Sirtuins, also called Class III HDACs, consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD+) to remove acetyl group from lysine residues and to produce nicotinamide and O-acetyl-ADP-ribose. This intriguing class of enzymes has been implicated in regulating various cellular events and has also been suggested to mediate the beneficial effects of calorie restriction (CR). Sirtuins have been intensely pursued by academia and pharmaceutical industry as therapeutic targets. However, controversies on sirtuin biology also peaked during the last few years because of conflicting results from different research groups. This is partly because these enzymes have been discovered recently, and the intricate interaction loops between sirtuins and other proteins make the characterization of them extremely difficult. One of the daunting tasks is to correlate sirtuin activity to disease pathogenesis. Current molecular biology and proteomics techniques report protein abundance rather than active sirtuin content. Innovative chemical tools that can directly probe the functional state of sirtuins are desperately needed. This grant application proposes to take a highly integrative approach to interrogate the functional state of sirtuins in complex biological samples. Our preliminary results demonstrate the feasibility of this strategy. We have obtained a set of powerful chemical probes that are capable of assessing the active content of sirtuins in model systems. In this proposal we plan to synthesize focused libraries of activity-based chemical probes. The scaffolds are designed for enhanced selectivity and labeling efficiency. The probes that score favorably in labeling of recombinant sirtuins will be subjected to the profiling of whole cell lysate. In complex native proteome, the probe should selectively ?highlight? the active sirtuin components. Combined with mass spectrometry based proteomics analysis, this strategy should unveil the functional profile of sirtuins under different physiological and pathological conditions. This will provide information on how abnormal enzyme activity will contribute to disease progression. Furthermore, cell permeable probes will also be employed in cellular imaging study. It will enable the simultaneous detection of functional state and localization and empower the direct analysis of sirtuin function in response to cellular and environmental cues.
Sirtuins are epigenetic modifying enzymes that regulate gene transcription through their deacetylase activity. In addition to histone proteins, sirtuins also modify other protein and enzyme targets, many of which are central players in metabolic reprogramming and aging process. Therefore, the mammalian sirtuins are emerging targets for the treatment of metabolic disorder and age-related diseases. This project aims at developing innovative chemical tools to elucidate the dynamic and essential changes of sirtuin activity in response to environmental and cellular stimuli, with the view to eventuate in sirtuin-targeted therapeutics.
|Wood, Marci; Rymarchyk, Stacia; Zheng, Song et al. (2018) Trichostatin A inhibits deacetylation of histone H3 and p53 by SIRT6. Arch Biochem Biophys 638:8-17|
|Graham, Elysian; Rymarchyk, Stacia; Wood, Marci et al. (2018) Development of Activity-Based Chemical Probes for Human Sirtuins. ACS Chem Biol 13:782-792|
|Tran, Ai; Yokose, Ryota; Cen, Yana (2018) Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside. Org Biomol Chem 16:3662-3671|