Molecular architecture of the human protein deacetylase Sirt1 and its regulation by AROS and resveratrol

Abstract

Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses and ageing processes. Among the seven mammalian Sirtuins, Sirt1 is the physiologically best-studied isoform. It regulates nuclear functions such as chromatin remodelling and gene transcription, and it appears to mediate beneficial effects of a low calorie diet which can partly be mimicked by the Sirt1 activating polyphenol resveratrol. The molecular details of Sirt1 domain architecture and regulation, however, are little understood. It has a unique N-terminal domain and CTD (C-terminal domain) flanking a conserved Sirtuin catalytic core and these extensions are assumed to mediate Sirt1-specific features such as homo-oligomerization and activation by resveratrol. To analyse the architecture of human Sirt1 and functions of its N- and C-terminal extensions, we recombinantly produced Sirt1 and Sirt1 deletion constructs as well as the AROS (active regulator of Sirt1) protein. We then studied Sirt1 features such as molecular size, secondary structure and stimulation by small molecules and AROS. We find that Sirt1 is monomeric and has extended conformations in its flanking domains, likely disordered especially in the N-terminus, resulting in an increased hydrodynamic radius. Nevertheless, both termini increase Sirt1 deacetylase activity, indicating a regulatory function. We also find an unusual but defined conformation for AROS protein, which fails, however, to stimulate Sirt1. Resveratrol, in contrast, activates the Sirt1 catalytic core independent of the terminal domains, indicating a binding site within the catalytic core and suggesting that small molecule activators for other isoforms might also exist.

The N-Terminal Domain of SIRT1 Is a Positive Regulator of Endogenous SIRT1-Dependent Deacetylation and Transcriptional Outputs

“…The self-association of overexpressed NTERM domains through an interaction of motifs A and B is consistent with SIRT1 dimerization (Guo et al, 2012), although recombinant SIRT1 proteins purified from E. coli or Sf9 insect cells are monomeric (our results, Lakshminarasimhan et al, 2013b). SIRT1 oligomerization may require additional posttranslational modifications, or partner proteins that assemble into a multiprotein complex.…”

An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

“…Human Sirt1 residues 1-747 and 225-664, Sirt2-34-356, Sirt3-114-399 and 114-380, Sirt4-25-314, Sirt5-34-302, Sirt6-13-308 and 13-355, and Sirt7-1-400, 59-356, and 14-367 were expressed with N-terminal His-tag in E. coli 29,44,45 . Proteins were purified through affinity and size exclusion chromatography (see also Supplementary Methods).…”

AROS has a context‐dependent effect on SIRT1

“…The same heat shock pathway is activated by SIRT1 overexpression [34]. However, in a very recently published study, AROS failed to activate SIRT1 [35].…”