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. 2013 Oct 4;288(40):29036-45.
doi: 10.1074/jbc.M113.486753. Epub 2013 Aug 13.

Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix

Gregory R Wagner  1 , R Mark Payne
Affiliations

Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix

Gregory R Wagner et al. J Biol Chem. .

Abstract

Alterations in mitochondrial protein acetylation are implicated in the pathophysiology of diabetes, the metabolic syndrome, mitochondrial disorders, and cancer. However, a viable mechanism responsible for the widespread acetylation in mitochondria remains unknown. Here, we demonstrate that the physiologic pH and acyl-CoA concentrations of the mitochondrial matrix are sufficient to cause dose- and time-dependent, but enzyme-independent acetylation and succinylation of mitochondrial and nonmitochondrial proteins in vitro. These data suggest that protein acylation in mitochondria may be a chemical event facilitated by the alkaline pH and high concentrations of reactive acyl-CoAs present in the mitochondrial matrix. Although these results do not exclude the possibility of enzyme-mediated protein acylation in mitochondria, they demonstrate that such a mechanism may not be required in its unique chemical environment. These findings may have implications for the evolutionary roles that the mitochondria-localized SIRT3 deacetylase and SIRT5 desuccinylase have in the maintenance of metabolic health.

Keywords: Acetyl Coenzyme A; Acetylation; Metabolic Diseases; Metabolic Regulation; Mitochondrial Metabolism; Nonenzymatic; Sirtuins; Succinyl Coenzyme A; Succinylation; pH Regulation.

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Figures

FIGURE 1.
FIGURE 1.
A, schematic highlighting the unique chemical environment of the mitochondrial matrix. Both the pH and the acetyl-CoA concentration of the mitochondrial matrix are considerably higher than the cytosol and nucleus (pH 8.0 versus pH 7.2, and 0.1–1.5 mm versus 3–30 μm. B, nonenzymatic acetylation is mechanistically analogous to other endogenous nonenzymatic lysine modifications such as glycation and carbamylation. In each example, the mechanism involves a primary amine performing a nucleophilic attack on the carbonyl carbon of the endogenous metabolite.
FIGURE 2.
FIGURE 2.
Nonenzymatic Nϵ-acetylation in the chemical conditions of the mitochondrial matrix. A, acetyl-CoA concentration-dependent increases in mitochondrial protein acetylation under native and denatured conditions. The mouse liver mitochondrial extracts (30 μg) were incubated over a range of acetyl-CoA concentrations previously determined to occur in mitochondria (15, 16) (0.1–1.5 mm, and 1 μl of matrix volume/mg of mitochondrial protein) for 6 h at 37 °C with gentle mixing. For denatured samples, mitochondrial extracts (30 μg) were heated at 95 °C for 10 min prior to the addition of acetyl-CoA. IB, immunoblot; ac-lysine, acetyl-lysine. B, quantification of densitometry data in A normalized to Complex III. The control reaction with the smaller arbitrary value was set to zero, and the remaining data points were transformed accordingly. AU, arbitrary units. C, time-dependent incorporation of acetyl groups into native and denatured mitochondrial protein lysine residues. D, quantification of the densitometry data in C normalized to Complex III. E, time-dependent incorporation of acetyl groups into native mitochondrial protein over a broader range of time points. F, acetylation is not caused by the denaturation process and is pH-dependent. In A, C, E, and F, the nitrocellulose membrane used for immunoblotting was stained with the nonspecific protein marker Ponceau S to show equal loading as indicated.
FIGURE 3.
FIGURE 3.
The acetyl transfer mechanism is not competitively inhibited by coenzyme A, and its rate is directly proportional to hydroxide ion concentration. A, CoA does not competitively inhibit the observed acetyl transfer reaction. Mitochondrial extracts were prepared from liver. IB, immunoblot; ac-lysine, acetyl-lysine. B, the physiologic pH (8.0) and acetyl-CoA concentration (1.5 mm) of the mitochondrial matrix are sufficient to induce widespread lysine acetylation of a nonmitochondrial protein (BSA) under native conditions. C, lysine acetylation rate is directly proportional to the concentration of hydroxide ions, consistent with specific base catalysis. AU, arbitrary units.
FIGURE 4.
FIGURE 4.
Protein succinylation is abundant within mitochondria and also occurs nonenzymatically at physiological pH. A, fractionation of mouse liver tissue into whole cell lysate (WCL), cytoplasm (cyto), and mitochondrial (mito) fractions followed by Western blotting with an anti-succinyl-lysine (Suc-lysine) antibody. Gapdh and respiratory Complex III Rieske protein antibodies were used to confirm cytoplasmic and mitochondrial subfractions, respectively. IB, immunoblot. B, acetyl-CoA and succinyl-CoA are both inherently reactive CoA thioesters. C, the physiologic pH (8.0) and succinyl-CoA concentration (0.1–0.6 mm) (15) of the mitochondrial matrix are sufficient to induce lysine succinylation of protein under native conditions.
FIGURE 5.
FIGURE 5.
Schematic illustrating the specific base catalyzed acyl transfer mechanism. The alkaline environment of the mitochondrial matrix reflects an increased concentration of hydroxide ions (OH−) free to abstract protons from accessible lysine residues (panel 1). The deprotonated lysine can then perform a nucleophilic attack on the terminal carbonyl carbon of the acyl-CoA (panel 2). A putative tetrahedral intermediate is formed (panel 3), which then collapses, displacing the thioester bond and leaving an acylated (acetylated) lysine, CoASH, and hydroxide (panel 4). This mechanism was adapted from that of the GCN5 acetyltransferase, which employs a general base catalyst to deprotonate lysine residues and initiate acetyl transfer (19).
FIGURE 6.
FIGURE 6.
The SIRT3 deacetylase attenuates acetyl-CoA-induced acetylation of mitochondrial proteins. A, acetylation of SIRT3 target proteins in the presence of acetyl-CoA at pH 8.0. IP, immunoprecipitation; IB, immunoblot; Ac-lysine, acetyl-lysine; LCAD, long-chain acyl-CoA dehydrogenase. B, recombinant SIRT3 reverses acetyl-CoA-induced acetylation of mitochondrial proteins at pH 8.0. A SIRT3 antibody was used to demonstrate the presence of recombinant (59 kDa) and endogenous (28 kDa) SIRT3. Mitochondrial extracts were prepared from liver. NAD+, nicotinamide adenine dinucleotide; NAM, nicotinamide (Sirtuin inhibitor); hSIRT3, human SIRT3.
FIGURE 7.
FIGURE 7.
Bioinformatics analysis of protein microenvironment and acetylation. A, acetyl sites undergoing the greatest increases in acetylation in response to SIRT3 deletion have significantly more proximal positively charged residues than sites undergoing smaller increases in acetylation (Wilcoxon rank sum test). B, iceLogo analysis of site-specific amino acid abundance between the two sequence sets in A. Amino acids with greater frequency at a specific site appear at the top, and those with lower frequency appear at the bottom. Positively charged residues are highlighted in blue, and negatively charged residues are highlighted in red. C, scatter plot of the predicted lysine ϵ-amino group acid dissociation constants calculated using structural data from five mitochondrial proteins encompassing 74 acetylation sites and their corresponding acetyl-lysine -fold changes in response to SIRT3 deletion as detailed in Hebert et al. (4). PROPKA 3.1 pKa prediction accounts for hydrogen bonding, desolvation effects, and Coulombic interactions on both internal and surface residues, but does not account for conformational flexibility (23).
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