Aging and Metabolism
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