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PreprintCited by 28Redox biology and oxidative stressUbiquitin and proteasome pathwaysCancer-related gene regulation

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Acetylation-dependent coupling between G6PD activity and apoptotic signaling

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Wu, Fang, Muskat, Natali H., Dvilansky, Inbar, Koren, Omri, Shahar, Anat, Gazit, Roi, Elia, Natalie, Arbely, Eyal·2023·Nature Communications
DOI 10.1101/2023.02.19.529048PMC PMC10556143

Preprint — published as 10.1038/s41467-023-41895-2

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Abstract

Lysine acetylation has been discovered in thousands of non-histone human proteins, including most metabolic enzymes. Deciphering the functions of acetylation is key to understanding how metabolic cues mediate metabolic enzyme regulation and cellular signaling. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is acetylated on multiple lysine residues. Using site-specifically acetylated G6PD, we show that acetylation can activate (AcK89) and inhibit (AcK403) G6PD. Acetylation-dependent inactivation is explained by structural studies showing distortion of the dimeric structure and active site of G6PD. We provide evidence for acetylation-dependent K95/97 ubiquitylation of G6PD and Y503 phosphorylation, as well as interaction with p53 and induction of early apoptotic events. Notably, we found that the acetylation of a single lysine residue coordinates diverse acetylation-dependent processes. Our data provide an example of the complex roles of acetylation as a posttranslational modification that orchestrates the regulation of enzymatic activity, posttranslational modifications, and apoptotic signaling

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Fig. 1 Site-specific acetylation modulates the catalytic activity of G6PD in cultured mammalian cells. a Inhibition of KDACs downregulates G6PD activity. Bars represent the relative Vmax of endogenous G6PD measured in cleared lysates of HEK293T and HCT116 cells cultured in the presence or absence of KDACi. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 biologically independent samples. b Crystal structure of dimeric human G6PD (PDB ID: 6E08). Putative lysine acetylation sites selected for this study are presented in sticks model. G6P (predicted position) and structural NADP+ are displayed as spheres. The main focus of this study is on residues K89 and K403, which are highlighted in bold. c Acetylation of specific lysine residues is dynamic and differentially regulated by KDACs. Immunoblots show the acetylation level of immunopurified acetylated G6PD-Flag variants, expressed in HEK293T cells cultured in the presence (+) or absence (−) of KDACi. d Site-specific acetylation modulates G6PD activity. Bars represent the relative Vmax of exogenous full-length G6PD measured in cell lysate. Indicated acetylated G6PD variants were expressed in HEK293T (left) and HCT116 (right) cells cultured in the absence (top) or presence (bottom) of KDACi. G6PD activity was normalized to immunoblot intensities of expressed G6PD and displayed relative to AcK414 (pWT G6PD). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 4 or 6 (HEK293T), 3, 4, or 5 (HCT116) biologically independent samples. ND, not determined. Source data are provided as a Source Data file.
Fig. 1 Site-specific acetylation modulates the catalytic activity of G6PD in cultured mammalian cells. a Inhibition of KDACs downregulates G6PD activity. Bars represent the relative Vmax of endogenous G6PD measured in cleared lysates of HEK293T and HCT116 cells cultured in the presence or absence of KDACi. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 biologically independent samples. b Crystal structure of dimeric human G6PD (PDB ID: 6E08). Putative lysine acetylation sites selected for this study are presented in sticks model. G6P (predicted position) and structural NADP+ are displayed as spheres. The main focus of this study is on residues K89 and K403, which are highlighted in bold. c Acetylation of specific lysine residues is dynamic and differentially regulated by KDACs. Immunoblots show the acetylation level of immunopurified acetylated G6PD-Flag variants, expressed in HEK293T cells cultured in the presence (+) or absence (−) of KDACi. d Site-specific acetylation modulates G6PD activity. Bars represent the relative Vmax of exogenous full-length G6PD measured in cell lysate. Indicated acetylated G6PD variants were expressed in HEK293T (left) and HCT116 (right) cells cultured in the absence (top) or presence (bottom) of KDACi. G6PD activity was normalized to immunoblot intensities of expressed G6PD and displayed relative to AcK414 (pWT G6PD). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 4 or 6 (HEK293T), 3, 4, or 5 (HCT116) biologically independent samples. ND, not determined. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 2 Lysine 403 acetylation inhibits and destabilizes G6PD in vitro and in cells. a Purified site-specifically acetylated full-length G6PD expressed in E. coli. b Site-specific acetylation significantly affects the catalytic activity of recombinant G6PD. Bars represent the mean apparent kinetic parameters (obtained by fitting the data to the Michaelis–Menten equation) ± fitting error; n = 8 (Vmax), or 4 (KM^app) independent experiments (Supplementary Fig. 4a, b). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. * p < 1 × 10−4. c Acetylation of K403 destabilizes the dimeric structure of G6PD. Indicated purified G6PD variants were cross-linked, resolved by SDS-PAGE, and visualized by immunoblotting using an antibody against the C-terminal 6×His tag. Bars represent the percentage of dimeric G6PD out of the total G6PD population [monomeric (Mr: ~59 KDa), and dimeric (Mr: ~120 KDa)]. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 independent experiments. d Lysine 403 acetylation reduces the thermodynamic stability of G6PD. Bars represent mean T1/2 values ± SD, measured at increasing concentrations of NADP+ (Supplementary Fig. 5); n = 3 (WT, AcK89, AcK386), or 4 (AcK403, AcK408, AcK432, AcK497). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. e Lysine 403 acetylation increases the in vitro aggregation propensity of G6PD. Curves represent the accumulation of G6PD aggregates as a function of time and indicated concentration of NADP+. Aggregation was monitored by measuring the increase in scattered 340 nm light. Data are the mean ± SD, n = 3 independent experiments. f Lysine 403 acetylation increases the proteolytic degradation rate of G6PD in vitro. Western blots show the amount of indicated G6PD variants following chymotrypsin (Chy) digestion as a function of time (top) or as a function of chymotrypsin concentration (bottom). g Lysine 403 acetylation destabilizes G6PD in cells. Curves represent the relative amounts of G6PD variants in HCT116 cells as a function of time after CHX treatment. Data are the mean ± SD, n = 3 (WT, AcK403) or 5 (pWT, AcK89) biologically independent samples. ND not determined. Source data are provided as a Source Data file.
Fig. 2 Lysine 403 acetylation inhibits and destabilizes G6PD in vitro and in cells. a Purified site-specifically acetylated full-length G6PD expressed in E. coli. b Site-specific acetylation significantly affects the catalytic activity of recombinant G6PD. Bars represent the mean apparent kinetic parameters (obtained by fitting the data to the Michaelis–Menten equation) ± fitting error; n = 8 (Vmax), or 4 (KM^app) independent experiments (Supplementary Fig. 4a, b). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. * p < 1 × 10−4. c Acetylation of K403 destabilizes the dimeric structure of G6PD. Indicated purified G6PD variants were cross-linked, resolved by SDS-PAGE, and visualized by immunoblotting using an antibody against the C-terminal 6×His tag. Bars represent the percentage of dimeric G6PD out of the total G6PD population [monomeric (Mr: ~59 KDa), and dimeric (Mr: ~120 KDa)]. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 independent experiments. d Lysine 403 acetylation reduces the thermodynamic stability of G6PD. Bars represent mean T1/2 values ± SD, measured at increasing concentrations of NADP+ (Supplementary Fig. 5); n = 3 (WT, AcK89, AcK386), or 4 (AcK403, AcK408, AcK432, AcK497). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. e Lysine 403 acetylation increases the in vitro aggregation propensity of G6PD. Curves represent the accumulation of G6PD aggregates as a function of time and indicated concentration of NADP+. Aggregation was monitored by measuring the increase in scattered 340 nm light. Data are the mean ± SD, n = 3 independent experiments. f Lysine 403 acetylation increases the proteolytic degradation rate of G6PD in vitro. Western blots show the amount of indicated G6PD variants following chymotrypsin (Chy) digestion as a function of time (top) or as a function of chymotrypsin concentration (bottom). g Lysine 403 acetylation destabilizes G6PD in cells. Curves represent the relative amounts of G6PD variants in HCT116 cells as a function of time after CHX treatment. Data are the mean ± SD, n = 3 (WT, AcK403) or 5 (pWT, AcK89) biologically independent samples. ND not determined. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 3 Lysine 89 acetylation promotes partial unwinding of helix αb’ and ubiquitylation of residues K95/97. a Lysine 89 acetylation promotes ubiquitylation of G6PD. Representative Western blots showing the ubiquitylation level of immunoprecipitated Flag-tagged G6PD variants, co-expressed with HA-tagged ubiquitin in HEK293T cells treated with 5 μM MG132 for 16 h. b Mutation of lysine 95 and 97 to arginine does not affect G6PD activity. Bars represent the relative Vmax of pWT, AcK89, and indicated Lys-to-Arg G6PD mutants, measured in lysates of cells incubated in the absence of KDACi, and displayed relative to pWT G6PD. Data are the mean ± SD, n = 3 biologically independent samples. c AcK89 G6PD is ubquitylated on residues K95/97. Representative Western blots showing the ubiquitylation level of immunoprecipitated Flag-tagged G6PD variants, co-expressed with HA-tagged ubiquitin in HEK293T cells treated with 5 μM MG132 for 16 h. d Lysine 89 acetylation has minimal effect on the three-dimensional structure of monomeric G6PD. Superposition of AcK89 G6PD (green) and non-acetylated G6PD (cyan, PDB ID: 6E08). Structural NADP+ and K89 are shown in sticks model. e Lysine 89 acetylation promotes local conformational changes. Left: zoom in on K89 and nearby helices, showing the position of AcK89 (green) relative to the position of K89 (cyan, PDB ID: 6E08). Right: electron density map around residue AcK89. The 2Fo–Fc map was contoured at 1 σ. f Lysine 89 acetylation promotes partial unwinding of helix αb'. Local conformational changes around AcK89 with the partial unwinding of helix αb' (green), relative to the structure of non-acetylated G6PD (cyan, PDB ID: 6E08). Source data are provided as a Source Data file.
Fig. 3 Lysine 89 acetylation promotes partial unwinding of helix αb’ and ubiquitylation of residues K95/97. a Lysine 89 acetylation promotes ubiquitylation of G6PD. Representative Western blots showing the ubiquitylation level of immunoprecipitated Flag-tagged G6PD variants, co-expressed with HA-tagged ubiquitin in HEK293T cells treated with 5 μM MG132 for 16 h. b Mutation of lysine 95 and 97 to arginine does not affect G6PD activity. Bars represent the relative Vmax of pWT, AcK89, and indicated Lys-to-Arg G6PD mutants, measured in lysates of cells incubated in the absence of KDACi, and displayed relative to pWT G6PD. Data are the mean ± SD, n = 3 biologically independent samples. c AcK89 G6PD is ubquitylated on residues K95/97. Representative Western blots showing the ubiquitylation level of immunoprecipitated Flag-tagged G6PD variants, co-expressed with HA-tagged ubiquitin in HEK293T cells treated with 5 μM MG132 for 16 h. d Lysine 89 acetylation has minimal effect on the three-dimensional structure of monomeric G6PD. Superposition of AcK89 G6PD (green) and non-acetylated G6PD (cyan, PDB ID: 6E08). Structural NADP+ and K89 are shown in sticks model. e Lysine 89 acetylation promotes local conformational changes. Left: zoom in on K89 and nearby helices, showing the position of AcK89 (green) relative to the position of K89 (cyan, PDB ID: 6E08). Right: electron density map around residue AcK89. The 2Fo–Fc map was contoured at 1 σ. f Lysine 89 acetylation promotes partial unwinding of helix αb'. Local conformational changes around AcK89 with the partial unwinding of helix αb' (green), relative to the structure of non-acetylated G6PD (cyan, PDB ID: 6E08). Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 4 Lysine 403 acetylation is regulated by Sirt1 and Sirt2. a The effect of acetylation on G6PD activity is maintained following the inhibition of endogenous KDACs. Bars represent relative Vmax of acetylated G6PD variants expressed in HEK293T cells cultured in the presence or absence of indicated KDACi. The activity was measured in cleared total cell lysates, corrected to G6PD expression level, and normalized to the activity of pWT expressed without KDACi treatment. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 4 (AcK171), 5 or 6 (AcK403), 6 (AcK89), or 9 (pWT) biologically independent samples. b Lysine 403 acetylation is stabilized upon inhibition of endogenous NAD+-dependent deacetylases. Representative Western blots showing the acetylation level of immunoprecipitated AcK89 and AcK403 G6PD, expressed in HEK293T cells cultured in the presence or absence of indicated KDACi. c The inhibitory effect of K403 acetylation is alleviated by NAD+-dependent deacetylases in vitro. Bars represent the relative Vmax of recombinant G6PD incubated with cleared lysates of HEK293T cells pretreated with indicated KDACi [mean ± SD; n = 4 (AcK89), or 5 (AcK403) independent experiments]. Western blots show the amount of G6PD (anti 6×His) and acetylation level following incubation with cleared cell lysates. d Lysine 403-acetylated G6PD is a substrate of Sirt1 and Sirt2. Western blots show the acetylation level of recombinant WT, AcK89, and AcK403 G6PD, following incubation with immunoprecipitated Sirt1, Sirt2, or HDAC6, in the presence or absence of indicated KDACi. e Sirt1 and Sirt2 reactivate AcK403 G6PD. Bars represent the relative Vmax of recombinant WT and AcK403 G6PD, following incubation with deacetylases as described in d. Data are the mean ± SD; n = 3 (Sirt1 treatment), or 7 (Sirt2, HDAC6 treatment) independent experiments. f Sirt1 and Sirt2 deacetylate AcK403 in a time-dependent manner. Representative Western blots showing K403 acetylation level as a function of incubation time with immunoprecipitated Sirt1, Sirt2, or HDAC6. Source data are provided as a Source Data file.
Fig. 4 Lysine 403 acetylation is regulated by Sirt1 and Sirt2. a The effect of acetylation on G6PD activity is maintained following the inhibition of endogenous KDACs. Bars represent relative Vmax of acetylated G6PD variants expressed in HEK293T cells cultured in the presence or absence of indicated KDACi. The activity was measured in cleared total cell lysates, corrected to G6PD expression level, and normalized to the activity of pWT expressed without KDACi treatment. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 4 (AcK171), 5 or 6 (AcK403), 6 (AcK89), or 9 (pWT) biologically independent samples. b Lysine 403 acetylation is stabilized upon inhibition of endogenous NAD+-dependent deacetylases. Representative Western blots showing the acetylation level of immunoprecipitated AcK89 and AcK403 G6PD, expressed in HEK293T cells cultured in the presence or absence of indicated KDACi. c The inhibitory effect of K403 acetylation is alleviated by NAD+-dependent deacetylases in vitro. Bars represent the relative Vmax of recombinant G6PD incubated with cleared lysates of HEK293T cells pretreated with indicated KDACi [mean ± SD; n = 4 (AcK89), or 5 (AcK403) independent experiments]. Western blots show the amount of G6PD (anti 6×His) and acetylation level following incubation with cleared cell lysates. d Lysine 403-acetylated G6PD is a substrate of Sirt1 and Sirt2. Western blots show the acetylation level of recombinant WT, AcK89, and AcK403 G6PD, following incubation with immunoprecipitated Sirt1, Sirt2, or HDAC6, in the presence or absence of indicated KDACi. e Sirt1 and Sirt2 reactivate AcK403 G6PD. Bars represent the relative Vmax of recombinant WT and AcK403 G6PD, following incubation with deacetylases as described in d. Data are the mean ± SD; n = 3 (Sirt1 treatment), or 7 (Sirt2, HDAC6 treatment) independent experiments. f Sirt1 and Sirt2 deacetylate AcK403 in a time-dependent manner. Representative Western blots showing K403 acetylation level as a function of incubation time with immunoprecipitated Sirt1, Sirt2, or HDAC6. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 5 Lysine 403 acetylation promotes structural changes at the dimer interface and the active site. a Dimeric structure of AcK403 G6PD. The expected position of catalytic NADP+ (light blue), structural NADP+ (light orange), and G6P (light purple) are presented as transparent spheres, based on superposition of AcK403 G6PD structure with structures of WT and mutant G6PD (PDB ID: 6VA7, 6E08, and 2BHL)47,49,73. Acetylated lysine residue 403 is highlighted in red. b Lysine 403 acetylation affects the dimeric structure of G6PD. Space-filling model of dimeric AcK403 G6PD (left) and WT G6PD (center, PDB ID: 6E08), with residue Glu93 highlighted in red. Right: The orientation of the lower monomers (darker shades) relative to the upper monomers (lighter shades) is compared by superimposing the upper monomers of each dimeric structure. The distance between Cα atoms of Glu93 residues is shown. c Lysine 403 acetylation is accompanied by distortion of βN and unwinding of αl. Superposition of AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 6E08), with a close-up view of the structural NADP+ binding site. Lysine 403 is presented in sticks model. d Close-up view of helices αf and αa' in AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 6E08). Backbone hydrogen bonds between residues V5–L7 and Y428–K432 that form a β-sheet-like structure are shown in gray. Residues L16, L20, and L442 that form a hydrophobic core between helices αa' and αm are rendered in sticks model. Also shown is residue K205, which rotates by ~180° following K403 acetylation and unfolding of helix αf'. e Lysine 403 acetylation promotes long-range conformational changes in the active site. Close-up view of the G6P binding site in AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 2BHL). The position of the substrate G6P in WT G6PD is shown, together with polar interactions with residues H201, Y202, and K205 (gray).
Fig. 5 Lysine 403 acetylation promotes structural changes at the dimer interface and the active site. a Dimeric structure of AcK403 G6PD. The expected position of catalytic NADP+ (light blue), structural NADP+ (light orange), and G6P (light purple) are presented as transparent spheres, based on superposition of AcK403 G6PD structure with structures of WT and mutant G6PD (PDB ID: 6VA7, 6E08, and 2BHL)47,49,73. Acetylated lysine residue 403 is highlighted in red. b Lysine 403 acetylation affects the dimeric structure of G6PD. Space-filling model of dimeric AcK403 G6PD (left) and WT G6PD (center, PDB ID: 6E08), with residue Glu93 highlighted in red. Right: The orientation of the lower monomers (darker shades) relative to the upper monomers (lighter shades) is compared by superimposing the upper monomers of each dimeric structure. The distance between Cα atoms of Glu93 residues is shown. c Lysine 403 acetylation is accompanied by distortion of βN and unwinding of αl. Superposition of AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 6E08), with a close-up view of the structural NADP+ binding site. Lysine 403 is presented in sticks model. d Close-up view of helices αf and αa' in AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 6E08). Backbone hydrogen bonds between residues V5–L7 and Y428–K432 that form a β-sheet-like structure are shown in gray. Residues L16, L20, and L442 that form a hydrophobic core between helices αa' and αm are rendered in sticks model. Also shown is residue K205, which rotates by ~180° following K403 acetylation and unfolding of helix αf'. e Lysine 403 acetylation promotes long-range conformational changes in the active site. Close-up view of the G6P binding site in AcK403 G6PD (green) and WT G6PD (cyan, PDB ID: 2BHL). The position of the substrate G6P in WT G6PD is shown, together with polar interactions with residues H201, Y202, and K205 (gray).CC BY · PubMed Central
Fig. 6 p53 modulates Fyn-dependent phosphorylation as a function of K403 acetylation. a Y401 and Y503 are major Fyn phosphorylation sites in pWT and AcK403 G6PD, respectively. Representative Western blots showing tyrosine phosphorylation of immunoprecipitated G6PD co-expressed with Fyn. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and presented as mean values ± SD; n = 3 biologically independent samples. b G6PD is phosphorylated by Fyn in vitro. Bars represent the relative phosphorylation level of recombinant G6PD, following in vitro incubation with Fyn. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 5 independent experiments. c Acetylation of G6PD K403 promotes the interaction with p53. Top: Co-IP using HA-tagged p53. Bottom: Reciprocal binding using 6×His-tagged G6PD. d AcK403 G6PD stabilizes p53 in cells. Bars represent the relative immunoblot intensities of endogenous p53, 120 min post-CHX treatment (Supplementary Fig. 10b). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 (pWT, AcK89, AcK171), or 4 (GFP, AcK403) biologically independent samples. e, f G6PD K403 acetylation-dependent interaction with p53' regulates Fyn-mediated G6PD phosphorylation at substoichiometric amounts of p53'. Representative Western blot (e) and quantification (f) of G6PD and p53' phosphorylation levels following in vitro incubation with active Fyn at indicated p53':G6PD molar ratios. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 6 or 7 independent experiments (Supplementary Fig. 10e). g p53' impairs Fyn-dependent G6PD phosphorylation at stoichiometric p53':G6PD ratio. Representative Western blot showing p53' and G6PD phosphorylation levels, following incubation with active Fyn. h G6PD K403 acetylation results in increased Bax levels and decreased Bcl2 levels in HEK293T cells. i G6PD K403 acetylation correlates with increased cleaved caspase-3 levels in HEK293T cells. j Annexin V/DAPI double staining of HEK293T cells expressing pWT or AcK403. Data were analyzed using two-sided t-test and are presented as mean values ± SD; n = 3 biologically independent samples. ns not significant, ND not detected. Source data are provided as a Source Data file.
Fig. 6 p53 modulates Fyn-dependent phosphorylation as a function of K403 acetylation. a Y401 and Y503 are major Fyn phosphorylation sites in pWT and AcK403 G6PD, respectively. Representative Western blots showing tyrosine phosphorylation of immunoprecipitated G6PD co-expressed with Fyn. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and presented as mean values ± SD; n = 3 biologically independent samples. b G6PD is phosphorylated by Fyn in vitro. Bars represent the relative phosphorylation level of recombinant G6PD, following in vitro incubation with Fyn. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 5 independent experiments. c Acetylation of G6PD K403 promotes the interaction with p53. Top: Co-IP using HA-tagged p53. Bottom: Reciprocal binding using 6×His-tagged G6PD. d AcK403 G6PD stabilizes p53 in cells. Bars represent the relative immunoblot intensities of endogenous p53, 120 min post-CHX treatment (Supplementary Fig. 10b). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 3 (pWT, AcK89, AcK171), or 4 (GFP, AcK403) biologically independent samples. e, f G6PD K403 acetylation-dependent interaction with p53' regulates Fyn-mediated G6PD phosphorylation at substoichiometric amounts of p53'. Representative Western blot (e) and quantification (f) of G6PD and p53' phosphorylation levels following in vitro incubation with active Fyn at indicated p53':G6PD molar ratios. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test and are presented as mean values ± SD; n = 6 or 7 independent experiments (Supplementary Fig. 10e). g p53' impairs Fyn-dependent G6PD phosphorylation at stoichiometric p53':G6PD ratio. Representative Western blot showing p53' and G6PD phosphorylation levels, following incubation with active Fyn. h G6PD K403 acetylation results in increased Bax levels and decreased Bcl2 levels in HEK293T cells. i G6PD K403 acetylation correlates with increased cleaved caspase-3 levels in HEK293T cells. j Annexin V/DAPI double staining of HEK293T cells expressing pWT or AcK403. Data were analyzed using two-sided t-test and are presented as mean values ± SD; n = 3 biologically independent samples. ns not significant, ND not detected. Source data are provided as a Source Data file.CC BY · PubMed Central
Fig. 7 Suggested model for the effect of K89 and K403 acetylation on activity and cellular functions of G6PD. Left: Acetylation of K89 enhances the catalytic activity of G6PD and promotes its ubiquitylation on residues K95/K97. Sirt1 and Sirt2 can potentially deacetylate AcK89. Right: K403 is acetylated by KAT9 (Wang et al.34) and CBP (this work) and deacetylated by Sirt2 (Wang et al.34 and this work) and Sirt1 (this work). Acetylation of K403 inhibits G6PD activity and promotes the interaction with p53, leading to the stabilization of p53 and induction of pro-apoptotic signaling. K403 acetylation also promotes Fyn-dependent phosphorylation of Y503, which can be partially inhibited by the interaction between K403-acetylated G6PD and p53. Y503 phosphorylation has no effect on K403 deacetylation by Sirt1 and Sirt2. Created with BioRender.com.
Fig. 7 Suggested model for the effect of K89 and K403 acetylation on activity and cellular functions of G6PD. Left: Acetylation of K89 enhances the catalytic activity of G6PD and promotes its ubiquitylation on residues K95/K97. Sirt1 and Sirt2 can potentially deacetylate AcK89. Right: K403 is acetylated by KAT9 (Wang et al.34) and CBP (this work) and deacetylated by Sirt2 (Wang et al.34 and this work) and Sirt1 (this work). Acetylation of K403 inhibits G6PD activity and promotes the interaction with p53, leading to the stabilization of p53 and induction of pro-apoptotic signaling. K403 acetylation also promotes Fyn-dependent phosphorylation of Y503, which can be partially inhibited by the interaction between K403-acetylated G6PD and p53. Y503 phosphorylation has no effect on K403 deacetylation by Sirt1 and Sirt2. Created with BioRender.com.CC BY · PubMed Central

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Cites

Research works printed but not linked: 71

  1. 10.1016/j.molcel.2013.07.002 · DOI · Paper page
  2. 10.1016/j.molcel.2011.04.028 · DOI
  3. 10.1073/pnas.1505995112 · DOI
  4. 10.1073/pnas.1914072117 · DOI
  5. 10.1126/science.1179687 · DOI · Paper page
  6. 10.1126/science.1179689 · DOI · Paper page
  7. 2018, Dynamic Acetylation of Phosphoenolpyruvate Carboxykinase Toggles Enzyme Activity between Gluconeogenic and Anaplerotic Reactions, Mol. Cell, 71, 718, 10.1016/j.molcel.2018.07.031 · DOI
  8. 10.1074/jbc.M111.317404 · DOI
  9. 10.1016/j.bj.2020.08.001 · DOI
  10. 2012, Overexpression of G6PD is associated with poor clinical outcome in gastric cancer, Tumour Biol, 33, 95, 10.1007/s13277-011-0251-9 · DOI
  11. 2015, Overexpression of G6PD is associated with high risks of recurrent metastasis and poor progression-free survival in primary breast carcinoma, World J. Surg. Oncol, 13, 1
  12. 10.1110/ps.035352.108 · DOI
  13. 2022, Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity, Proc. Natl. Acad. Sci. USA, 119, e2119695119, 10.1073/pnas.2119695119 · DOI
  14. 10.1038/ncomms9468 · DOI
  15. 2017, Polo-like kinase 1 coordinates biosynthesis during cell cycle progression by directly activating pentose phosphate pathway, Nat. Commun, 8, 1506, 10.1038/s41467-017-01647-5 · DOI
  16. 10.1074/jbc.M007505200 · DOI
  17. Meng, Q. et al. Recent findings in the regulation of G6PD and its role in diseases. Front. Pharmacol. 13, 932154 (2022). · DOI
  18. 10.1126/science.1175371 · DOI · Paper page
  19. 10.1074/mcp.O114.047555 · DOI
  20. 10.1016/j.celrep.2013.07.024 · DOI
  21. 10.1016/j.molcel.2012.01.026 · DOI
  22. 10.1038/nmeth.2518 · DOI
  23. 10.1016/j.molcel.2015.05.006 · DOI
  24. 10.1074/mcp.M112.019547 · DOI
  25. 10.1016/j.cell.2018.04.033 · DOI
  26. 10.1016/j.celrep.2012.07.006 · DOI
  27. 2021, COXIV and SIRT2-mediated G6PD deacetylation modulate ROS homeostasis to extend pupal lifespan, FEBS J, 288, 2436, 10.1111/febs.15592 · DOI
  28. 10.1074/mcp.M117.067587 · DOI
  29. Ahn, D. et al. An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection. Cell Rep. 35, 109196 (2021). · DOI
  30. 10.1002/pmic.201200001 · DOI
  31. Weinert, B. T. et al. Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation. Sci. Signal. 4, ra48–ra48 (2011). · DOI
  32. 2016, Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites, Mol. Med. Rep, 14, 1726, 10.3892/mmr.2016.5449 · DOI
  33. 10.1002/embj.201387224 · DOI
  34. 2021, TSP50 promotes hepatocyte proliferation and tumour formation by activating glucose-6-phosphate dehydrogenase (G6PD), Cell Prolif, 54, e13015, 10.1111/cpr.13015 · DOI
  35. 10.1038/s41598-016-0001-8 · DOI · Paper page
  36. 2021, Silent information regulator 2 promotes clear cell renal cell carcinoma progression through deacetylation and small ubiquitin-related modifier 1 modification of glucose 6-phosphate dehydrogenase, Cancer Sci, 112, 4075, 10.1111/cas.15085 · DOI
  37. Garcia, A. A. et al. Stabilization of glucose-6-phosphate dehydrogenase oligomers enhances catalytic activity and stability of clinical variants. J. Biol. Chem. 298, 101610 (2022). · DOI
  38. 10.1016/j.molcel.2009.07.027 · DOI
  39. 10.1016/j.bbrc.2008.04.164 · DOI
  40. 10.1016/j.febslet.2012.01.029 · DOI
  41. 10.1073/pnas.1105028108 · DOI
  42. 10.1002/cbic.201500681 · DOI
  43. 10.1038/nmeth.3701 · DOI
  44. 2000, Human mutations in glucose 6-phosphate dehydrogenase reflect evolutionary history, FASEB J, 14, 485, 10.1096/fasebj.14.3.485 · DOI
  45. 10.1038/ncomms3327 · DOI
  46. 10.1038/s41467-018-02974-x · DOI · Paper page
  47. 2017, Mutations in the tetramer interface of human glucose-6-phosphate dehydrogenase reveals kinetic differences between oligomeric states, FEBS Lett, 591, 1278, 10.1002/1873-3468.12638 · DOI
  48. 10.1073/pnas.2023989118 · DOI
  49. 10.1016/j.bbadis.2009.05.003 · DOI
  50. 2017, Coupling between protein stability and catalytic activity determines pathogenicity of g6pd variants, Cell Rep, 18, 2592, 10.1016/j.celrep.2017.02.048 · DOI
  51. 2020, Revealing Dynamic Protein Acetylation across Subcellular Compartments, J. Proteome. Res, 19, 2404, 10.1021/acs.jproteome.0c00088 · DOI
  52. 10.1126/sciadv.abd5393 · DOI
  53. 2022, Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43, Nat. Commun, 13, 1223, 10.1038/s41467-022-28822-7 · DOI · Paper page
  54. Alessandro, M. et al. Fyn specifically Regulates the activity of red cell glucose-6-phosphate-dehydrogenase. Redox Biol. 36, 101639 (2020). · DOI
  55. 10.1038/ncb2172 · DOI
  56. 10.1093/nar/gkz319 · DOI
  57. 10.1126/science.1092734 · DOI · Paper page
  58. 1995, Increased induction of apoptosis in mononuclear cells of a glucose-6-phosphate dehydrogenase deficient patient, J. Mol. Med. (Berl, 73, 47, 10.1007/BF00203619 · DOI
  59. 10.1038/s41467-019-09078-0 · DOI
  60. 10.1016/j.cell.2009.04.037 · DOI · Paper page
  61. 10.1016/j.ab.2009.02.009 · DOI
  62. 10.1016/j.molcel.2014.06.020 · DOI
  63. 10.1021/bi4000244 · DOI
  64. 10.1083/jcb.200105017 · DOI
  65. 10.1016/j.molcel.2014.04.028 · DOI · Paper page
  66. Nabbi, A. & Riabowol, K . Rapid isolation of nuclei from cells in vitro. Cold Spring Harb. Protoc. 2015, 769–772 (2015). · DOI
  67. 10.1016/0003-2697(69)90025-6 · DOI
  68. 10.1107/S0907444909052925 · DOI · Paper page
  69. 10.1093/nar/gkg509 · DOI
  70. 10.1093/nar/gky1016 · DOI · Paper page
  71. 10.1107/S0907444905002350 · DOI

Metadata

Authors
Wu, F., Muskat, N. H., Dvilansky, I., Koren, O., Shahar, A., Gazit, R., Elia, N., & Arbely, E.
Affiliations
Ben-Gurion University of the Negev (IL)
Corresponding author
Arbely, Eyal
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2023-02-19
Volume
14
Issue
1
Pages
6208-6208
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2023.02.19.529048
PMC
PMC10556143
OpenAlex
W4387372445
Cited by
28
References
1
Citations by year
2026: 8 · 2025: 16 · 2024: 4
FWCI
2.49
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Commission (HORIZON2020)
  • Israel Science Foundation (678461)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cancer-related gene regulation
Keywords
lysine acetylation, glucose-6-phosphate dehydrogenase, pentose phosphate pathway, enzyme regulation, post-translational modification, ubiquitination, p53, apoptosis, protein dimer structure, phosphorylation, metabolic signaling
MeSH terms
Acetylation, Humans, Lysine, Protein Processing, Post-Translational

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