Abstract
Sirtuin 6 (SIRT6), a member of the mammalian sirtuin family, is a nuclear deacetylase with substrate-specific NAD(+)-dependent activity. SIRT6 has emerged as a critical regulator of diverse processes, including DNA repair, gene expression, telomere maintenance, and metabolism. However, our knowledge regarding its interactions and regulation remains limited. Here, we present a comprehensive proteomics-based analysis of SIRT6 protein interactions and their dependence on SIRT6 catalytic activity. We also identify evolutionarily conserved SIRT6 phosphorylations, including four within a proline-rich disordered region, and show that the conserved S338 phosphorylation can modulate selected SIRT6 interactions. By integrating molecular biology tools, microscopy, immunoaffinity purifications, label-free quantitative mass spectrometry, and bioinformatic analyses, we have established the first large-scale SIRT6 interaction network. Relative protein abundances and gene ontology functional assessment highlighted proteins involved in transcription regulation, chromatin organization, nuclear transport, telomerase function, and RNA processing. Independent immunoisolations under increased stringency distinguished the most stable SIRT6 interactions. One prominent interaction with Ras-GTPase-activating protein-binding protein 1 (G3BP1) was further validated by microscopy, reciprocal purifications, and isolations in different cell types and of endogenous SIRT6. Interestingly, a subset of specific interactions, including G3BP1, were significantly reduced or abolished in isolations of catalytically deficient SIRT6 mutant, revealing previously unknown interplay between SIRT6 activity and its associations. Overall, our study reveals putative means of regulation of SIRT6 functions via interactions and modifications, providing an important resource for future studies on the molecular mechanisms underlying sirtuin functions.
Highlights
Mammalian sirtuins, SIRT1–7, are a family of seven evolutionarily conserved enzymes with homologues throughout all kingdoms of life [1, 2]
Dependent sirtuin 6 (SIRT6) Interactions plays a central role in its known functions, as the enzyme is targeted to specific chromatin regions to maintain chromatin integrity [25, 28, 29] or regulate gene expression via recruitment by transcription factors (e.g. NFkB, HIF1␣) [26, 30, 31]
HEK293 cells were selected because SIRT6 is endogenously expressed in mammalian kidney tissue [20, 51, 52], and these cells have been commonly used in SIRT6 studies [9, 36, 51, 53]
Summary
SIRT1–7, are a family of seven evolutionarily conserved enzymes with homologues throughout all kingdoms of life [1, 2]. Dependent SIRT6 Interactions plays a central role in its known functions, as the enzyme is targeted to specific chromatin regions (e.g. telomeres) to maintain chromatin integrity [25, 28, 29] or regulate gene expression via recruitment by transcription factors (e.g. NFkB, HIF1␣) [26, 30, 31] This catalytic activity was shown to be diminished upon the mutation of histidine 133 (H133), a residue conserved among all seven human sirtuins [1, 9, 25, 32]. Our findings provide a new perspective on the likely means of regulation of the diverse range of SIRT6 functions in health and disease processes
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