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Identifying the functional contribution of the defatty-acylase activity of SIRT6

Abstract

Mammalian sirtuin 6 (SIRT6) exhibits many pivotal functions and multiple enzymatic activities, but the contribution of each activity to the various functions is unclear. We identified a SIRT6 mutant (G60A) that possesses efficient defatty-acylase activity but has substantially decreased deacetylase activity in vitro and no detectable deacetylase activity in cells. The G60A mutant has a decreased ability to bind NAD+, but the presence of fatty-acyl lysine peptides restores NAD+ binding, explaining the retention of the defatty-acylase activity. Using this mutant, we found that the defatty-acylase activity of SIRT6 regulates the secretion of numerous proteins. Notably, many ribosomal proteins were secreted via exosomes from Sirt6 knockout mouse embryonic fibroblasts, and these exosomes increased NIH 3T3 cell proliferation compared with control exosomes. Our data indicate that distinct activities of SIRT6 regulate different pathways and that the G60A mutant is a useful tool to study the contribution of defatty-acylase activity to SIRT6's various functions.

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Figure 1: In vitro deacetylation and defatty acylation activities of WT SIRT6 and mutants.
Figure 2: Mechanistic study on how SIRT6 G60A maintains defatty acylation but loses deacetylation.
Figure 3: In-cell validation of the deacetylase and defatty-acylase activities of WT and mutant SIRT6.
Figure 4: Analysis of secreted proteins by SILAC.
Figure 5: Validation of secreted proteins regulated by SIRT6.
Figure 6: SIRT6 defatty-acylase activity inhibits ribosomal protein sorting to the exosomes.

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Acknowledgements

We thank R. Mostoslavsky (Massachusetts General Hospital) for providing the Sirt6 WT and KO MEFs, P. Chen (Cornell University) for the use of the fluorescence spectrophotometer and S. Zhang (proteomic and MS facility of Cornell University) for help with the SILAC experiments. SILAC experiments performed on a mass spectrometer were supported by a US National Institutes of Health (NIH) SIG grant 1S10 OD017992-01. Imaging data were acquired in the Cornell BRC imaging facility using a Zeiss LSM880 confocal/multiphoton microscope funded by NYSTEM (CO29155) and NIH (S10OD018516). This work is supported by a grant from the NIH NIGMS (R01 GM098596 to H.L.).

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Authors and Affiliations

Authors

Contributions

X.Z. and H.L. designed the research and wrote the manuscript; X.Z. performed biochemical and cellular studies; S.K. synthesized peptides for the in vitro deacylation study; H.J. made SIRT6 WT and TNF-α plasmids; M.A.A. performed exosome fractionation experiments; X.C. made HEK293T SIRT6 KO cells; N.A.S. and J.H.S. synthesized Alk14 and BODIPY-N3; X.Z., R.A.C. and H.L. analyzed the results; H.L. directed the study.

Corresponding author

Correspondence to Hening Lin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–5 and Supplementary Figures 1–18. (PDF 4223 kb)

Supplementary Data Set 1

Secretion SILAC in Sirt6 WT and KO MEFs. (XLSX 124 kb)

Supplementary Data Set 2

Proteins detected in all three SILACs. (XLSX 57 kb)

Supplementary Data Set 3

Secretion SILAC in Sirt6 KO MEFs and Sirt6 KO MEFsexpressing SIRT6 WT. (XLSX 118 kb)

Supplementary Data Set 4

Secretion SILAC in Sirt6 KO MEFs and Sirt6 KO MEFsexpressing SIRT6 G60A. (XLSX 108 kb)

Supplementary Data Set 5

Secretion SILAC in Sirt6 KO MEFs and Sirt6 KO MEFsexpressing SIRT6 H133Y. (XLSX 110 kb)

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Zhang, X., Khan, S., Jiang, H. et al. Identifying the functional contribution of the defatty-acylase activity of SIRT6. Nat Chem Biol 12, 614–620 (2016). https://doi.org/10.1038/nchembio.2106

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