Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl–coenzyme A hydratase/3-hydroxyacyl–coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.

SUMMARY Caloric restriction (CR) extends the lifespan and healthspan of a variety of species, and slows the progression of age-related hearing loss (AHL), a common age-related disorder associated with oxidative stress. Here we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice, but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced to oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR, and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.

Heterozygous mutations in the gene encoding isocitrate dehydrogenase-1 (IDH1) occur in certain human brain tumors, but their mechanistic role in tumor development is unknown. We have shown that tumor-derived IDH1 mutations impair the enzyme’s affinity for its substrate and dominantly inhibit wild-type IDH1 activity through the formation of catalytically inactive heterodimers. Forced expression of mutant IDH1 in cultured cells reduces formation of the enzyme product,α-ketoglutarate (α-KG), and increases the levels of hypoxia-inducible factor subunit HIF-1α, a transcription factor that facilitates tumor growth when oxygen is low and whose stability is regulated by α-KG. The rise in HIF-1α levels was reversible by an α-KG derivative. HIF-1α levels were higher in human gliomas harboring an IDH1 mutation than in tumors without a mutation. Thus, IDH1 appears to function as a tumor suppressor that, when mutationally inactivated, contributes to tumorigenesis in part through induction of the HIF-1 pathway.

Summary Calorie restriction (CR) extends lifespan in diverse species. Mitochondria play a key role in CR adaptation, however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR vs. control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites −2,193 from mitochondrial proteins rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD+) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD+-dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD+ supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD+ bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.

Background: NAD ϩ -dependent deacetylase SIRT3 is essential for the prevention of age-related hearing loss during caloric

Summary Emerging evidence suggests that protein acetylation is a broad-ranging regulatory mechanism. Here we utilize acetyl-peptide arrays and metabolomic analyses to identify substrates of mitochondrial deacetylase Sirt3. We identified ornithine transcarbamoylase (OTC) from the urea cycle, and enzymes involved in β-oxidation. Metabolomic analyses of fasted mice lacking Sirt3 (sirt3−/−) revealed alterations in β-oxidation and the urea cycle. Biochemical analysis demonstrated that Sirt3 directly deacetylates OTC and stimulates its activity. Mice under caloric restriction (CR) increased Sirt3 protein levels, leading to deacetylation and stimulation of OTC activity. In contrast, sirt3−/− mice failed to deacetylate OTC in response to CR. Inability to stimulate OTC under CR led to a failure to reduce orotic acid levels, a known outcome of OTC deficiency. Thus, Sirt3 directly regulates OTC activity and promotes the urea cycle during CR, and the results suggest that under low energy input, Sirt3 modulates mitochondria by promoting amino-acid catabolism and β-oxidation.

Metabolic reprogramming has been widely recognized as a hallmark of malignancy. The uptake and metabolism of amino acids are aberrantly upregulated in many cancers that display addiction to particular amino acids. Amino acids facilitate the survival and proliferation of cancer cells under genotoxic, oxidative, and nutritional stress. Thus, targeting amino acid metabolism is becoming a potential therapeutic strategy for cancer patients. In this review, we will systematically summarize the recent progress of amino acid metabolism in malignancy and discuss their interconnection with mammalian target of rapamycin complex 1 (mTORC1) signaling, epigenetic modification, tumor growth and immunity, and ferroptosis. Finally, we will highlight the potential therapeutic applications.