title: In vivo measurement of the acetylation state of sirtuin substrates as a proxy for sirtuin activity.
abstract: Evaluating the precise catalytic activity of sirtuin proteins in vivo is a challenging endeavor. Enzymological methods, including those employed in commercially available kits, require the isolation of immunopurified protein from cells or tissues, which can perturb regulatory protein-protein interactions as well as remove the enzyme from the reaction-altering effects of intracellular NAD(+), nicotinamide, and O-acetyl-ADP ribose concentrations. As such, the measurement of the steady state acetylation status of select sirtuin substrates in vivo remains an important tool for evaluating changes in sirtuin activity. Here, we describe how to perform the analysis of the acetylation status of key SIRT1 and SIRT3 targets in rodent tissues and cultured cells.
authors.affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
authors.forename: John
authors.initials: J
authors.lastname: Dominy
authors.forename: Pere
authors.initials: P
authors.lastname: Puigserver
authors.forename: Carles
authors.initials: C
authors.lastname: Cantó
mesh_terms: D000107:Acetylation; D000818:Animals; D001859:Bone Neoplasms / Q000378:metabolism*; D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008239:Lysine / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism*; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011499:Protein Processing, Post-Translational; D056564:Sirtuin 1 / Q000378:metabolism*; D056566:Sirtuin 3 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C485290:Ppargc1a protein, mouse; D014157:Transcription Factors; C447939:SIRT1 protein, human; C466093:SIRT3 protein, human; D056564:Sirtuin 1; D056566:Sirtuin 3; D042967:Electron Transport Complex I; C571164:NDUFA9 protein, human; D008239:Lysine