John C. Newman

SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5(-/-) animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways

Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD+-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis.

Ketone bodies as signaling metabolites

Traditionally, the ketone body β-hydroxybutyrate (βOHB) has been looked upon as a carrier of energy from liver to peripheral tissues during fasting or exercise. However, βOHB also signals via extracellular receptors and acts as an endogenous inhibitor of histone deacetylases (HDACs). These recent findings support a model in which βOHB functions to link the environment, in this case the diet, and gene expression via chromatin modifications. We review the regulation and functions of ketone bodies, the relationship between ketone bodies and calorie restriction, and the implications of HDAC inhibition by the ketone body βOHB in the modulation of metabolism and in diseases of aging.

Mitochondrial Protein Acylation and Intermediary Metabolism: Regulation by Sirtuins and Implications for Metabolic Disease

The sirtuins are a family of NAD+-dependent protein deacetylases that regulate cell survival, metabolism, and longevity. Three sirtuins, SIRT3–5, localize to mitochondria. Expression of SIRT3 is selectively activated during fasting and calorie restriction. SIRT3 regulates the acetylation level and enzymatic activity of key metabolic enzymes, such as acetyl-CoA synthetase, long-chain acyl-CoA dehydrogenase, and 3-hydroxy-3-methylglutaryl-CoA synthase 2, and enhances fat metabolism during fasting. SIRT5 exhibits demalonylase/desuccinylase activity, and lysine succinylation and malonylation are abundant mitochondrial protein modifications. No convincing enzymatic activity has been reported for SIRT4. Here, we review the emerging role of mitochondrial sirtuins as metabolic sensors that respond to changes in the energy status of the cell and modulate the activities of key metabolic enzymes via protein deacylation.

Mitochondrial sirtuins: regulators of protein acylation and metabolism

Sirtuins are NAD(+)-dependent protein deacetylases and have been implicated in the regulation of metabolism, stress responses, and aging. Three sirtuins are located in mitochondria: SIRT3, 4, and 5. SIRT3 deacetylates and regulates the enzymatic activity of many metabolic enzymes in mitochondria, whereas SIRT5 removes two novel post-translational modifications, lysine malonylation and succinylation. Here, we review the current knowledge of how mitochondrial sirtuins function in metabolism and metabolic diseases, and offer a conceptual model how they may regulate mitochondrial function through distinct deacylation activities (deacetylation, demalonylation, or desuccinylation).

β-hydroxybutyrate: Much more than a metabolite

The ketone body β-hydroxybutyrate (βOHB) is a convenient carrier of energy from adipocytes to peripheral tissues during fasting or exercise. However, βOHB is more than just a metabolite, having important cellular signaling roles as well. βOHB is an endogenous inhibitor of histone deacetylases (HDACs) and a ligand for at least two cell surface receptors. In addition, the downstream products of βOHB metabolism including acetyl-CoA, succinyl-CoA, and NAD+ (nicotinamide adenine dinucleotide) themselves have signaling activities. These regulatory functions of βOHB serve to link the outside environment to cellular function and gene expression, and have important implications for the pathogenesis and treatment of metabolic diseases including type 2 diabetes.

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome.

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.

Strategies and Challenges in Clinical Trials Targeting Human Aging

Interventions that target fundamental aging processes have the potential to transform human health and health care. A variety of candidate drugs have emerged from basic and translational research that may target aging processes. Some of these drugs are already in clinical use for other purposes, such as metformin and rapamycin. However, designing clinical trials to test interventions that target the aging process poses a unique set of challenges. This paper summarizes the outcomes of an international meeting co-ordinated by the NIH-funded Geroscience Network to further the goal of developing a translational pipeline to move candidate compounds through clinical trials and ultimately into use. We review the evidence that some drugs already in clinical use may target fundamental aging processes. We discuss the design principles of clinical trials to test such interventions in humans, including study populations, interventions, and outcomes. As examples, we offer several scenarios for potential clinical trials centered on the concepts of health span (delayed multimorbidity and functional decline) and resilience (response to or recovery from an acute health stress). Finally, we describe how this discussion helped inform the design of the proposed Targeting Aging with Metformin study.

The conserved Cockayne syndrome B-piggyBac fusion protein (CSB-PGBD3) affects DNA repair and induces both interferon-like and innate antiviral responses in CSB-null cells

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome.

Salicylate, diflunisal and their metabolites inhibit CBP/p300 and exhibit anticancer activity

Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity in vitro by direct competition with acetyl-Coenzyme A at the catalytic site. We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs. DOI: http://dx.doi.org/10.7554/eLife.11156.001