Sirtuin-regulated lipoylation is an evolutionarily conserved mediator of metabolic health and disease

Abstract

Lipoylation is a rare, but highly conserved lysine posttranslational modification. To date, it is known to occur on only four multimeric metabolic enzymes in mammals, yet these proteins are staples in the core metabolic landscape. The dysregulation of these mitochondrial proteins is linked to a range of human metabolic disorders. Perhaps most striking is that lipoylation itself, the proteins that add or remove the modification, as well as the proteins it decorates are all evolutionarily conserved from bacteria to humans, highlighting the importance of this essential cofactor. My thesis work investigates the biological significance of protein lipoylation, and provides a better understanding of its regulation in health and disease states. We discovered mitochondrial sirtuin 4 (SIRT4) as the first known cellular lipoamidase in mammals, removing lipoyl modifications from the pyruvate dehydrogenase complex (PDH). PDH is a metabolic linchpin, facilitating the flow of carbon into the TCA cycle following glycolysis. Further expanding our knowledge of cellular lipoamidases, we next demonstrated that this enzymatic activity is conserved in bacterial sirtuins, which share close sequence homology to SIRT4. In both studies, we used a multidisciplinary approach, integrating in vitro and in vivo assays, including biochemical, mass spectrometry-based proteomic, and functional assays. Underscoring the metabolic importance of lipoylation, we showed that bacterial sirtuins inhibit PDH and the alpha-ketoglutarate dehydrogenase complex (KDH) in both gram-negative and gram-positive bacteria. KDH is another carbon entry point into the TCA cycle. We established the dynamic response of this enzymatic activity to its cellular environment, showing that sirtuin lipoamidase activity responds to nutrient availability. Finally, we investigated the role of lipoylation during disease states. Aberrant function of lipoylated complexes, like PDH, have been implicated in metabolic disorders, cancer, and Alzheimer’s disease. We previously discovered that human SIRT4 acts against DNA virus infections, while the bacterial sirtuin CobB acts against bacteriophage infections. Using human cytomegalovirus (HCMV), we examined the antiviral role of SIRT4, and the mechanism through which HCMV inhibits SIRT4 functions. Altogether, this work demonstrates that understanding sirtuin-regulated lipoylation provides a unique and specific target to manipulate the activities of these metabolic complexes when studying disease or developing therapeutics.