Mitochondrial Protein Hyperacetylation Research Articles

Biotin starvation causes mitochondrial protein hyperacetylation and partial rescue by the SIRT3-like deacetylase Hst4p

The essential vitamin biotin is a covalent and tenaciously attached prosthetic group in several carboxylases that play important roles in the regulation of energy metabolism. Here we describe increased acetyl-CoA levels and mitochondrial hyperacetylation as downstream metabolic effects of biotin deficiency. Upregulated mitochondrial acetylation sites correlate with the cellular deficiency of the Hst4p deacetylase, and a biotin-starvation-induced accumulation of Hst4p in mitochondria supports a role for Hst4p in lowering mitochondrial acetylation. We show that biotin starvation and knockout of Hst4p cause alterations in cellular respiration and an increase in reactive oxygen species (ROS). These results suggest that Hst4p plays a pivotal role in biotin metabolism and cellular energy homeostasis, and supports that Hst4p is a functional yeast homologue of the sirtuin deacetylase SIRT3. With biotin deficiency being involved in various metabolic disorders, this study provides valuable insight into the metabolic effects biotin exerts on eukaryotic cells.

Chronic ethanol consumption induces mitochondrial protein acetylation and oxidative stress in the kidney

In this study, we present the novel findings that chronic ethanol consumption induces mitochondrial protein hyperacetylation in the kidney and correlates with significantly increased renal oxidative stress. A major proteomic footprint of alcoholic liver disease (ALD) is an increase in hepatic mitochondrial protein acetylation. Protein hyperacetylation has been shown to alter enzymatic function of numerous proteins and plays a role in regulating metabolic processes. Renal mitochondrial targets of hyperacetylation include numerous metabolic and antioxidant pathways, such as lipid metabolism, oxidative phosphorylation, and amino acid metabolism, as well as glutathione and thioredoxin pathways. Disruption of protein lysine acetylation has the potential to impair renal function through metabolic dysregulation and decreased antioxidant capacity. Due to a significant elevation in ethanol-mediated renal oxidative stress, we highlight the acetylation of superoxide dismutase, peroxiredoxins, glutathione reductase, and glutathione transferase enzymes. Since oxidative stress is a known factor in ethanol-induced nephrotoxicity, we examined biochemical markers of protein hyperacetylation and oxidative stress. Our results demonstrate increased protein acetylation concurrent with depleted glutathione, altered Cys redox potential, and the presence of 4-HNE protein modifications in our 6-week model of early-stage alcoholic nephrotoxicity. These findings support the hypothesis that ethanol metabolism causes an influx of mitochondrial metabolic substrate, resulting in mitochondrial protein hyperacetylation with the potential to impact mitochondrial metabolic and antioxidant processes.

Abstract 210: Progressive Mitochondrial Protein Acetylation and Microsteatosis is Associated with Diastolic Dysfunction in a Hypertrophic Cardiomyopathy Model

We tested the hypothesis that increased mitochondrial protein acetylation is associated with impaired fatty acid metabolism and diastolic dysfunction in Friedreich’s Ataxia (FRDA). FRDA results from deficiency of the mitochondrial protein, frataxin (FXN), and causes hypertrophic cardiomyopathy. FRDA hearts show decreased ATP production. We previously showed that FXN loss results in loss of activity of the NAD+-dependent mitochondrial deacetylase, sirtuin 3 (SIRT3), and cardiac mitochondrial protein hyperacetylation in a mouse model of FRDA. Long-chain and medium chain acyl CoA dehydrogenases (LCAD, MCAD) are targets of SIRT3, suggesting that abnormal acetylation may alter fatty acid metabolism. A cardiac specific mouse model with conditional deletion of FXN in heart and skeletal muscle (FXN MCK-Cre-/-) was compared to healthy controls (FXNfl/fl). Mice underwent echocardiogram and left heart catheterization in vivo at age 30 and 65 days. Heart lysate was examined for overall lysine acetylation at ages 30, 45 and 70 days, and acetylated LCAD and MCAD. Myocardial cells were stained with oil red to detect lipids. Hearts of FXN MCK-Cre-/- animals showed mitochondrial hyperacetylation, increased LCAD and MCAD acetylation, and microsteatosis compared to controls. Diastolic dysfunction was evident in FXN MCK-Cre-/- at day 65 by increased mitral valve E/A ratios and IVRT (p<0.04 and p<0.03, respectively, n=7), prolonged tau, and decreased -dP/dt (p<0.02 and p <0.03, respectively, n=6). Cardiac mechanics at day 65 showed systolic failure with reduced EF and FS (p<0.002 and p<0.003, respectively, n=7). Comparison of FXN deficient mice and controls at day 65 trended toward flattened ESPVR and left shifted EDPVR curves, but differences were not significant. Early diastolic dysfunction was evident at day 30 in FXN MCK-Cre-/- by high E/A ratio and EDP compared to day 65 controls (p <0.03, n=7, and p<0.03, n=6, respectively). We conclude that progressive mitochondrial protein acetylation and abnormal fatty acid metabolism is associated with early diastolic dysfunction and later systolic failure. This metabolic dysregulation and pathophysiology may share characteristics with other metabolic heart disorders, such as diabetes and metabolic syndrome.

Loss of SIRT3 leads to a compensatory shift in cellular metabolism promoting cancer cell growth

Background Mechanisms involved in regulating metabolic reprogramming in cancer cells are not fully understood. Acetylation is emerging as a major regulator of mitochondrial metabolism and may contribute to metabolic derangements that occur in cancer cells. Sirtuin-3, (SIRT3), is the main mitochondrial deacetylase and it serves to maintain mitochondrial energy homeostasis by deacetylating and activating mitochondrial proteins. Loss of SIRT3 leads to altered cellular metabolism including reduced ATP production and decreased fatty acid oxidation [1]. Remarkably, reduced SIRT3 expression is associated with cancer in patients and Sirt3 knockout mice [2]. However, the mechanism of mitochondrial protein hyperacetylation and the sub-sequent increased susceptibility to tumor formation remains unknown.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development

Sirt3 is a mitochondrial NAD+-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR −/−) and LDLR/Sirt3 double-knockout (Sirt3 −/− LDLR −/−) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR −/− mice. Sirt3 deficiency does not affect atherosclerosis in LDLR −/− mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0399-0) contains supplementary material, which is available to authorized users.

HINT2 and fatty liver disease: Mitochondrial protein hyperacetylation gives a hint?

HINT2 and fatty liver disease: Mitochondrial protein hyperacetylation gives a hint?

SIRT3 Weighs Heavily in the Metabolic Balance: A New Role for SIRT3 in Metabolic Syndrome

Eating a "Western diet" high in fat and sugars is associated with accelerated development of age-related metabolic diseases such as obesity, insulin resistance, and diabetes while incidences of these diseases are decreased on a low-calorie diet. The mitochondrial NAD(+)-dependent protein deacetylase SIRT3 has previously been shown to be important in adapting to metabolic stress brought on by fasting and calorie restriction. During times of metabolic stress, SIRT3 is upregulated and maintains homeostasis following nutrient deprivation by turning on pathways such as fatty acid oxidation, antioxidant production, and the urea cycle. New studies now demonstrate that SIRT3 is regulated during nutrient excess. During high-fat diet feeding, SIRT3 is downregulated leading to mitochondrial protein hyperacetylation. The consequence of this hyperacetylation is the accelerated development of metabolic syndrome. Thus, SIRT3 is emerging as an important metabolic sensor working to restore metabolic homeostasis during times of stress.

Mitochondrial Acetylome Analysis in a Mouse Model of Alcohol-Induced Liver Injury Utilizing SIRT3 Knockout Mice

Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD.

Mitochondrial SIRT3: A New Potential Therapeutic Target for Metabolic Syndrome

In this issue of Molecular Cell, Hirschey etal. demonstrate that loss of the NAD(+)-dependent deacetylase SIRT3 and resultant mitochondrial protein hyperacetylation play a critical role in the pathogenesis of metabolic syndrome, providing new insights into the therapeutic potential of SIRT3.

SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

4-Hydroxynonenal Inhibits SIRT3 via Thiol-Specific Modification

4-Hydroxynonenal (4-HNE) is an endogenous product of lipid peroxidation known to play a role in cellular signaling through protein modification and is a major precursor for protein carbonyl adducts found in alcoholic liver disease (ALD). In the present study, a greater than 2-fold increase in protein carbonylation of sirtuin 3 (SIRT3), a mitochondrial class III histone deacetylase, is reported in liver mitochondrial extracts of ethanol-consuming mice. The consequence of this in vivo carbonylation on SIRT3 deacetylase activity is unknown. Interestingly, mitochondrial protein hyperacetylation was observed in a time-dependent increase in a model of chronic ethanol consumption; however, the underlying mechanisms for this remain unknown. Tandem mass spectrometry was used to identify and characterize the in vitro covalent modification of rSIRT3 by 4-HNE at Cys(280), a critical zinc-binding residue, and the resulting inhibition of rSIRT3 activity via pathophysiologically relevant concentrations of 4-HNE. Computational-based molecular modeling simulations indicate that 4-HNE modification alters the conformation of the zinc-binding domain inducing minor changes within the active site, resulting in the allosteric inhibition of SIRT3 activity. These conformational data are supported by the calculated binding energies derived from molecular docking studies suggesting the substrate peptide of acetyl-CoA synthetase 2 (AceCS2-K(ac)) and display a greater affinity for native SIRT3 as compared with the 4-HNE adducted protein. The results of this study characterize altered mitochondrial protein acetylation in a mouse model of chronic ethanol ingestion and thiol-specific allosteric inhibition of rSIRT3 resulting from 4-HNE adduction.

Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation

Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD(+)-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD(+) levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3⁻(/)⁻ mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.

Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation.

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.