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首页> 外文期刊>Redox Biology >Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators
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Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators

机译：改进的基于质谱的活性测定法揭示了氧化和代谢应激作为sirtuin-1调节剂

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Sirtuin-1 (SirT1) catalyzes NADsup+/sup-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.
机译：Sirtuin-1（SirT1）催化NAD + 依赖的蛋白质赖氨酸脱乙酰基反应，是能量和脂质代谢，线粒体生物发生，凋亡和衰老的关键调节剂。 SirT1的激活可减轻与糖尿病和肥胖症相关的代谢紊乱。药理分子，细胞氧化还原和营养状态可以调节SirT1活性。衡量内源性SirT1活性的技术障碍限制了疾病中SirT1的表征及其被小分子激活的能力。本文中，我们开发了一种基于相对定量质谱的技术，以生物素标记的乙酰化p53衍生肽为底物，测量细胞和组织匀浆中的内源性SirT1活性（RAMSSAY /相对活性Sirt1活性测定）。我们证明氧化和代谢应激会减少肝细胞系HepG2中的SirT1活性。此外，包括烟酰胺和EX-527在内的药理分子会减弱SirT1活性。 SirT1的激活剂，多酚S17834，多酚白藜芦醇或非多酚Sirtris化合物SRT1720未能显着激活内源性SirT1。此外，我们提供的证据表明，给小鼠喂食高脂肪高蔗糖饮食（HFHS）会抑制小鼠肝脏中的内源性SirT1活性。总而言之，我们介绍了一种稳健，特异性和灵敏的质谱分析方法，用于在细胞和组织裂解物中使用生物素标记的肽来检测和定量内源性SirT1活性。通过这种测定，我们可以确定药理分子以及代谢和氧化应激如何调节内源性SirT1活性。该测定法还可适用于其他瑟土因同种型。

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