the Siah activity could also result in a more potent degradation of E2, which would limit its availability and activity. A decrease in the activity of the OGDHC complex is reported to take place in neurological disorders, including Alzheimer and Parkinson patients. Whether the reported changes in OGDHC are the result of an elevated activity of the Siah family of ubiquitin ligases has yet to be determined. Because Siah proteins are virtually undetectable in Western blots owing to their strong E3 ligase activity, which also limits their own availability, a simple analysis such as immunostaining is not possible. Of note, Siah2 expression and activity increase in hypoxia (15), suggesting that certain hypoxia and stress conditions may increase Siah2 activity, which may decrease OGDHC activity.
An alternate antecedent one can envision for the effect of Siah on OGDHC-E2 occurs when OGDHC-E2 is released from the mitochondria. The latter takes place on disruption of the mitochondrial membrane, as is often seen during programmed cell death. Indeed, treating cells with compounds that cause apoptosis results in the presence of OGDHC-E2 within the cytoplasm, which can be observed in Siah2 Ϫ/Ϫ cells even in the absence of proteasome inhibitors. This finding suggests that for cases in which there is a decrease in the activity of Siah2, conditions may result in the accumulation of OGDHC-E2 entering into or being released from the mitochondria. PDHC-E2, OGDHC-E2, and BCOADHC-E2 are expressed abnormally in the apical region of BEC and are major targets of autoantibodies to mitochondria proteins in PBC patients (19,22). In situ hybridization of these autoantigens did not reveal an increase in mRNA transcription in the BEC cells of PBC patients (24), indicating that abnormal expression of these autoantigens in BEC cells is a post-translational event, consistent with the possibility that impaired degradation of OGDHC-E2 could be the cause of this condition.
Taken together, our findings provide the first insight into the ubiquitination-dependent degradation of a mitochondrial protein, OGDHC-E2. Siah2-mediated degradation of OGDHC-E2 attenuates the activity of this key mitochondrial enzyme and is therefore expected to affect the energy-metabolic function of the mitochondria. Such changes are not expected to occur under normal physiologic conditions but rather when Siah expression and/or activities are modified. Because changes in the expression and activity of OGDHC-E2 are often seen in several pathological disorders, our findings may point to a mechanism that underlies impaired mitochondrial function in neurodegenerative disorders as well as in autoimmune disorders associated with primary biliary liver cirrhosis.