Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators

Autor: Di Shao, Richard A. Cohen, Mark E. McComb, Chunxiang Yao, Maya H. Kim, Markus Bachschmid, Jessica L. Fry, Reiko Matsui, Francesca Seta, Catherine E. Costello
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0301 basic medicine
Male
endocrine system diseases
GSH
reduced glutathione
Clinical Biochemistry
Endogeny
Resveratrol
medicine.disease_cause
Biochemistry
B6J
C57BL/6J mouse strain
Mass Spectrometry
chemistry.chemical_compound
Mice
HPHG
high palmitate high glucose medium
0302 clinical medicine
SRT1720
Sirtuin 1
NAD+
nicotinamide adenine dinucleotide
Drug Discovery
lcsh:QH301-705.5
lcsh:R5-920
biology
Chemistry
GSSG
oxidized glutathione
food and beverages
Hep G2 Cells
HFHS
high fat high sucrose diet
HEK-293
human embryonic kidney cell-293
p53
tumor suppressor p53
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
HPLC
high performance liquid chromatography
Sirtuin
HepG2
human hepatocellular carcinoma cell line
IAM
iodoacetamide
lipids (amino acids
peptides
and proteins)
IP
immuno-precipitation
lcsh:Medicine (General)
hormones
hormone substitutes
and hormone antagonists
IgG
immunoglobulin G
Antineoplastic Agents
Mice
Transgenic
ND
chow diet
Article
RONS
reactive oxygen and nitrogen species
03 medical and health sciences
Stress
Physiological
medicine
Animals
Humans
Metabolomics
CysNO
S-nitrosocysteine
DBC-1
deleted in breast cancer 1
Organic Chemistry
SirT1
Sirtuin-1
Enzyme Activation
Oxidative Stress
enzymes and coenzymes (carbohydrates)
030104 developmental biology
Mitochondrial biogenesis
LacZ
beta-galactosidase
MS
mass spectrometry
lcsh:Biology (General)
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
biology.protein
BSA
bovine serum albumin
NAD+ kinase
SirBACO
Sirtuin-1 Bacterial Artificial Chromosome Overexpressor
030217 neurology & neurosurgery
Oxidative stress
Zdroj: Redox Biology, Vol 22, Iss, Pp-(2019)
Redox Biology
ISSN: 2213-2317
Popis: Sirtuin-1 (SirT1) catalyzes NAD+-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regulate SirT1 activity. Technical barriers against measuring endogenous SirT1 activity have limited characterization of SirT1 in disease and its activation by small molecules. Herein, we developed a relative quantitative mass spectrometry-based technique for measuring endogenous SirT1 activity (RAMSSAY/RelAtive Mass Spectrometry Sirt1 Activity assaY) in cell and tissue homogenates using a biotin-labeled, acetylated p53-derived peptide as a substrate. We demonstrate that oxidative and metabolic stress diminish SirT1 activity in the hepatic cell line HepG2. Moreover, pharmacologic molecules including nicotinamide and EX-527 attenuate SirT1 activity; purported activators of SirT1, the polyphenol S17834, the polyphenol resveratrol, or the non-polyphenolic Sirtris compound SRT1720, failed to activate endogenous SirT1 significantly. Furthermore, we provide evidence that feeding a high fat high sucrose diet (HFHS) to mice inhibits endogenous SirT1 activity in mouse liver. In summary, we introduce a robust, specific and sensitive mass spectrometry-based assay for detecting and quantifying endogenous SirT1 activity using a biotin-labeled peptide in cell and tissue lysates. With this assay, we determine how pharmacologic molecules and metabolic and oxidative stress regulate endogenous SirT1 activity. The assay may also be adapted for other sirtuin isoforms.
Highlights • Fast, sensitive, and specific MALDI-TOF based sirtuin-1 activity assay applicable to cell and tissue lysates. • Oxidative and metabolic stress inhibit Sirtuin-1 deacetylase activity. • Purported activators of SirT1failed to significantly activate endogenous SirT1. • The activity assay is adaptable to other sirtuin isoforms using specific synthetic peptides and assay conditions.
Databáze: OpenAIRE
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