SIRT6 is an epigenetic modification enzyme that regulates gene transcription through its deacetylase activity. In addition to histone protein, SIRT6 also modify other proteins and enzymes, some of which are central players in metabolic reprogramming and aging process. Therefore, SIRT6 has emerged as a therapeutic target for the treatment of metabolic disorder and age-related diseases. Here, we report that SIRT6 deacetylates lysine 382 of p53 in short synthetic peptide sequence and in full length p53. Further studies showed that the deacetylation of H3K9Ac and p53K382Ac are insensitive to nicotinamide inhibition, but are sensitive to trichostatin A (TSA) inhibition. Detailed kinetic analysis revealed that TSA competes with the peptide substrate for inhibition, and this inhibition is unique to SIRT6 in the sirtuin family. Taken together, this study not only suggests potential roles of SIRT6 in regulating apoptosis and stress resistance via direct deacetylation of p53, but also provides lead compound for the development of potent and selective SIRT6 inhibitors.
Sirtuins consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD+) to remove an acetyl group from lysine residues. These enzymes have been implicated in regulating various cellular events and have also been suggested to mediate the beneficial effects of calorie restriction (CR). However, controversies on sirtuin biology also peaked during the past 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. 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. We have obtained a set of powerful activity-based chemical probes that are capable of assessing the active content of sirtuins in model systems. These probes consist of a chemical “warhead” that binds to the active site of active enzyme and a handle that can be used for the visualization of these enzymes by fluorescence. In complex native proteome, the probes can selectively “highlight” the active sirtuin components. Furthermore, these probes were also able to probe the dynamic change of sirtuin activity in response to cellular stimuli. These chemical probes and the labeling strategies will provide transformative technology to allow the direct linking of sirtuin activity to distinct physiological processes. They will create new opportunities to investigate how sirtuins provide health benefits in adapting cells to environmental cues and provide critical information to dissect sirtuin regulatory networks.
The C. jejuni CG8421 challenge model provides a safe and effective tool, without the risk of Guillain-Barré syndrome. The model demonstrates high attack rates after lower doses of challenge inoculum, provides further understanding of immunologic responses, and permits future investigation of candidate Campylobacter vaccines.
SIRT1 is the most extensively studied human sirtuin with a broad spectrum of endogenous targets. It has been implicated in the regulation of a myriad of cellular events, such as gene transcription, mitochondria biogenesis, insulin secretion as well as glucose and lipid metabolism. From a mechanistic perspective, nicotinamide (NAM), a byproduct of a sirtuin-catalyzed reaction, reverses a reaction intermediate to regenerate NAD+ through “base exchange”, leading to the inhibition of the forward deacetylation. NAM has been suggested as a universal sirtuin negative regulator. Sirtuins have evolved different strategies in response to NAM regulation. Here, we report the detailed kinetic analysis of SIRT1-catalyzed reactions using endogenous substrate-based synthetic peptides. A novel substrate-dependent sensitivity of SIRT1 to NAM inhibition was observed. Additionally, SIRT1 demonstrated pH-dependent deacetylation with normal solvent isotope effects (SIEs), consistent with proton transfer in the rate-limiting step. Base exchange, in contrast, was insensitive to pH changes with no apparent SIEs, indicative of lack of proton transfer in the rate-limiting step. Consequently, NAM inhibition was attenuated at a high pH in proteated buffers. Our study provides new evidence for “activation by de-repression” as an effective sirtuin activation strategy.
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