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. 2017 Aug 10;474(16):2829-2839.
doi: 10.1042/BCJ20170389.

Potential Mechanisms Linking SIRT Activity and Hypoxic 2-hydroxyglutarate Generation: No Role for Direct Enzyme (De)acetylation

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Free PMC article

Potential Mechanisms Linking SIRT Activity and Hypoxic 2-hydroxyglutarate Generation: No Role for Direct Enzyme (De)acetylation

Sergiy M Nadtochiy et al. Biochem J. .
Free PMC article

Abstract

2-Hydroxyglutarate (2-HG) is a hypoxic metabolite with potentially important epigenetic signaling roles. The mechanisms underlying 2-HG generation are poorly understood, but evidence suggests a potential regulatory role for the sirtuin family of lysine deacetylases. Thus, we hypothesized that the acetylation status of the major 2-HG-generating enzymes [lactate dehydrogenase (LDH), isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH)] may govern their 2-HG-generating activity. In vitro acetylation of these enzymes, with confirmation by western blotting, mass spectrometry, reversibility by recombinant sirtuins and an assay for global lysine occupancy, yielded no effect on 2-HG-generating activity. In addition, while elevated 2-HG in hypoxia is associated with the activation of lysine deacetylases, we found that mice lacking mitochondrial SIRT3 exhibited hyperacetylation and elevated 2-HG. These data suggest that there is no direct link between enzyme acetylation and 2-HG production. Furthermore, our observed effects of in vitro acetylation on the canonical activities of IDH, MDH and LDH appeared to contrast with previous findings wherein acetyl-mimetic lysine mutations resulted in the inhibition of these enzymes. Overall, these data suggest that a causal relationship should not be assumed between acetylation of metabolic enzymes and their activities, canonical or otherwise.

Keywords: acetylation; epigenetics; hypoxia; ischemia; sirtuins.

Conflict of interest statement

DISCLOSURES

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Cellular Hypoxia, LDH Acetylation and Canonical vs. 2-HG Generating Activity
(A): HEK293 cells were exposed to hypoxia with or without the SIRT1 inhibitor splitomicin (Sp), followed by measurement of 2-HG levels. Data are means ± SEM, n=5. *p<0.05 vs. control (Ctrl.) p<0.05 vs. hypoxia alone (Hypx.) (B): Purified LDH was acetylated in-vitro with acetyl-CoA (see methods), followed by western blot probe with anti-acetyl-lysine (K-Ac) antibody. Ponceau S stained membrane below indicates protein loading. Images representative of at least 4 independent experiments. (C): Canonical activity of naïve and acetylated LDH (Ac-LDH) with pyruvate and NADH as substrates was measured spectrophotometrically as NADH consumption (left panel) or by LC-MS/MS as lactate production (right panel). (D): Non-canonical activity of LDH with α-KG and NADH as substrates was measured spectrophotometrically as NADH consumption (left panel) or by LC-MS/MS as 2-HG production (right panel). All enzyme rate data are means ± SEM, n=4–7. Reactions monitored are shown above each graph, with the metabolite measured shown in red font.
Figure 2
Figure 2. IDH2 Acetylation and Canonical vs. 2-HG Generating Activity
(A): Purified IDH2 was acetylated in-vitro with acetyl-CoA (see methods), followed by western blot probe with anti-acetyl-lysine (K-Ac) antibody. Ponceau S stained membrane below indicates protein loading. Images representative of at least 4 independent experiments. (B): Canonical activity of naïve and acetylated IDH2 (Ac-IDH2) with isocitrate and NADP+ as substrates was measured spectrophotometrically as NADPH production (left panel) or by LC-MS/MS as α-KG production (right panel). (C): Non-canonical reductive activity of IDH2 with α-KG and NADPH as substrates was measured spectrophotometrically as NADPH consumption (left panel) or by LC-MS/MS as 2-HG production (right panel). All enzyme rate data are means ± SEM, n=4. *p<0.05 between naïve and Ac-IDH2. Reactions monitored are shown above each graph, with the metabolite measured shown in red font.
Figure 3
Figure 3. MDH2 Acetylation and Canonical vs. 2-HG Generating Activity
(A): Purified MDH2 was acetylated in-vitro with acetyl-CoA (see methods), followed by western blot probe with anti-acetyl-lysine (K-Ac) antibody. Ponceau S stained membrane below indicates protein loading. Images representative of at least 4 independent experiments. (B/C/D): Naïve and acetylated MDH2 (Ac-MDH2) were digested with trypsin and resultant peptides analyzed by mass spectrometry to identify acetylation sites. 76% sequence coverage was obtained (panel C, red font) including the peptide containing the proposed K239 acetylation site (underlined). Sample spectra are shown in B, with panel D listing identified peptides corresponding to bn+ ions (red) and yn+ ions (blue). A difference of 42 mass units between y+1 ion in MDH2 vs. Ac-MDH2 (189.123 – 147.113) indicates acetylation. (E): Canonical activity of naïve and acetylated MDH2 with OAA and NADH as substrates was measured spectrophotometrically as NADH consumption (left panel) or by LC-MS/MS as malate production (right panel). (F): Non-canonical activity of MDH2 with α-KG and NADH as substrates was measured spectrophotometrically as NADH consumption (left panel) or by LC-MS/MS as 2-HG production (right panel). All enzyme rate data are means ± SEM, n=4–6. Reactions monitored are shown above each graph, with the metabolite measured shown in red font.
Figure 4
Figure 4. Acetylation, 2-HG Generation and pH in Sirt3−/− mice, and the Effects of SIRT Inhibition on Cytosolic pH
(A): Western blot shows absence of SIRT3 protein in cardiac tissue from Sirt3−/− mice (vs. WT). (B): Western blot shows protein acetylation (pan anti-acetyl-lysine antibody) in cardiac mitochondria from WT and Sirt3−/− mice. Ponceau S stained membrane indicates protein loading. Blots are representative of at least 4 independent experiments. (C): LC-MS/MS based metabolite profiling of WT vs. Sirt3−/− cardiac tissue. Data are expressed as the metabolite level in Sirt3−/− relative to paired WT samples, with individual data points (n=6 pairs) alongside means ± SEM. *p<0.05 between WT and Sirt3−/−. (D): L-2-HG dehydrogenase (L-2-HGDH) activity in cardiac mitochondria from WT and Sirt3−/− mice was measured spectrophotometrically via formazan-linked assay (left panel) or by LC-MS/MS as α-KG formation (right panel). Enzyme rate data are means ± SEM, n=5. Reactions monitored are shown above each graph, with the metabolite measured shown in red font. (E/F): Cytosolic pH measurements in primary cultured adult cardiomyocytes from WT and Sirt3−/− mice. Alongside representative images of cellular pH (E), graph shows quantitation of pH from 4 independent cell preparations (F). Data are means ± SEM. (G/H): Cytosolic pH measurements in primary cultured adult cardiomyocytes from WT mice subjected to simulated ischemia (see methods), with or without the SIRT1 inhibitor splitomicin (Sp). Alongside representative images of cellular pH (G), graph shows quantitation of pH from 4–6 independent cell preparations (H). Data are means ± SEM. *p<0.05 vs. control (Ctrl.) p<0.05 vs. ischemia alone (Isch.).

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