title: A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity.
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.
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544.
authors.forename: Yana V
authors.initials: YV
authors.lastname: Miteva
authors.forename: Ileana M
authors.initials: IM
authors.lastname: Cristea
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D004265:DNA Helicases; D005786:Gene Expression Regulation; D006801:Humans; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000235:genetics*; D040901:Proteomics; D020365:RNA Helicases; D000067776:RNA Recognition Motif Proteins; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000067776:RNA Recognition Motif Proteins; C516592:SIRT6 protein, human; D037761:Sirtuins; D004265:DNA Helicases; C116070:G3BP1 protein, human; D020365:RNA Helicases