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

Nature Chemical Biology volume 12, pages 614–620 (2016)Cite this article

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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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References

  1. Imai, S., Armstrong, C.M., Kaeberlein, M. & Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800 (2000).

    Article  CAS  Google Scholar 

  2. Mostoslavsky, R. et al. Genomic instability and aging-like phenotype in the absence of mammalian SIRT6. Cell 124, 315–329 (2006).

    Article  CAS  Google Scholar 

  3. Michishita, E. et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature 452, 492–496 (2008).

    Article  CAS  Google Scholar 

  4. Mao, Z. et al. SIRT6 promotes DNA repair under stress by activating PARP1. Science 332, 1443–1446 (2011).

    Article  CAS  Google Scholar 

  5. Kaidi, A., Weinert, B.T., Choudhary, C. & Jackson, S.P. Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science 329, 1348–1353 (2010).

    Article  CAS  Google Scholar 

  6. Sebastián, C. et al. The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism. Cell 151, 1185–1199 (2012).

    Article  Google Scholar 

  7. Zhong, L. et al. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 140, 280–293 (2010).

    Article  CAS  Google Scholar 

  8. Kawahara, T.L. et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-κB-dependent gene expression and organismal life span. Cell 136, 62–74 (2009).

    Article  CAS  Google Scholar 

  9. Kanfi, Y. et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature 483, 218–221 (2012).

    Article  CAS  Google Scholar 

  10. Michishita, E. et al. Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6. Cell Cycle 8, 2664–2666 (2009).

    Article  CAS  Google Scholar 

  11. Schwer, B. et al. Neural sirtuin 6 (Sirt6) ablation attenuates somatic growth and causes obesity. Proc. Natl. Acad. Sci. USA 107, 21790–21794 (2010).

    Article  CAS  Google Scholar 

  12. Yang, B., Zwaans, B.M., Eckersdorff, M. & Lombard, D.B. The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promote genomic stability. Cell Cycle 8, 2662–2663 (2009).

    Article  CAS  Google Scholar 

  13. Feldman, J.L., Baeza, J. & Denu, J.M. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J. Biol. Chem. 288, 31350–31356 (2013).

    Article  CAS  Google Scholar 

  14. Jiang, H. et al. SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine. Nature 496, 110–113 (2013).

    Article  CAS  Google Scholar 

  15. Liszt, G., Ford, E., Kurtev, M. & Guarente, L. Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J. Biol. Chem. 280, 21313–21320 (2005).

    Article  CAS  Google Scholar 

  16. Gil, R., Barth, S., Kanfi, Y. & Cohen, H.Y. SIRT6 exhibits nucleosome-dependent deacetylase activity. Nucleic Acids Res. 41, 8537–8545 (2013).

    Article  CAS  Google Scholar 

  17. Du, J., Jiang, H. & Lin, H. Investigating the ADP-ribosyltransferase activity of sirtuins with NAD analogues and 32P-NAD. Biochemistry 48, 2878–2890 (2009).

    Article  CAS  Google Scholar 

  18. Pan, P.W. et al. Structure and biochemical functions of SIRT6. J. Biol. Chem. 286, 14575–14587 (2011).

    Article  CAS  Google Scholar 

  19. Tennen, R.I., Berber, E. & Chua, K.F. Functional dissection of SIRT6: identification of domains that regulate histone deacetylase activity and chromatin localization. Mech. Ageing Dev. 131, 185–192 (2010).

    Article  CAS  Google Scholar 

  20. Al-Nedawi, K., Meehan, B. & Rak, J. Microvesicles: messengers and mediators of tumor progression. Cell Cycle 8, 2014–2018 (2009).

    Article  CAS  Google Scholar 

  21. Antonyak, M.A. et al. Cancer cell-derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells. Proc. Natl. Acad. Sci. USA 108, 4852–4857 (2011).

    Article  CAS  Google Scholar 

  22. Costa-Silva, B. et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat. Cell Biol. 17, 816–826 (2015).

    Article  CAS  Google Scholar 

  23. Ewald, C.Y., Landis, J.N., Porter Abate, J., Murphy, C.T. & Blackwell, T.K. Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity. Nature 519, 97–101 (2015).

    Article  CAS  Google Scholar 

  24. Zhu, A.Y. et al. Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine. ACS Chem. Biol. 7, 155–159 (2012).

    Article  CAS  Google Scholar 

  25. Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).

    Article  CAS  Google Scholar 

  26. Jiang, H., Kim, J.H., Frizzell, K.M., Kraus, W.L. & Lin, H. Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases. J. Am. Chem. Soc. 132, 9363–9372 (2010).

    Article  CAS  Google Scholar 

  27. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  28. Jedrusik-Bode, M. et al. The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals. J. Cell Sci. 126, 5166–5177 (2013).

    Article  CAS  Google Scholar 

  29. Huang, Z. et al. Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene 31, 3709–3720 (2012).

    Article  CAS  Google Scholar 

  30. Soo, C.Y. et al. Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology 136, 192–197 (2012).

    Article  CAS  Google Scholar 

Download references

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

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York, USA

    Xiaoyu Zhang, Saba Khan, Hong Jiang, Xiao Chen, Nicole A Spiegelman, Jonathan H Shrimp, Richard A Cerione & Hening Lin

  2. Department of Molecular Medicine, Cornell University, Ithaca, New York, USA

    Marc A Antonyak & Richard A Cerione

  3. Howard Hughes Medical Institute, Cornell University, Ithaca, New York, USA

    Hening Lin

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  1. Xiaoyu Zhang
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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.

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Correspondence to Hening Lin.

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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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