Over Expression of Wild Type or a Catalytically Dead Mutant of SIRTUIN 6 Does Not Influence NFκB Responses

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.

[1]  A. Seluanov,et al.  SIRT6 Promotes DNA Repair Under Stress by Activating PARP1 , 2011, Science.

[2]  Howard Y. Chang,et al.  Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks , 2011, PLoS genetics.

[3]  Dan S. Tawfik,et al.  The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters. , 2011, Biochemistry.

[4]  P. Pan,et al.  Structure and Biochemical Functions of SIRT6* , 2011, The Journal of Biological Chemistry.

[5]  S. Jackson,et al.  Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation , 2010, Science.

[6]  Ruth I. Tennen,et al.  Functional dissection of SIRT6: Identification of domains that regulate histone deacetylase activity and chromatin localization , 2010, Mechanisms of Ageing and Development.

[7]  Orian S. Shirihai,et al.  The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1α , 2010, Cell.

[8]  Or Gozani,et al.  Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6 , 2009, Cell cycle.

[9]  Bo Yang,et al.  The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promote genomic stability , 2009, Cell cycle.

[10]  W. MacNee,et al.  Accelerated lung aging: a novel pathogenic mechanism of chronic obstructive pulmonary disease (COPD). , 2009, Biochemical Society transactions.

[11]  J. Zlatanova,et al.  Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications , 2009, Nucleic acids research.

[12]  Howard Y. Chang,et al.  SIRT6 Links Histone H3 Lysine 9 Deacetylation to NF-κB-Dependent Gene Expression and Organismal Life Span , 2009, Cell.

[13]  S. Guan,et al.  SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair , 2009, Aging.

[14]  P. Elliott,et al.  Sirtuins — novel therapeutic targets to treat age-associated diseases , 2008, Nature Reviews Drug Discovery.

[15]  A. Sauve,et al.  Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase. , 2008, Biochemistry.

[16]  Howard Y. Chang,et al.  SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin , 2008, Nature.

[17]  Brad T. Sherman,et al.  Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources , 2008, Nature Protocols.

[18]  E. Stylianou,et al.  The Relationship between Intranuclear Mobility of the NF-κB Subunit p65 and Its DNA Binding Affinity* , 2006, Journal of Biological Chemistry.

[19]  K. Glaser,et al.  Corrigendum to “Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction” [Anal. Biochem. 332 (2004) 90–99] , 2006 .

[20]  Pingfang Liu,et al.  Genomic Instability and Aging-like Phenotype in the Absence of Mammalian SIRT6 , 2006, Cell.

[21]  Brian C. Smith,et al.  Sir2 protein deacetylases: evidence for chemical intermediates and functions of a conserved histidine. , 2006, Biochemistry.

[22]  Izumi Horikawa,et al.  Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. , 2005, Molecular biology of the cell.

[23]  P. Distefano,et al.  Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin. , 2005, Methods.

[24]  L. Guarente,et al.  Mouse Sir2 Homolog SIRT6 Is a Nuclear ADP-ribosyltransferase* , 2005, Journal of Biological Chemistry.

[25]  Seon-Young Kim,et al.  PAGE: Parametric Analysis of Gene Set Enrichment , 2005, BMC Bioinform..

[26]  Arthur S Slutsky,et al.  DNA microarray analysis of gene expression in alveolar epithelial cells in response to TNFα, LPS, and cyclic stretch , 2004 .

[27]  K. Glaser,et al.  Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction. , 2004, Analytical biochemistry.

[28]  J. Denu,et al.  Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases. , 2004, Biochemistry.

[29]  R. Frye,et al.  Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. , 2000, Biochemical and biophysical research communications.

[30]  T. Yoshimura,et al.  Transcriptional Regulation of The Human Monocyte Chemoattractant Protein-1 Gene , 1997, The Journal of Biological Chemistry.

[31]  T. Yoshimura,et al.  Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of two NF-kappaB sites and NF-kappaB/Rel subunit specificity. , 1997, The Journal of biological chemistry.

[32]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .