Tier 4 — mechanistic

A proteomic perspective of Sirtuin 6 (SIRT6) phosphorylation and interactions and their dependence on its catalytic activity

Yana V. Miteva, Ileana M. Cristea
Molecular & Cellular Proteomics 2014 Volume 13, issue 1, pages 168–183

Bibliography

PubMed
PMID 24163442
PubMed Central
PMC3879612
DOI
10.1074/mcp.M113.032847
Funding
US National Institute on Drug Abuse grants DP1DA026192, R21AI102187, and R21HD073044 to I.M.C., and Human Frontier Science Program Organization award RGY0079/2009-C to I.M.C.
Competing interests
Not explicitly stated in the published manuscript.

Study snapshot

DesignInteractome and phosphoproteomic profiling using stable isotope-free immunoaffinity purification coupled to nano-LC-MS/MS. SIRT6-EGFP wild-type, catalytically deficient H133Y mutant, and S338A phospho-null mutant compared against EGFP-only controls. Reciprocal endogenous immunoprecipitations and orthogonal validation by confocal microscopy, immunogold electron microscopy, and Western blot.
ModelHEK293 human embryonic kidney cells and CEMT human T-cell lymphoblasts, each stably expressing EGFP-tagged SIRT6 (wild-type, H133Y catalytically deficient, or S338A phospho-null).
SampleTwo biological replicates plus one technical replicate per wild-type and H133Y condition; three biological replicates per S338A condition. Additional endogenous SIRT6 immunoprecipitations from whole-cell lysate and nuclear fractions.
InterventionImmunoaffinity purification of SIRT6-EGFP complexes under an optimised mild-stringency lysis buffer and a high-stringency lysis buffer, followed by tryptic digestion and mass spectrometry. Comparison of protein interactions and phosphorylation sites between wild-type and catalytically deficient SIRT6.
DurationNot applicable — biochemical steady-state analysis.
EndpointsNumber and identity of SIRT6-interacting proteins passing SAINT specificity criteria; Identity and site-mapping of SIRT6 phosphorylation events; Effect of H133Y catalytic disruption on the SIRT6 interactome; Effect of S338A phospho-null mutation on nuclear-pore-complex interactions; Validation of SIRT6-G3BP1 interaction across cell types and endogenous SIRT6

What the study showed, in plain terms

This is not a supplement or an animal study. It is a large-scale protein chemistry analysis using human cell lines, and it laid the groundwork for understanding how SIRT6 is switched on and off inside a cell.

The Princeton team wanted to answer two connected questions. Which proteins does SIRT6 physically interact with inside the nucleus, and does SIRT6 itself carry chemical tags that turn its activity up or down? To find out, they engineered human kidney cells to make a slightly modified SIRT6 that could be pulled out of the cell along with anything it was holding on to, then identified every attached protein by mass spectrometry.

They found seventy-four different SIRT6 partners falling into recognisable groups — proteins that control gene transcription, proteins that repair damaged DNA, proteins that organise chromatin, and proteins that shuttle molecules in and out of the nucleus. They also mapped five sites on SIRT6 that carry phosphate tags, four of them clustered in a floppy region at the tail end of the protein. Two of these tags, on positions serine 10 and serine 338, later became central to the story of how SIRT6 is regulated.

Most strikingly, when the team broke SIRT6's catalytic machinery, many of the strongest interactions disappeared — including a prominent partnership with a protein called G3BP1. In other words, SIRT6 does not simply do its enzyme job and then move on. It uses its enzyme activity to hold on to other proteins. This linkage between what SIRT6 does and who it works with reshaped how researchers think about SIRT6 regulation.

Key findings

  • Seventy-four SIRT6-interacting proteins were identified with high confidence (SAINT score above 0.85) across replicate experiments in HEK293 cells, spanning transcription regulation, DNA repair, chromatin organisation, nuclear transport, telomerase regulation, mRNA and rRNA processing, and neurofilament network formation.
  • Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) emerged as the most abundant SIRT6 partner across every experimental condition, including high-stringency isolations, immunoprecipitations of endogenous SIRT6, and CEMT T-cell lymphoblasts confirming that the interaction is not cell-type-specific.
  • Five evolutionarily conserved SIRT6 phosphorylation sites were mapped by mass spectrometry: S10 at the amino terminus, and T294, S303, S330, and S338 clustered within a proline-rich, natively disordered region at the carboxy terminus.
  • Disruption of SIRT6 catalytic activity by the H133Y mutation abolished or downregulated more than 50-fold a specific subset of interactions including G3BP1, YLPM1, USP7, and several nucleoporin family members, while other partners such as lamins, histones H2AFX and H2AFY, TP53, and MKI67 remained bound at wild-type levels.
  • The S338A phospho-null SIRT6 mutant showed reduced association with nuclear-pore-complex proteins compared with wild-type SIRT6, indicating that phosphorylation at S338 modulates a subset of SIRT6 interactions relevant to nucleoplasmic localisation.
  • Immunogold electron microscopy confirmed that a subset of SIRT6 localises to the vicinity of nuclear pores, consistent with the biochemically observed nucleoporin interactions and suggesting a putative role for the nuclear pore in organising SIRT6-dependent chromatin regulation.

What this study can and cannot tell us

This study uses cultured human cell lines and does not test whether the SIRT6 interactions or phosphorylation sites identified here have functional consequences in tissue, animal, or human physiology. Cell-type-specific SIRT6 partners in liver, brain, or muscle may differ from those catalogued in HEK293 kidney cells.

Overexpression of EGFP-tagged SIRT6 at roughly three-fold above endogenous levels is used as the primary discovery tool. While endogenous SIRT6 immunoprecipitations partially replicate the interaction network, the confidence for lower-abundance interactions relies on the tagged-overexpression system. The EGFP tag itself, although small, may perturb selected interactions that depend on the SIRT6 amino or carboxy terminus.

The paper identifies SIRT6 phosphorylation sites and demonstrates that S338 phosphorylation modulates specific interactions, but it does not identify the kinases responsible for these modifications or the physiological signals that trigger them. Subsequent work (Van Meter et al. 2016) later identified c-Jun N-terminal kinase as the S10 phosphorylator under oxidative stress; the kinases for S303, S330, and S338 remain to be elucidated.

The catalytically deficient H133Y mutant is used to argue that SIRT6 catalytic activity is required to hold specific interactions such as G3BP1. However, the H133Y mutation disrupts NAD-binding pocket geometry as well as catalysis, so an inability to bind NAD or subtle conformational effects cannot be entirely excluded as contributors to the observed loss of interactions.

Editorial review

Reviewed by the Biohack Blueprint research team

Last verified

Dr. Khaled Mahmoud, MBBChCritical Care Medicine, Longevity Science & Editorial Direction Dr. Amr Yasser, PhDCardiometabolic, Musculoskeletal, Gut & Bioavailability Dr. Abdulla Mostafa, PhDCellular Senescence, Neurological, Mitochondrial & Brain-Ageing Biology