SIRT 3‐dependent GOT 2 acetylation status affects the malate–aspartate NADH shuttle activity and pancreatic tumor growth
2015; Springer Nature; Volume: 34; Issue: 8 Linguagem: Inglês
10.15252/embj.201591041
ISSN1460-2075
AutoresHui Yang, Lisha Zhou, Qian Shi, Yuzheng Zhao, Huaipeng Lin, Mengli Zhang, Shimin Zhao, Yi Yang, Zhi‐Qiang Ling, Kun‐Liang Guan, Yue Xiong, Dan Ye,
Tópico(s)Autophagy in Disease and Therapy
ResumoArticle9 March 2015free access SIRT3-dependent GOT2 acetylation status affects the malate–aspartate NADH shuttle activity and pancreatic tumor growth Hui Yang Hui Yang State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Lisha Zhou Lisha Zhou State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Qian Shi Qian Shi State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yuzheng Zhao Yuzheng Zhao School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Huaipeng Lin Huaipeng Lin State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Mengli Zhang Mengli Zhang State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Shimin Zhao Shimin Zhao State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yi Yang Yi Yang School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Zhi-Qiang Ling Zhi-Qiang Ling Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China Search for more papers by this author Kun-Liang Guan Corresponding Author Kun-Liang Guan State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Yue Xiong Corresponding Author Yue Xiong State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Dan Ye Corresponding Author Dan Ye State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Hui Yang Hui Yang State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Lisha Zhou Lisha Zhou State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Qian Shi Qian Shi State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yuzheng Zhao Yuzheng Zhao School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Huaipeng Lin Huaipeng Lin State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Mengli Zhang Mengli Zhang State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Shimin Zhao Shimin Zhao State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Yi Yang Yi Yang School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Zhi-Qiang Ling Zhi-Qiang Ling Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China Search for more papers by this author Kun-Liang Guan Corresponding Author Kun-Liang Guan State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA Search for more papers by this author Yue Xiong Corresponding Author Yue Xiong State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Dan Ye Corresponding Author Dan Ye State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China Search for more papers by this author Author Information Hui Yang1,‡, Lisha Zhou1,‡, Qian Shi1, Yuzheng Zhao2, Huaipeng Lin1, Mengli Zhang1, Shimin Zhao1, Yi Yang2, Zhi-Qiang Ling3, Kun-Liang Guan 1,4, Yue Xiong 1,5 and Dan Ye 1 1State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Molecular and Cell Biology Lab, Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, China 2School of Pharmacy, East China University of Science and Technology, Shanghai, China 3Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China 4Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA 5Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 8582461482; E-mail: [email protected] *Corresponding author. Tel: +1 9199622142; Fax: +1 9199668799; E-mail: [email protected] *Corresponding author. Tel: +86 2154237834; Fax: +86 2154237450; E-mail: [email protected] The EMBO Journal (2015)34:1110-1125https://doi.org/10.15252/embj.201591041 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The malate–aspartate shuttle is indispensable for the net transfer of cytosolic NADH into mitochondria to maintain a high rate of glycolysis and to support rapid tumor cell growth. The malate–aspartate shuttle is operated by two pairs of enzymes that localize to the mitochondria and cytoplasm, glutamate oxaloacetate transaminases (GOT), and malate dehydrogenases (MDH). Here, we show that mitochondrial GOT2 is acetylated and that deacetylation depends on mitochondrial SIRT3. We have identified that acetylation occurs at three lysine residues, K159, K185, and K404 (3K), and enhances the association between GOT2 and MDH2. The GOT2 acetylation at these three residues promotes the net transfer of cytosolic NADH into mitochondria and changes the mitochondrial NADH/NAD+ redox state to support ATP production. Additionally, GOT2 3K acetylation stimulates NADPH production to suppress ROS and to protect cells from oxidative damage. Moreover, GOT2 3K acetylation promotes pancreatic cell proliferation and tumor growth in vivo. Finally, we show that GOT2 K159 acetylation is increased in human pancreatic tumors, which correlates with reduced SIRT3 expression. Our study uncovers a previously unknown mechanism by which GOT2 acetylation stimulates the malate–aspartate NADH shuttle activity and oxidative protection. Synopsis Acetylation of oxaloacetate transaminase (GOT2) promotes its binding with malate dehydrogenase (MDH2), thereby regulating the malate–aspartate shuttle activity, cytosol-to-mitochondrion transfer of NADH, oxidative protection and tumor growth. GOT2 acetylation at three lysine residues enhances its association with MDH2. SIRT3 is the major deacetylase of GOT2. Acetylation of GOT2 modulates mitochondrial NADH/NAD+ redox status, energy production, and cell survival during oxidative stress. GOT2 K159 acetylation is increased in human pancreatic tumors, correlating with reduced SIRT3 expression. Introduction The glycolytic pathway is of great importance for energy production in the cell. During the breakdown of glucose to pyruvate, the coenzyme NAD+ (nicotinamide adenine dinucleotide, oxidized form) is reduced to NADH, and rapid oxidation of NADH to regenerate NAD+ is equally important for the cell to ensure glycolysis to proceed. Oxidation of cytosolic NADH can be achieved by anaerobic respiration that drives the reduction of pyruvate to lactate, or by aerobic respiration and the transfer of electrons via the mitochondrial electron transport chain (ETC). However, the transfer of NADH into mitochondria is hindered by the impermeability of the inner mitochondrial membrane to NADH (Lehninger, 1951). Therefore, the malate–aspartate shuttle plays an important role in transferring reducing equivalents across the mitochondrial membrane for energy production. Supporting this notion, the malate–aspartate shuttle has been shown to be the major way for the net transfer of cytosolic NADH into mitochondria in rat heart and liver (Williamson et al, 1970, 1971; Safer et al, 1971). Moreover, the malate–aspartate shuttle has been reported to oxidize 20–80% of cytosolic NADH in Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma and L1210 cells (Dionisi et al, 1974; Greenhouse & Lehninger, 1977). The malate–aspartate shuttle is operated by two pairs of enzymes, mitochondrially and cytoplasmically localized glutamate oxaloacetate transaminases (GOT) and malate dehydrogenases (MDH), which act in concert to transfer reducing equivalents across the mitochondrial membrane, with no net movement of carbon or nitrogen (Barron et al, 1998). As shown in Fig 1A, cytoplasmic MDH1 reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate is then transported to the interior of the mitochondrion in exchange for α-ketoglutarate (α-KG). Inside the mitochondrion, malate is oxidized once again to OAA by mitochondrial MDH2, giving rise to NADH which enters the ETC to produce ATP. In return, mitochondrial GOT2 converts OAA inside the mitochondrion to aspartate by transamination, with the amino group being donated by glutamate, giving rise to α-KG. Aspartate and α-KG then enter the cytosol, wherein aspartate is converted to OAA, while α-KG is converted to glutamate mediated by GOT1. Earlier studies have suggested that GOT may interact with MDH in both the cytosol and the mitochondrion (Backman & Johansson, 1976). Moreover, it has been proposed that GOT and MDH may form some substrate channelling for the transit of OAA in cells (Bryce et al, 1976; Arrio-Dupont et al, 1985). This may explain the absence of high OAA accumulation in rat liver cells (Williamson et al, 1969; Parrilla et al, 1975; Siess et al, 1977). However, very little is known about the formation of the GOT-MDH complex in response to metabolic changes and about the physiological significance of such a regulation. Figure 1. Acetylation of GOT2 at K159, K185, and K404 enhances its protein interaction with MDH2 A. Cartoon representation of the malate–aspartate shuttle. As shown, this shuttle is operated by two pairs of enzymes, cytosolic GOT1 and MDH1 as well as mitochondrial GOT2 and MDH2, which act in concert to transfer reducing equivalents across the mitochondrial membrane. B, C. GOT2 can be acetylated. Flag-tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag-bead-purified GOT2 were determined by Western blot analysis using a pan-anti-acetyllysine antibody (α-Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag. D. GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag-GOT2 and Myc-MDH2 were co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag-bead-purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis. E. Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag-GOT2 was co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag-GOT2 and its association with endogenous MDH2 were determined by Western blot analysis. F. Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag-MDH2 was co-overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag-MDH2 and endogenous GOT2 was determined by Western blot analysis. G. Cartoon representation of GOT2 structure (PDB ID: 3PDB) (Han et al 2011) made by Pymol (www.pymol.org). Three putative lysine residues (i.e., K159, K185, and K404) were labeled in red. See also Supplementary Fig S3. H. Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K-to-R mutants were examined for their protein association with ectopically expressed Myc-MDH2 by Western blot analysis. I. GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag-GOT2 was overexpressed in HEK293T cells without or with co-overexpression of Myc-MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag-GOT2 from the Myc-MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag-GOT2 by Western blot analysis. J. The 3KQ/R mutations do not change GOT2 enzyme activity. Wild-type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in “4”. Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison. Download figure Download PowerPoint We and others have previously discovered that lysine acetylation is an evolutionarily conserved post-translational modification in the regulation of a wide range of cellular processes, particularly in nuclear transcription and cytoplasmic metabolism (Kim et al, 2006; Choudhary et al, 2009; Zhao et al, 2010). To date, more than 4,500 acetylated proteins have been identified (Lundby et al, 2012). Among these acetylated proteins is MDH2, in which as many as four lysine residues are acetylated, and acetylation has been shown to activate MDH2 (Zhao et al, 2010). The other mitochondrial component of the malate–aspartate shuttle, GOT2, is also acetylated (Choudhary et al, 2009; Zhao et al, 2010). In this report, we investigated GOT2 acetylation and its functional significance in controlling the malate–aspartate shuttle activity in the cell. Results Acetylation of GOT2 at K159, K185 and K404 enhances its binding with MDH2 Previous proteomic analyses based on mass spectrometry have identified a large number of acetylated proteins, including GOT2 (Choudhary et al, 2009; Zhao et al, 2010). To confirm this, we transfected Flag-GOT2 into HEK293T cells and examined the acetylation of GOT2 by Western blotting using a pan-anti-acetyllysine antibody. We found that GOT2 was indeed acetylated and its acetylation was enhanced by approximately 2.5-fold after 4 h of treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases (Bitterman et al, 2002; Avalos et al, 2005) (Fig 1B). Treatment with Trichostatin A (TSA), an inhibitor of histone deacetylase HDAC I and II (Furumai et al, 2001), did not substantially affect GOT2 acetylation (Fig 1B), and additional treatment with TSA did not further change the acetylation level of GOT2 in cells co-treated with NAM (Fig 1C). These results indicate that GOT2 is acetylated and is regulated by a member of the SIRT family of deacetylases. A previous study has shown that GOT may interact with MDH both in the cytosol and in the mitochondrion (Backman & Johansson, 1976). We thus tested the protein association between ectopically expressed Flag-GOT2 and Myc-MDH2 in HEK293T cells. We found the protein interaction between Flag-GOT2 and Myc-MDH2 was extremely weak, hardly to be detected (Fig 1D). Notably, the GOT2–MDH2 association was substantially enhanced by NAM treatment (Fig 1D). Likewise, the association of Flag-GOT2 with endogenous MDH2 and that of Flag-MDH2 with endogenous GOT2 were hardly detectable in HEK293T, but were greatly enhanced by NAM treatment (Fig 1E and F). These results suggest that GOT2–MDH2 association is promoted by acetylation and negatively regulated by a SIRT-mediated deacetylation. As noted in the introduction, we have previously identified four major regulatory sites targeted by acetylation in MDH2, including K185, K301, K307, and K314 (4K) (Zhao et al, 2010). When Flag-tagged wild-type and the acetylation-deficient 4KR mutant MDH2 were overexpressed in HEK293T cells, we found that both wild-type and 4KR mutant MDH2 hardly interacted with endogenous GOT2 (Supplementary Fig S1). Interestingly, the 4KR mutant MDH2 displayed negligible response in changing acetylation level after NAM treatment, but still exhibited enhanced protein interaction with endogenous GOT2 (Supplementary Fig S1), suggesting that the observed effect of NAM on promoting GOT2–MDH2 association is most likely accomplished by increasing the acetylation of GOT2, but not MDH2. Given that GOT2 is a conserved protein among the species of Homo sapiens, Mus musculus, Xenopus tropicalis, and Danio rerio (Supplementary Fig S2), we speculated that important regulatory sites targeted by acetylation might also be conserved. Sequence alignments from diverse species revealed that the 14 putative acetylated lysine residues are invariant (Supplementary Fig S2) (Choudhary et al, 2009). To determine which lysine residue(s) plays a major role in the regulation of GOT2 and/or its association with MDH2, we divided these 14 putative acetylated residues into six groups according to their position in the structure of GOT2 (Fig 1G; Supplementary Fig S3A–E). We mutated each group of putative acetylated lysine (K) sites to arginine (R) or glutamine (Q) and examined the protein association of each group of GOT2 mutants with ectopically expressed Myc-MDH2. The K-to-R mutation retains a positive charge and is often used as a deacetylated mimic, whereas the K-to-Q mutation abolishes the positive charge and may act as a surrogate of acetylation (Megee et al, 1990). In agreement with our earlier finding in Fig 1D, the interaction between wild-type GOT2 and MDH2 was significantly enhanced by 5 h of NAM treatment (Fig 1H). Strikingly, simultaneous mutation of three putative acetylation lysine residues, K159, K185, and K404, to similarly positively charged arginine (3KR) in GOT2 disrupted its binding with MDH2 in cells even after NAM treatment (Fig 1H). The association with MDH2 was not or only mildly affected by single or double K-to-R mutation targeting K159, K185, or K404 in the GOT2 protein (Supplementary Fig S4A and B). To provide further evidence supporting the role of acetylation in promoting GOT2–MDH2 association, we generated an antibody specifically recognizing the K159-acetylated GOT2 [α-acGOT2(K159)] (Supplementary Fig S5A–C). We then transfected HEK293T cells either singularly with plasmid expressing Flag-GOT2 or both Flag-GOT2 and Myc-MDH2 plasmids. Transfected cells were treated with SIRT inhibitor NAM for 5 h prior to lysis to increase acetylated GOT2. Lysates from both cells were subjected to double immunoprecipitations, first with the Myc antibody and then with the Flag antibody in supernatants depleted by the Myc antibody. We found that Flag-GOT2 was detected in Myc precipitates from Flag-GOT2 and Myc-MDH2 co-transfected cells (top panel, lane 2, Fig 1I), but not in cells transfected with Flag-GOT2 alone (top panel, lane 1), demonstrating the specificity of GOT2–MDH2 association. Importantly, K159-acetylated GOT2 was readily detected in Myc precipitates from Flag-GOT2 and Myc-MDH2 co-transfected cells, but was not seen in supernatants of the same cell lysate after Myc depletion. This result indicates that nearly all K159-acetylated GOT2 is in the complex with MDH2. To determine whether acetylation of these three sites (K159, K185, and K404) would affect the enzyme activity of GOT2, we employed an expression system genetically encoding Nε-acetyllysine to prepare recombinant K159-acetylated GOT2 protein in E. coli (Neumann et al, 2008, 2009), which produced recombinant protein with complete acetylation at the targeted lysine residue. Using the α-acGOT2(K159) antibody, we verified the incorporation of acetyllysine at K159 (Supplementary Fig S5D). By monitoring the production of glutamate, we found that the purified GOT2K159ac displayed identical enzyme activity as compared to wild-type proteins (Supplementary Fig S5D). We then ectopically expressed and purified from E. coli both 3KR and 3KQ mutant GOT2 proteins and examined their enzymatic activity. We found that 3K mutations did not change GOT2 enzyme activity (Fig 1J). Taken together, these results suggest that GOT2 3K acetylation can enhance the protein association between GOT2 and MDH2 without affecting GOT2 enzyme activity. Glucose and glutamine promote GOT2 acetylation and GOT2–MDH2 association Both glucose and glutamine are the major carbon and energy sources for cultured mammalian cells. When Panc-1 cells were treated with high glucose or glutamine, we observed a significant increase in the mitochondrial NADH level (Supplementary Fig S6A and B). This raises the possibility that glucose or glutamine may affect the activity of the malate–aspartate shuttle activity, thereby influencing the net transfer of cytosolic NADH into mitochondria. Supporting this notion, a previous study has shown that the activity of the malate–aspartate shuttle in the rat heart was greatly elevated by glutamate, the deaminated product of glutamine (Digerness & Reddy, 1976). Moreover, a recent study has reported that inhibition of the malate–aspartate shuttle by aminooxyacetate (AOA) can hinder the effect of high glucose on increasing mitochondrial NADH (Zhao et al, 2011). These findings suggest that the malate–aspartate NADH shuttle may act as a sensor of energy status that maintains cellular energy homeostasis. We next set out to investigate whether acetylation of GOT2 at 3K is involved in the regulation of the malate–aspartate shuttle activity in cells exposed to high glucose and glutamine. In HEK293T cells transiently co-overexpressing Flag-GOT2 and Myc-MDH2, we found that glucose increased the K159 acetylation level of Flag-GOT2 in a dose-dependent manner and that the increased K159 acetylation of GOT2 was in parallel with enhanced GOT2–MDH2 association after glucose treatment (Fig 2A). Likewise, high glutamine increased the K159 acetylation level of Flag-GOT2 in a dose-dependent manner (Fig 2B), and again, the increased K159 acetylation of GOT2 was associated with enhanced GOT2–MDH2 interaction after glutamine treatment (Fig 2B). These results suggest that GOT2 3K acetylation can regulate the malate–aspartate shuttle activity through modulating GOT2–MDH2 association under physiological conditions. Figure 2. Glucose and glutamine promote GOT2 acetylation and GOT2–MDH2 association A, B. Glucose and glutamine increase GOT2 K159 acetylation and GOT2–MDH2 association in HEK293T cells. Flag-GOT2 and Myc-MDH2 were overexpressed in cells treated with increased concentrations of glucose (A) or glutamine (B) for 6 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 and its protein association with Myc-MDH2 were determined by Western blot analysis. C, D. Glucose and glutamine increase GOT2 K159 acetylation in Panc-1 cells. Flag-GOT2 was overexpressed in cells treated with different concentrations of glucose (C) and glutamine (D) for 4 h. GOT2 proteins were purified by Flag beads, and the K159 acetylation level of GOT2 was determined by Western blot analysis. Relative GOT2 K159 acetylation levels were normalized against Flag protein levels. E, F. Quantification of the percentage of K159-acetylated endogenous GOT2 in Panc-1 cells. Recombinant fully K159-acetylated GOT2 was loaded onto the same gel, together with endogenous GOT2 from Panc-1 cells treated without or with glucose (12 mM) (E) or glutamine (2 mM) (F) for 4 h. GOT2 protein and K159 acetylation were detected by Western blot. The percentages of K159 acetylation in GOT2 were calculated after normalizing against GOT2 protein levels. G, H. 3K mutant GOT2 displays negligible response in changing protein association with MDH2 after glucose or glutamine treatment. Panc-1 cells with GOT2 knockdown and re-expression of wild-type or 3K mutant GOT2 were treated with glucose (G) or glutamine (H) at the indicated concentrations for 4 h. The protein association between Flag-tagged wild-type or 3K mutant GOT2 and endogenous MDH2 was determined by Western blot analysis. Download figure Download PowerPoint Pancreatic ductal adenocarcinoma cancer (PDAC) is highly sensitive to glucose and glutamine deprivation (Ying et al, 2012; Son et al, 2013). In addition to a well-known glutamine metabolism pathway mediated by glutamate dehydrogenase (GLUD1), PDAC relies on a novel pathway for glutamine metabolism in which glutamate transaminases GOT1 and GOT2 are involved (Son et al, 2013). We next determined the regulation of GOT2 K159 acetylation by glucose and glutamine in Panc-1, a pancreatic cancer cell line. We found that the level of K159 acetylation in Flag-GOT2 was increased by as much as 3.67-fold and 3.02-fold in Panc-1 cells treated with glucose (12 mM) and glutamine (2 mM), respectively (Fig 2C and D). By using the recombinant GOT2K159ac protein purified from E. coli as the standard, we found that 14–16% of endogenous GOT2 was acetylated at K159 in Panc-1 cells in culture medium containing no glucose and glutamine, while the K159 acetylation level of endogenous GOT2 was increased to 43 and 48% when the cells were maintained with glucose (12 mM) and glutamine (2 mM), respectively (Fig 2E and F). We then generated GOT2 knockdown Panc-1 cells, in which we stably expressed GOT2 variants (Supplementary Fig S7), and found that glucose or glutamine treatment significantly increased the association of wild-type GOT2 with MDH
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