Publications

• Sirtuin Lipoamidase Activity Is Conserved in Bacteria as a Regulator of Metabolic Enzyme Complexes.

  Rowland E.A., Greco T.M., Snowden C.K., McCabe A.L., Silhavy T.J., Cristea I.M.

  Lipoic acid is an essential metabolic cofactor added as a posttranslational modification on several multimeric enzyme complexes. These protein complexes, evolutionarily conserved from bacteria to humans, are core regulators of cellular metabolism. While the multistep enzymatic process of adding li ... >> More

  Lipoic acid is an essential metabolic cofactor added as a posttranslational modification on several multimeric enzyme complexes. These protein complexes, evolutionarily conserved from bacteria to humans, are core regulators of cellular metabolism. While the multistep enzymatic process of adding lipoyl modifications has been well characterized in <i>Escherichia coli</i>, the enzyme required for the removal of these lipoyl moieties (i.e., a lipoamidase or delipoylase) has not yet been identified. Here, we describe our discovery of sirtuins as lipoamidases in bacteria and establish their conserved substrates. Specifically, by using a series of knockout, overexpression, biochemical, <i>in vitro</i>, proteomic, and functional assays, we determined the substrates of sirtuin CobB in <i>E. coli</i> as components of the pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KDH), and glycine cleavage (GCV) complexes. <i>In vitro</i> assays provided direct evidence for this specific CobB activity and its NAD⁺ dependence, a signature of all sirtuins. By designing a targeted quantitative mass spectrometry method, we further measured sirtuin-dependent, site-specific lipoylation on these substrates. The biological significance of CobB-modulated lipoylation was next established by its inhibition of both PDH and KDH activities. By restricting the carbon sources available to <i>E. coli</i>, we demonstrated that CobB regulates PDH and KDH under several growth conditions. Additionally, we found that SrtN, the sirtuin homolog in Gram-positive <i>Bacillus subtilis</i>, can also act as a lipoamidase. By demonstrating the evolutionary conservation of lipoamidase activity across sirtuin homologs, along with the conservation of common substrates, this work emphasizes the significance of protein lipoylation in regulating central metabolic processes.<b>IMPORTANCE</b> Here, we demonstrate that sirtuin lipoamidase activity exists in both Gram-positive and Gram-negative bacteria and establishing its conservation from bacteria to humans. Specifically, we discovered that CobB and SrtN act as lipoamidases in <i>E. coli</i> and <i>B. subtilis</i>, respectively. Intriguingly, not only is this sirtuin enzymatic activity conserved, but also the lipoylated substrates and functions are conserved, as bacterial sirtuins negatively regulate the lipoylation levels and activities of PDH and KDH. Considering that PDH and KDH regulate two carbon entry points into the tricarboxylic acid cycle, our finding highlights lipoylation as a conserved molecular toggle that regulates central metabolic pathways. Indeed, our findings from tests in which we limited nutrient availability support this. Furthermore, this study illustrates how the integration of technologies from different disciplines provides avenues to uncover enzymatic activities at the core of cellular metabolism regulation. << Less

  mBio 8:e01096-17(2017) [PubMed] [EuropePMC]

• Mitochondrial Function, Metabolic Regulation, and Human Disease Viewed through the Prism of Sirtuin 4 (SIRT4) Functions.

  Betsinger C.N., Cristea I.M.

  As cellular metabolic hubs, mitochondria are the main energy producers for the cell. These organelles host essential energy producing biochemical processes, including the TCA cycle, fatty acid oxidation, and oxidative phosphorylation. An accumulating body of literature has demonstrated that a majo ... >> More

  As cellular metabolic hubs, mitochondria are the main energy producers for the cell. These organelles host essential energy producing biochemical processes, including the TCA cycle, fatty acid oxidation, and oxidative phosphorylation. An accumulating body of literature has demonstrated that a majority of mitochondrial proteins are decorated with diverse posttranslational modifications (PTMs). Given the critical roles of these proteins in cellular metabolic pathways and response to environmental stress or pathogens, understanding the role of PTMs in regulating their functions has become an area of intense investigation. A major family of enzymes that regulate PTMs within the mitochondria are sirtuins (SIRTs). Albeit until recently the least understood sirtuin, SIRT4 has emerged as an enzyme capable of removing diverse PTMs from its substrates, thereby modulating their functions. SIRT4 was shown to have ADP-ribosyltransferase, deacetylase, lipoamidase, and deacylase enzymatic activities. As metabolic dysfunction is linked to human disease, SIRT4 levels and activities have been implicated in modulating susceptibility to hyperinsulinemia and diabetes, liver disease, cancer, neurodegeneration, heart disease, aging, and pathogenic infections. Therefore, SIRT4 has emerged as a possible candidate for targeted therapeutics. Here, we discuss the diverse enzymatic activities and substrates of SIRT4 and its roles in human health and disease. << Less

  J Proteome Res 18:1929-1938(2019) [PubMed] [EuropePMC]

• Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

  Mathias R.A., Greco T.M., Oberstein A., Budayeva H.G., Chakrabarti R., Rowland E.A., Kang Y., Shenk T., Cristea I.M.

  Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellula ... >> More

  Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH). Importantly, SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity. PDH, which converts pyruvate to acetyl-CoA, has been known to be primarily regulated by phosphorylation of its E1 component. We determine that SIRT4 enzymatically hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity. We demonstrate SIRT4-mediated regulation of DLAT lipoyl levels and PDH activity in cells and in vivo, in mouse liver. Furthermore, metabolic flux switching via glutamine stimulation induces SIRT4 lipoamidase activity to inhibit PDH, highlighting SIRT4 as a guardian of cellular metabolism. << Less

  Cell 159:1615-1625(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.