Regulation of G6PD acetylation by KAT9/SIRT2 modulates NADPH homeostasis and cell survival during oxidative stress
2014; Springer Nature; Linguagem: Inglês
10.1002/embj.201387224
ISSN1460-2075
AutoresYiping Wang, Lishi Zhou, Yuzheng Zhao, Shiwen Wang, L.-L. Chen, Lina Liu, Zhi Ling, HU Fuyou, Y.-P. Sun, J.-Y. Zhang, Chen Yang, Yi Yang, Yue Xiong, Kun‐Liang Guan, D. Ye,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArticle25 April 2014free access Regulation of G6PD acetylation by SIRT2 and KAT9 modulates NADPH homeostasis and cell survival during oxidative stress† Yi-Ping Wang Yi-Ping Wang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Li-Sha Zhou Li-Sha Zhou Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Yu-Zheng Zhao Yu-Zheng Zhao School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Shi-Wen Wang Shi-Wen Wang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Lei-Lei Chen Lei-Lei Chen Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Li-Xia Liu Li-Xia Liu Key Laboratory of Synthetic Biology, Bioinformatics Center and Laboratory of Systems Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 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 Fu-Jun Hu Fu-Jun Hu Department of Radiotherapy, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China Search for more papers by this author Yi-Ping Sun Yi-Ping Sun Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Jing-Ye Zhang Jing-Ye Zhang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Chen Yang Chen Yang Key Laboratory of Synthetic Biology, Bioinformatics Center and Laboratory of Systems Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 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 Yue Xiong Yue Xiong Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Kun-Liang Guan Kun-Liang Guan Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, 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 Dan Ye Corresponding Author Dan Ye Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Yi-Ping Wang Yi-Ping Wang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Li-Sha Zhou Li-Sha Zhou Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Yu-Zheng Zhao Yu-Zheng Zhao School of Pharmacy, East China University of Science and Technology, Shanghai, China Search for more papers by this author Shi-Wen Wang Shi-Wen Wang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Lei-Lei Chen Lei-Lei Chen Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Li-Xia Liu Li-Xia Liu Key Laboratory of Synthetic Biology, Bioinformatics Center and Laboratory of Systems Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 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 Fu-Jun Hu Fu-Jun Hu Department of Radiotherapy, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China Search for more papers by this author Yi-Ping Sun Yi-Ping Sun Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Jing-Ye Zhang Jing-Ye Zhang Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Chen Yang Chen Yang Key Laboratory of Synthetic Biology, Bioinformatics Center and Laboratory of Systems Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 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 Yue Xiong Yue Xiong Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Kun-Liang Guan Kun-Liang Guan Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, 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 Dan Ye Corresponding Author Dan Ye Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China Search for more papers by this author Author Information Yi-Ping Wang1, Li-Sha Zhou1, Yu-Zheng Zhao2, Shi-Wen Wang1, Lei-Lei Chen1, Li-Xia Liu3, Zhi-Qiang Ling4, Fu-Jun Hu5, Yi-Ping Sun1, Jing-Ye Zhang1, Chen Yang3, Yi Yang2, Yue Xiong1,6, Kun-Liang Guan1,7 and Dan Ye 1 1Key Laboratory of Molecular Medicine of Ministry of Education and Institutes of Biomedical Sciences, Shanghai Medical College, College of Life Science, Fudan University, Shanghai, China 2School of Pharmacy, East China University of Science and Technology, Shanghai, China 3Key Laboratory of Synthetic Biology, Bioinformatics Center and Laboratory of Systems Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 4Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China 5Department of Radiotherapy, Zhejiang Province Cancer Hospital, Zhejiang Cancer Center, Hangzhou, China 6Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA 7Department of Pharmacology and Moores Cancer Center, University of California San Diego, La Jolla, CA, USA *Corresponding author. Tel: +86 21 5423 7834; Fax: +86 21 5423 7450; E-mail: [email protected] The EMBO Journal (2014)33:1304-1320https://doi.org/10.1002/embj.201387224 †Correction added on 26 May 2014, after first online publication. In the article title, "KAT9/SIRT2" was corrected to "SIRT2 and KAT9". ‡See also: LE Wu & DA Sinclair (June 2014) 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 Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway (PPP) and plays an essential role in the oxidative stress response by producing NADPH, the main intracellular reductant. G6PD deficiency is the most common human enzyme defect, affecting more than 400 million people worldwide. Here, we show that G6PD is negatively regulated by acetylation on lysine 403 (K403), an evolutionarily conserved residue. The K403 acetylated G6PD is incapable of forming active dimers and displays a complete loss of activity. Knockdown of G6PD sensitizes cells to oxidative stress, and re-expression of wild-type G6PD, but not the K403 acetylation mimetic mutant, rescues cells from oxidative injury. Moreover, we show that cells sense extracellular oxidative stimuli to decrease G6PD acetylation in a SIRT2-dependent manner. The SIRT2-mediated deacetylation and activation of G6PD stimulates PPP to supply cytosolic NADPH to counteract oxidative damage and protect mouse erythrocytes. We also identified KAT9/ELP3 as a potential acetyltransferase of G6PD. Our study uncovers a previously unknown mechanism by which acetylation negatively regulates G6PD activity to maintain cellular NADPH homeostasis during oxidative stress. Synopsis The pentose phosphate pathway plays an important role in the oxidative stress response by supplying the reductant NADPH. SIRT2-mediated deacetylation and activation of the glucose-6-phosphate dehydrogenase, the rate-limiting enzyme in this pathway, stimulates the production of cytosolic NADPH to counteract oxidative damage. K403 acetylation decreases G6PD activity by inhibiting dimer formation. SIRT2 and KAT9/ELP3 regulate G6PD K403 acetylation. Regulation of G6PD K403 acetylation modulates NADPH homeostasis and cell survival during oxidative stress. Introduction Nicotinamide adenine dinucleotide phosphate (NADPH) is a functionally important metabolite that is commonly used for reductive biosynthesis and maintenance of cellular redox potential. It is a required cofactor in reductive biosynthesis of fatty acids, isoprenoids, and aromatic amino acids (Turner & Turner, 1980; Graeve et al, 1994; Hauschild & von Schaewen, 2003). NADPH is also used to keep glutathione in its reduced form. Reduced glutathione (GSH) acts as a scavenger for dangerous oxidative metabolites in the cell, and it converts harmful hydrogen peroxide to water with the help of glutathione peroxidase (GSHPx) (Margis et al, 2008). Perturbed NADPH production increases sensitivity to reactive oxygen species (ROS) and provokes apoptosis (Kim et al, 2007). Despite the functional importance of NADPH, mechanisms of maintaining cellular NADPH homeostasis are not fully understood. Numerous pathways are known to maintain cellular NADPH levels. The major NADPH-producing enzymes in the cell are glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in the pentose phosphate pathway (PPP), malic enzyme (ME) in the pyruvate cycling pathway, and isocitrate dehydrogenase (IDH) in the tricarboxylic acid (TCA) cycle (Salati & Amir-Ahmady, 2001). Activity of IDH1, ME1, and 6PGD remains unchanged during oxidative stress, while G6PD is the only NADPH-producing enzyme that is activated (Filosa et al, 2003). G6PD catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate and concomitantly reduces NADP+ to NADPH, which is the rate-limiting and primary control step of the NADPH-generating portion in the PPP. Thus, G6PD acts as a guardian of cellular redox potential during oxidative stress (Filosa et al, 2003). G6PD is highly conserved from yeast to mammalian species (Kletzien et al, 1994; Notaro et al, 2000). Yeast carries one G6PD gene, Zwf1, which when deleted causes phenotypes indicative of hypersensitivity to oxidative stress, including aerobic methionine auxotrophy and sensitivity to hydrogen peroxide (Juhnke et al, 1996; Lee et al, 1999; Blank et al, 2005). The critical function of G6PD in oxidative stress response is also conserved in mammals. Mouse embryonic stem cells with G6PD deletion display an approximately 50% reduction in the [NADPH]/[NADP+] ratio and are extremely sensitive to the lethal effects of external chemical oxidants (Pandolfi et al, 1995; Filosa et al, 2003). Furthermore, human G6PD deficiency is a common genetic abnormality found in 5–10% of the global population (Cappellini & Fiorelli, 2008). While the polymorphic mutations in G6PD affect amino acid residues throughout the enzyme and decrease the stability of the enzyme in the red blood cell, the severe mutations mostly affect residues at the dimer interface or those that interact with a structural NADP+ molecule that stabilizes the enzyme (Mason et al, 2007). The distinctive phenotype of patients with G6PD deficiency is chronic and drug- or food-induced hemolytic anemia, which is attributed to the inability to produce NADPH and withstand harmful oxidants in erythrocyte cells where other NADPH-producing enzymes are lacking (Vulliamy et al, 1993). Together, these findings further support the notion that G6PD is of central importance for NADPH homeostasis and redox regulation. 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). The acetylation state of a given protein results from the balanced action of lysine acetyltransferases (KATs) and deacetylases (KDACs), enzymes that catalyze the addition and removal, respectively, of an acetyl group from a lysine residue. In particular, KDACs including classical HDACs (histone deacetylases) and sirtuins (SIRTs) have received more and more attention not only for their physiological roles, but also for their involvement in disease states and, consequently, for being a therapeutic target (Haberland et al, 2009). Several recent acetylome proteomic studies have identified more than 4,500 acetylated proteins (Kim et al, 2006; Choudhary et al, 2009; Zhao et al, 2010; Lundby et al, 2012). Among these identified acetylated proteins is G6PD, implicating a novel regulatory mechanism of G6PD at the post-translational level. This study is directed toward identifying potential KAT and KDAC enzymes of G6PD and understanding how acetylation regulates G6PD activity to maintain cellular NADPH homeostasis and redox potential during oxidative stress. Results Lysine 403 is an important regulatory acetylation site in G6PD In a recent proteomic study (Choudhary et al, 2009), G6PD was identified to be acetylated on 7 lysine residues, including lysine 89 (K89), lysine 171 (K171), lysine 386 (K386), lysine 403 (K403), lysine 432 (K432), lysine 497 (K497), and lysine 514 (K514) (A Fig S1). Western blotting with a pan-anti-acetyllysine antibody demonstrated that G6PD was indeed acetylated and its acetylation was significantly elevated (up to ~2.5-fold) in HEK293T cells after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases (Bitterman et al, 2002; Avalos et al, 2005; Smith et al, 2008) (Fig 1A). The effect of NAM on increasing G6PD acetylation was found to be in a dose-dependent manner, while G6PD specific activity was decreased by as much as 40% after NAM treatment (Fig 1A). Treatment with trichostatin A (TSA), an inhibitor of histone deacetylase HDAC (Furumai et al, 2001), did not affect G6PD acetylation and activity (Supplementary Fig S2), and additional treatment with TSA did not further change either G6PD acetylation or its enzyme activity in cells co-treated with NAM (Fig 1A). When purified G6PD protein was incubated in vitro with bacterial deacetylase CobB (Zhao et al, 2004) in the presence of NAD+, G6PD acetylation was decreased by twofold and concomitantly its enzymatic activity was increased by as much as twofold (Fig 1B). These data suggest that acetylation negatively regulates G6PD activity. Figure 1. Acetylation negatively regulates G6PD activity G6PD acetylation inhibits its enzyme activity. Flag-tagged G6PD was expressed in HEK293T cells treated with NAM or NAM+TSA at the indicated concentrations. Acetylation levels and enzyme activity of Flag bead-purified G6PD were determined by Western blot analysis and enzyme assay, respectively. Acetylation levels were blotted with a pan-anti-acetyllysine antibody (α-Ac). Catalytic activity of affinity-purified G6PD was determined and normalized to protein levels. G6PD activity under no treatment condition was set as 100%. Shown are average values with standard deviation (s.d.) of triplicated experiments. IB and IP denote immunoblotting and immunoprecipitation, respectively. ** denotes P < 0.01 for cells treated with NAM/TSA for the indicated periods versus no NAM/TSA treatment; n.s. = not significant. G6PD acetylation levels were normalized against Flag. G6PD is activated by in vitro deacetylation. Affinity-purified Flag-tagged G6PD was incubated with recombinant CobB with or without NAD+ at 37°C for 2 h. G6PD acetylation and activity were determined. Shown are average values with standard deviation (s.d.) of triplicated experiments. ** denotes P < 0.01 for cells treated with CobB and/or NAD+ versus no treatment (NT); n.s. = not significant. G6PD acetylation levels were normalized against Flag. Mapping the major regulated sites of acetylation in G6PD. Wild-type (WT) G6PD and the indicated mutants were each expressed in HEK293T cells. Proteins were purified by IP, and specific G6PD activity was determined. Shown are average values with standard deviation (s.d.) of triplicated experiments. ** denotes P < 0.01 for cells expressing the indicated G6PD mutants versus cells expressing WT G6PD; n.s. = not significant. K403 is the important regulatory acetylation site in G6PD. Flag-tagged wild-type G6PD, the K171R, K386R, and K403R mutants were each expressed in HEK293T cells, followed by treatments with or without 15 mM NAM. Acetylation levels and activity of G6PD were determined. Shown are average values with standard deviation (s.d.) of triplicated experiments. ** denotes P < 0.01 for the indicated comparison; n.s. = not significant. G6PD acetylation levels were normalized against Flag. Download figure Download PowerPoint Because G6PD is a highly conserved protein (Kletzien et al, 1994), we speculated that important regulatory sites targeted by acetylation might be also conserved. Sequence alignments from diverse species revealed that five of the acetylated lysines (K89, K386, K432, K497, and K514) are not conserved, while two lysines (K171 and K403) are invariant (Supplementary Fig S1). To determine which lysine residue(s) plays a major role in the regulation of G6PD, we mutated each of the 7 putative acetylation sites to arginine (R) or glutamine (Q) and assayed their activity individually. The K to R mutation retains a positive charge and is often used as a deacetylated mimetic, whereas the K to Q mutation abolishes the positive charge and may act as a surrogate of acetylation (Megee et al, 1990). By continuously monitoring the generation of NADPH (Tian et al, 1998), we found that substitutions at K89, K432, K497, and K514 did not significantly affect G6PD activity as compared with wild-type G6PD (Fig 1C and D). Mutation of K171 to either arginine or glutamine led to a complete loss in G6PD catalytic activity (Fig 1C and D). Mutation of either K386 or K403 to glutamine, but not to arginine, resulted in a significant reduction in G6PD activity (Fig 1C and D). Moreover, the K386R mutant responded normally to NAM treatment regarding G6PD acetylation and enzyme activity (Fig 1E). However, the K403R mutant displayed negligible response in changing acetylation and enzyme activity upon NAM treatment (Fig 1E and Supplementary Fig S3). These results suggest that K403 is an important regulatory acetylation site which controls G6PD activity. Acetylation of K403 impairs the formation of dimeric G6PD and inhibits enzyme activity The G6PD enzyme exists as a mixture of monomer, dimer, tetramer, and hexamer, but only the dimeric and tetrameric forms are catalytic active (Cohen & Rosemeyer, 1969; Babalola et al, 1976). The structure of human G6PD reveals that each subunit contains two NADP+ binding sites, a catalytic NADP+ coenzyme-binding domain and a structural NADP+ binding domain (Au et al, 2000). The structural NADP+ binding site is distant from the catalytic site but close to the dimer interface. The interplay of the structural NADP+ and the dimer interface affects the stability and integrity of active enzyme (Au et al, 2000; Wang & Engel, 2009). A high proportion of the clinical mutations associated with severe G6PD deficiency in human clusters around the structural NADP+ site (Au et al, 2000; Vulliamy & Luzzatto, 2003), suggesting that the structural NADP+ is vital for G6PD activity and function. Notably, K171 lies in the catalysis pocket of G6PD and directly binds with both G6P and catalytic NADP+, while K386 and K403 lie close to the dimer interface (Supplementary Fig S4). In particular, K403 physically interacts with structural NADP+ (Fig 2A). Therefore, it is possible that K171Q mutation would abolish G6PD activity through directly influencing the substrate recognition and/or catalysis, while K386Q and K403Q mutations would inhibit G6PD activity through impairing the integrity of dimer interface and subsequently the formation of active dimers and higher forms of G6PD. Figure 2. K403 acetylation impairs the formation of dimeric G6PD and inhibits its enzyme activity A. Cartoon representation of G6PD structure (PDB ID: 2BH9) (Kotaka et al, 2005) made by using Pymol (www.pymol.org). Upper left, electrostatic (G6PD) and stick (NADP+) representation of the crystal structure of G6PD bound to NADP+. Upper right, a closer view showing the structural NADP+ bound to a positive charged groove of G6PD. Lower panel, shown is a ribbon (G6PD)-and-stick (structural NADP+) representation of G6PD bound to the structural NADP+. The protein is colored in light green and NADP+ in yellow, with interacting residues on G6PD colored in magenta. Arrow indicates the interaction between Lys 403 and the structural NADP+. B. Acetylation at K403 blocks the protein-protein binding between wild-type G6PD. Flag-tagged G6PD, KR/KQ mutants of K171, K386, and K403 were each expressed in HEK293T cells co-expressing GFP-tagged G6PD. The interaction between Flag-tagged and GFP-tagged proteins was determined by Western blotting. C. K403Q mutation, but not K403R, inhibits the formation of dimeric G6PD. Flag-tagged G6PD, G6PDK403R, and G6PDK403Q were each expressed in HEK293T cells, followed by treatments with or without 0.025% glutaraldehyde. The formation of G6PD monomer and dimer was determined by Western blotting. D. NAM treatment impairs the protein-protein interaction between WT G6PD, but not the K403R mutant. Flag-tagged G6PD and G6PDK403R were each expressed in HEK293T cells co-expressing GFP-tagged G6PD and G6PDK403R, respectively. Cells were treated with or without 15 mM NAM, and the interaction between Flag-tagged and GFP-tagged proteins was determined by Western blotting. E, F. In vitro site-specific incorporation of acetylated K403 in G6PD. His-tagged G6PD and G6PDK403ac were recombinantly expressed and detected by Western blot with an anti-His antibody as well as a site-specific anti-acetyllysine antibody [α-acG6PD(K403)]. G6PD activity assay was performed using the purified unacetylated G6PD and G6PDK403ac proteins (E). Moreover, the purified unacetylated G6PD and G6PDK403ac proteins were treated with or without 0.025% glutaraldehyde, and the formation of G6PD monomer and dimer was tested by Western blotting (F). Purified Flag-tagged G6PDK403Q was used as a control. ** denotes P < 0.01 for the purified G6PDK403ac protein versus the unacetylated G6PD protein. Download figure Download PowerPoint To test this hypothesis, we determined the interaction between two differentially tagged G6PD proteins, G6PD-Flag and GFP-G6PD, in HEK293T cells. We found that mutation of K171 to either R (K171R) or Q (K171Q) did not affect the interaction between G6PD subunits (Fig 2B). In contrast, substitution of K386Q, but not K386R, impaired the interaction between G6PD subunits (Fig 2B). Strikingly, substitution of K403Q, but not K403R, entirely disrupted the interaction between G6PD subunits (Fig 2B). In addition, glutaraldehyde cross-linking assay demonstrated that the K403Q mutant displayed impaired ability to form dimers when compared to wild-type G6PD or the K403R mutant (Fig 2C). Moreover, NAM treatment decreased the binding by approximately 55% between the two differentially tagged proteins of wild-type G6PD in a dose-dependent manner (Fig 2D and Supplementary Fig S5). NAM treatment, however, failed to affect the interaction between Flag-tagged and GFP-tagged K403R mutant of G6PD (Fig 2D), further suggesting that K403 acetylation largely hinders the interaction between G6PD subunits. To unambiguously determine the effect of K403 acetylation on G6PD, we employed an expression system genetically encoding Nε-acetyllysine to prepare recombinant proteins in E. coli (Neumann et al, 2008, 2009). This expression system produced G6PD proteins with 100% acetylation at the targeted lysine residue. Only with Nε-acetyllysine in the growth medium was the full-length protein formed (Supplementary Fig S6). Moreover, we generated and verified an antibody specifically recognizing the K403 acetylated G6PD [α-acG6PD(K403)] (Supplementary Fig S7). The incorporated acetyllysine was confirmed by immunoblotting of the purified G6PDK403ac protein with this site-specific α-acG6PD(K403) antibody. Importantly, as compared to wild-type and K403R/Q mutants of G6PD, the recombinant G6PDK403ac protein displayed an identical pattern of proteolytic cleavage after treatments with proteases, chymotrypsin and clostripain (Supplementary Fig S8B). Moreover, the G6PDK403ac protein exhibited normal thermodynamic stability of protein folding when compared to wild-type and K403R/Q mutants of G6PD (Supplementary Fig S8C). This purified G6PDK403ac protein was catalytic inactive (Fig 2E and Supplementary Fig S8A), unequivocally demonstrating that acetylation of K403 inactivates G6PD. Furthermore, our data demonstrated that the G6PDK403ac protein was defective in dimer formation (Fig 2F). Together, these results clearly indicate that acetylation at K403 impairs the formation of dimeric G6PD and inhibits its enzyme activity. KAT9/ELP3 is involved in G6PD K403 acetylation and enzymatic inactivation Next, we set out to search for potential KAT(s) responsible for G6PD K403 acetylation. To this end, we generated a siRNA library with three siRNAs targeting each of the 19 human KATs (Allis et al, 2007), and then determined the activity of endogenous G6PD in HEK293T cells with transient knockdown of individual KAT gene. The knockdown efficiency of each siRNA was determined by quantitative RT-PCR of its target gene (Fig 3A). We found that knocking down most of the examined KAT genes did not substantially affect the enzyme activity of endogenous G6PD (Fig 3B). With one exception, knocking down KAT9 (also known as ELP3), which encodes the catalytic subunit of the histone acetyltransferase elongator complex and has previously been identified as an α-tubulin acetyltransferase in mouse neurons (Creppe et al, 2009), significantly stimulated the activity of endogenous G6PD (Fig 3B). Moreover, the degree of G6PD activation appeared to correlate with the KAT9 knockdown efficiency, as siRNA no. 2 and no. 3 were more potent in both KAT9 knockdown and G6PD activation than the siRNA no. 1. As expected, transient knockdown of KAT9 decreased the K403 acetylation levels of endogenous G6PD without changing its protein expression (Fig 3C and D), further supporting the notion that KAT9 is the potential acetyltransferase of G6PD. Figure 3. KAT9/ELP3 is the potential acetyltransferase of G6PD A. A siRNA library with three siRNAs targeting each of the 19 known HAT genes was generated. Each siRNA oligonucleotide was transiently transfected into HEK293T cells, and mRNA expression of HAT genes was determined by quantitative real-time PCR at 48 h post-transfection. B. HEK293T cells were transfected as described in (A) and then were subjected
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