Study On The Activation Mechanism Of Novel Peptide SIRT1 Activators

Sirtuins(SIRTs)are a class of deacylases that can remove acyl modifications at specific lysine sites of various protein substrates with the participation of NAD+and regulate many physiological functions such as transcription and metabolism,involved in the development of inflammation,cardiovascular diseases,acute kidney injury and neurodegenerative diseases.SIRT1 is a member of the SIRTs family and catalyzes the deacetylation of proteins,coupled with the breakdown of NAD~+into nicotinamide and2’-O-acetyl-ADP-ribose(OAADPr).In the present study,we confirmed that the tripeptide CWR significantly promotes SIRT1 enzymatic activity,and quantitative HPLC results confirmed that its activity is independent of the chemical moiety immediately C-terminal to the Acetyl-Lysine of the substrate.CWR uses a novel activation mechanism to promote SIRT1 enzyme activity:it bonds covalently to C1’on the ribose ring of the product OAADPr.We confirmed that CWR is highly selective with no effect on the enzymatic activity of SIRT2,SIRT3,SIRT5,and SIRT6.Molecular docking of CWR covalently bound to ADPr with SIRT1 yielded a model of their binding,and in this way successfully explained the source of the high selectivity of the CWR,namely,the distance between the arginine at position 274 in SIRT1(also a very conserved amino acid in SIRTs)and C1’on the ribose ring of OAADPr compared to the arginine in SIRT2,SIRT3,SIRT5 and SIRT6,is more distant.The distance affects the interaction of arginine with ADPr,which in turn affects the covalent bonding of CWR to ADPr.This was verified by the site-directed mutagenesis of SIRT3.We determined by enzyme kinetic analysis that CWR promoted the binding of acetylated peptides and SIRT1,but not the binding of NAD~+and SIRT1.Finally,we demonstrated that CWR reduced the acetylation level of p53 in cells by activating SIRT1 in a concentration-dependent manner.The elucidation of the activation mechanism of CWR can provide a new perspective for the design of SIRT1 activators that do not rely on the chemical moiety immediately C-terminal to the Acetyl-Lysine of the substrate,CWR and its structural analogs can be used for the basic research of age-related diseases.