Developing Chemical Tools for Studying Delipoylating Enzymes

Lysine lipoylation is the covalent attachment of a lipoyl group to lysine residues in proteins. Lysine lipoylation has been found in a number of important metabolic enzyme complexes, such as pyruvate deydrogenase (PDH). Literatures have shown that lysine lipoylation is essential for maintaining the activity of these enzyme complexes. A recent study discovered that SIRT4 could serve as lipoamidase and remove the lipoyl group from PDH complex, resulting in the loss of activity of PDH. Till date, research on lysine delipoylation has been very limited. In this project, we seek to develop novel chemical tools to investigate delipoylating enzymes. Such tools will expand our understanding of the roles of lipoamidases in various biological processes. We will first develop fluorescent probes that can report delipoylation activity. A panel of peptide-based fluorescent probes will be designed and synthesized to detect the delipoylation activity of enzymes. Upon incubating the probe with a suitable enzyme, delipoylation will occur and the fluorescence will be “turned on”. We will test the probes with various sirtuins. Through the study, we will generate reaction-based fluorescent probes to detect the activity of delipoylating enzymes for the first time. Our preliminary results demonstrated that SIRT1 and SIRT2 can recognize lipoyl-lysine residue as substrate. In the next step, we will develop mechanism-based inhibitors based on lipoyl-lysine substrate. Previous study has shown that thiolacetyl-lysine-containing peptides can inhibit sirtuin activity by forming a stable ADP ribose adduct. We hypothesize that thiollipoyl-lysine residue could be recognized by SIRT1/2 and form stable ADP ribose adduct as well. A library of inhibitors incorporating thiollipoyl-lysine moiety will be designed and synthesized. Subsequently the inhibitor libraries will be anchored onto microarray to investigate the binding specificity of sirtuins. Through the research, we hope to identify potent sirtuin inhibitors, which can be developed into useful anti-cancer agents.In the third part of the study, we will seek to generate chemical tools both to detect and to identify novel delipoylating enzymes in complex cellular environment. We will introduce a photo-cross-linker diazirine into our model probe. After being synthesized, the probes will first be used to detect the delipoylation activity in cellular lysates. They will then be applied to protein profiling experiments with various cancer cellular lysates. Through these experiments, novel endogenous delipoylating enzymes will be identified. Furthermore, we will also gain a deeper understanding of the expression levels of delipoylating enzymes in various biological samples.