Sirtuins are a family of nicotinamide adenine dinucleotide (NAD+)–dependent protein deacylases which have important roles in regulating cell stress resistance, metabolism, and aging. Three mammalian sirtuins, SIRT3, -4, and -5, are found primarily within mitochondria. SIRT3 exhibits robust deacetylase activity, while SIRT5 targets longer acylations with negative charges, including malonylation and succinylation. Given the central role for mitochondria in a broad range of cellular activities, the Verdin lab has done significant work in elucidating the biology of mitochondrial sirtuins and the lysine modifications they regulate.
Using antibodies specifically recognizing different lysine acylations, we found that proteins in SIRT3 knockout mouse tissues exhibit global hyperacetylation, while proteins in SIRT5 knockout mice exhibit a global increase in succinylation and malonylation. In collaboration with Dr. Brad Gibson, we performed label free quantitative mass spectrometry to characterize the landscape of lysine acetylation, succinylation, and malonylation in mouse livers, and further identified lysine residues targeted by SIRT3 or SIRT5. Our proteomic analyses have revealed hundreds of target proteins involved in a broad range of cellular activities, including major metabolic pathways such as fatty acid oxidation, TCA cycle, or glycolysis.
By deacetylating and activating long chain acyl-coA dehydrogenase (LCAD) and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), we showed that SIRT3 regulates processes such as fatty acid oxidation and ketogenesis. SIRT3 knockout mice exhibit deficient fatty acid oxidation and ketogenesis following fasting, for example. More importantly, mice lacking SIRT3 become more susceptible to the development of obesity, insulin resistance, hyperlipidemia, and steatohepatitis following high-fat diet. In humans, a single nucleotide polymorphism at the Sirt3 gene leads to reduced SIRT3 enzymatic activity and is associated with the metabolic syndrome. All together, this evidence suggests that SIRT3 plays very important roles in metabolic regulation by deacetylating proteins and regulating their activities. Other groups have also shown that SIRT3 is an important general metabolic regulator, with roles in the urea cycle, mitochondrial respiratory chain, and glycolysis.
Our more recent work on SIRT5 and its targeted lysine modifications have also revealed an important role for SIRT5 in metabolsm. SIRT5 positively regulates ketone body synthesis by desuccinylating key enzymes in this pathway, and we have further shown that hyper-succinylation of lysine sites near the catalytic pocket of the rate-limiting enzyme, HMGCS2, reduces enzymatic activity by blocking the substrate binding. SIRT5 knockout mice have deficient ketone body production during fasting, and loss of SIRT5 also impairs the functions of enzymes involved in fatty acid oxidation. Cell fractionation studies from our lab have shown both cytoplasmic and nuclear localization of SIRT5 protein, and in contrast to lysine succinylation (mainly abundant in mitochondria), we showed that lysine malonylation appears to be the more prevalent target in cytosolic proteins. For example, SIRT5 positively regulates glycolysis by demalonylating key enzymes in the glycolysis pathway, and loss of SIRT5 results in impaired glycolysis.
The biologically relevant enzymatic activity for SIRT4 remains controversial. We find that SIRT4 alters cellular ATP by modulating the adenine nucleotide translocator 2 (ANT2)-mediated oxidative phosphorylation efficiency. SIRT4 deficiency leads to increased oxygen consumption by uncoupling, mimicking energy deprivation, and thus initiates a homeostatic response involving AMPK and PGC1α. This work establishes an important role for SIRT4 in mediating a retrograde signaling from the mitochondrion to the nucleus to maintain energy homeostasis.
In the future, the Verdin lab is interested in continuing to explore the biology of sirtuins and the molecular mechanisms underlying their pleiotropic functions in metabolism and aging.
1. SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Hirschey MD, Shimazu T, Goetzman E, Jing E, Schwer B, Lombard DB, Grueter CA, Harris C, Biddinger S, Ilkayeva OR, Stevens RD, Li Y, Saha AK, Ruderman NB, Bain JR, Newgard CB, Farese RV Jr, Alt FW, Kahn CR, Verdin E. Nature. 2010 Mar 4;464(7285):121-5. doi:10.1038/nature08778. PMID: 20203611
2. SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome. Hirschey MD, Shimazu T, Jing E, Grueter CA, Collins AM, Aouizerat B, Stančáková A, Goetzman E, Lam MM, Schwer B, Stevens RD, Muehlbauer MJ, Kakar S, Bass NM, Kuusisto J, Laakso M, Alt FW, Newgard CB, Farese RV Jr, Kahn CR, Verdin E. Mol Cell. 2011 Oct 21;44(2):177-90. doi: 10.1016/j.molcel.2011.07.019. PMID: 21856199
3. Mitochondrial sirtuins: regulators of protein acylation and metabolism. He W, Newman JC, Wang MZ, Ho L, Verdin E. Trends Endocrinol Metab. 2012 Sep;23(9):467-76. doi: 10.1016/j.tem.2012.07.004. Epub 2012 Aug 16. Review. PMID:22902903 (review)
4. SIRT4 regulates ATP homeostasis and mediates a retrograde signaling via AMPK. Ho L, Titus AS, Banerjee KK, George S, Lin W, Deota S, Saha AK, Nakamura K, Gut P, Verdin E, Kolthur-Seetharam U. Aging (Albany NY). 2013 Nov;5(11):835-49. PMID: 24296486
5. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. Rardin MJ, He W, Nishida Y, Newman JC, Carrico C, Danielson SR, Guo A, Gut P, Sahu AK, Li B, Uppala R, Fitch M, Riiff T, Zhu L, Zhou J, Mulhern D, Stevens RD, Ilkayeva OR, Newgard CB, Jacobson MP, Hellerstein M, Goetzman ES, Gibson BW, Verdin E. Cell Metab. 2013 Dec 3;18(6):920-33. doi: 10.1016/j.cmet.2013.11.013. PMID: 24315375
6. SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target. Nishida Y, Rardin MJ, Carrico C, He W, Sahu AK, Gut P, Najjar R, Fitch M, Hellerstein M, Gibson BW, Verdin E. Mol Cell. 2015 Jul 16;59(2):321-32. doi: 10.1016/j.molcel.2015.05.022. Epub 2015 Jun 11. PMID: 26073543
We are broadly interested in how signals from the environment, such as what and how we eat, are transmitted into cells throughout the body to affect aging and disease. Some of these signals may offer windows into fundamental mechanisms of aging. Research targeting the process of aging holds out the hope of delaying or improving multiple chronic diseases simultaneously (such as diabetes, heart disease, and Alzheimer’s disease) while also having similar benefits on multifactorial geriatric syndromes like frailty or delirium. Interventions targeting the process of aging might therefore help older adults remain healthier and independent longer.
Model organism studies have identified a number of genes and interventions that simultaneously affect various aspects of aging and induce resistance to multiple stressors. The best-characterized longevity intervention to date is dietary restriction (DR), which in many species can prolong lifespan and delay a variety of age-related diseases. Many of the benefits of DR are due to specific nutrient-responsive pathways that can be manipulated independently of diet, such as insulin/insulin-like growth factor (IGF) pathways, target of rapamycin (mTOR) pathways, and the sirtuin NAD+-dependent deacetylases. For example, rapamycin and metformin extend lifespan when fed to normal mice, and SIRT3 mediates DR’s prevention of age-related hearing loss in mice.
A common feature of varied DR regimens is the production of ketone bodies, small lipid-derived molecules that serve as a source of energy in times of fasting. Our group recently found that the major ketone body in humans, b-hydroxybutyrate (BHB), inhibits histone deacetylases (HDACs) at concentrations reached during CR or fasting. Alterations in protein acetylation control many aspects of cellular function through epigenetic regulation of gene expression, as well as via post-translational protein modification. Inhibition of HDACs by BHB is one of several ways in which BHB is not just a simple carrier of energy, but acts as a signal as well (Figure 1).
We found that BHB inhibits class I deacetylases in vitro and causes histone hyperacetylation in vivo. Dietary states that elevate BHB, including CR and fasting, also cause histone hyperacetylation (Figure 2). BHB specifically causes histone hyperacetylation at the promoters of FOXO3 (a transcription factor implicated in longevity) and MT2 (an antioxidant protein). This results in up-regulation of FOXO3 and MT2 expression, and increases resistance to oxidative stress in the mouse kidney.
Interestingly, a link between modulation of HDAC function and aging is well-established in model organisms. A modest reduction in the activity of class I HDACs via genetic manipulation or drug treatment prolongs lifespan in yeast and flies, and reduces age-related cognitive decline in mice. HDACs control many cellular pathways relevant to aging. b-hydroxybutyrate is the only endogenous, small-molecule inhibitor of deacetylases so far identified in mammals. We hypothesize that ketone bodies, as endogenous HDAC inhibitors, may therefore be an epigenetic mediator of some of the benefits of DR by reprogramming gene expression and causing up-regulation of genes involved in pathways that promote healthspan and longevity (Figure 3).
1. Shimazu T, MD Hirschey, J Newman, W He, K Shirakawa, N Le Moan, CA Grueter, H Lim, LR Saunders, RD Stevens, CB Newgard, RV Farese, R de Cabo, S Ulrich, K Akassoglou, and E Verdin. Suppression of oxidative stress by b-hydroxybutyrate, an endogenous histone deacetylase inhibitor. Science. 339(6116):211-4 (2013) PMID: 23223453.
2. Newman JC and E Verdin. Ketone bodies as signaling metabolites. Trends Endocrinol Metab. 25(1):42-52 (2014). PMID: 24140022. (review)
3. Newman JC and E Verdin. b-hydroxybutyrate: Much more than a metabolite. Diabetes Res Clin Pract. 106(2):173-81 (2014). PMID: 25193333. (review)
4. John C. Newman, Anthony J. Covarrubias, Minghao Zhao, Xinxing Yu, Philipp Gut, Che-Ping Ng, Yu Huang, Saptarsi Haldar, Eric Verdin. Ketogenic diet reduces midlife mortality and improves memory in aging mice. Cell Metab. 2017 Sep 5;26(3):547-557.e8. PMID: 28877458