Zee, R. S., Pimentel, D. R. , Hou, X. L., Zang, M., Yao, C. X., O'Connor, Peter B., Bachschmid, M. M. and Cohen, R. A. (2008) Sirtuin-1 zinc thiolate center is a direct molecular target of oxidants. In: Society for Free Radical Biology and Medicine's 15th Annual Meeting, Indianapolis, IN, U.S.A., Nov 19-23, 2008. Published in: Free Radical Biology and Medicine, Vol.45 (Supplement). S76.

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Abstract

Sirtuin-1 (SIRT1) is an NAD+-dependent histone deacetylase (HDAC) that regulates metabolism. SIRT1 contains a Zn2+ atom coordinated by four cysteines, which is essential for enzyme activity. We hypothesized that the lower pKa of these thiolate center cysteines makes SIRT1 sensitive to reactive oxygen species, possibly lowering its activity in metabolic diseases. Methods: SIRT1 deacetylase activity was measured by cleavage of a fluorogenic p53 substrate peptide. Affinity of Zn2+ intact recombinant SIRT1 protein was determined on Zn2+-NTA beads, and thiolate cysteines were labeled in bovine aortic endothelial cells with biotinylated iodoacetamide (bIAM) after blocking free thiols and chelation of Zn2+. Results: the strong oxidant, peroxynitrite (10 to 1000 μM) dose-dependently inhibited SIRT1 binding to the Zn2+ NTA beads as well as SIRT1 activity. Both binding and activity were blocked by Zn2+ chelators. Treating endothelial cells with the peroxynitrite donor, SIN1, or with TNFα, decreased bIAM labeling of thiolate cysteines. Conclusions: the methods developed can detect the integrity of the Zn2+ thiolate center of recombinant and cellular SIRT1. Our results indicate that disruption of the Zn2+ thiolate domain of SIRT1 by a strong oxidant or metal chelator is associated with a negative effect on activity. Studies in endothelial cells indicate that oxidants added directly, or those induced by TNFα can disrupt a substantial fraction of the SIRT1 thiolate centers. This suggests that oxidants associated with metabolic diseases might directly regulate SIRT1.

Item Type:	Conference Item (Other)
Subjects:	Q Science > QD Chemistry Q Science > QP Physiology
Divisions:	Faculty of Science > Chemistry
Library of Congress Subject Headings (LCSH):	Histone deacetylase, Oxidizing agents -- Physiological effect, Transition metal complexes
Journal or Publication Title:	Free Radical Biology and Medicine
Publisher:	Elsevier Science Inc.
ISSN:	0891-5849
Date:	2008
Volume:	Vol.45
Number:	Supplement
Page Range:	S76
Identification Number:	10.1016/j.freeradbiomed.2008.10.011
Status:	Not Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Description:	Journal item abstract only
Conference Paper Type:	Other
Title of Event:	Society for Free Radical Biology and Medicine's 15th Annual Meeting
Type of Event:	Other
Location of Event:	Indianapolis, IN, U.S.A.
Date(s) of Event:	Nov 19-23, 2008
URI:	http://wrap.warwick.ac.uk/id/eprint/40613

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