Structural and functional study of human SIRT2 and SIRT1

Abstract

Posttranslational modification (PTM) refers to the functional groups that are covalently attached to histones and non-histone proteins after translation to alter protein binding partners, enzyme activities, cellular localizations and protein stabilities. Writers and erasers dynamically add or remove PTMs upon intra or extra cellular changes, which makes them important in almost every cell process. Sirtuins, responsible for removing acylation modifications, are classified as HDAC III due to their need of NAD+ as cofactor. They are important drug targets because of their essential roles in processes such as aging, inflammation, apoptosis and gene transcription. Knowing the structure and reaction mechanism of deacylation could provide useful information for Sirtuin enzyme activity modulation. However, the reaction of Sirtuin deacylation is a multi-step process and the reaction is quite fast. So up to now, most of the structures to disclose the reaction mechanism use analogs of NAD+ or use thioacylation to stall the reaction. It is hard to justify whether the captured structures represent true reaction intermediates or just an artificial effect. Here, in this thesis, I demonstrate the structures of several intermediates of SIRT2 demyristoylation with native substrates. The first structure is before the reaction, in which SIRT2 binds a H3K18 myristoylation peptide only, which shows a quite similar binding pattern of SIRT2 binding with other myristoylated peptides. The second structure is the one of SIRT2 with both modified peptide and intact NAD+, showing a productive NAD+ conformation. The next stage is intermediate I and II after nicotinamide cleavage, which is the first native intermediate still with nicotinamide in the active site. The last one is the structure with the product, 2’-O-myristoyl-ADP-ribose. Some of the structures presented here are the first native intermediates captured, truly helping in explaining how deacylation reaction progresses. Lipoylation, which is important in regulating metabolic enzymes’ activities, has been recently found to be the substrate of SIRT4. Here, in this thesis, I show another Sirtuin, SIRT2 is a rather more efficient lipoylation remover. Via molecule docking, the detailed interaction between lipoylation and SIRT2 is analyzed. Then biochemistry experiments demonstrate that SIRT2 has the highest de-lipoylation activity among all the Sirtuins. Further cellular experiments show that through delipoylation, SIRT2 can indeed influence PDH lipoylation status, thus regulating cell metabolism. Of all the Sirtuins, SIRT1 structure solving is the most difficult, due to its high flexibility. Up to now, there is only seven structures of SIRT1 solved, six of which are at low resolution, below 2.5Å . The only one high resolution is in the closed state. Here in this thesis, I show the SIRT1 structure with the highest resolution, 1.79Å in open form. In the structure, an N-terminal arginine from the symmetric molecule protrudes into the catalytic active site, mimicking an unmodified lysine, which would hamper the entry of small molecule inhibitors and may act as an auto regulation method of SIRT1. Those studies here provide deeper insights into reaction mechanism and functional aspects of Sirtuin family proteins, helping with drug design targeting Sirtuins.