REVERSIBLE MITOCHONDRIAL PROTEIN ACETYLATION AND METABOLIC REGULATION

描述（由申请人提供）：大量线粒体蛋白发生可逆的赖氨酸乙酰化，表明这种修饰在线粒体生物学中起着重要作用。在线粒体中已经鉴定了三种不同的NAD+依赖性蛋白脱乙酰酶，称为SIRT 3、4和5。该提案的PI广泛合作，以表征缺乏SIRT 3的敲除小鼠的表型。这些小鼠显示出显著的线粒体蛋白乙酰化。SIRT 3表达在禁食期间增加并激活肝脏中脂肪酸的β-氧化。SIRTS直接使脂肪酸氧化途径中的两种关键线粒体酶脱乙酰并增加其酶活性。缺乏SIRTS的小鼠的代谢组学分析显示，禁食期间酰基肉毒碱的水平显著高于野生型小鼠。SIRTS-/-小鼠还显示出降低的ATP水平和禁食后对冷暴露的不耐受性，这与它们的脂肪酸氧化缺陷一致。当SIRTS-/-小鼠被置于高脂肪饮食中时，它们显示出与人类代谢综合征密切相似的综合征的加速发展：肥胖、II型糖尿病、脂质异常、脂肪肝炎和肝细胞癌。这些发现确定乙酰化作为线粒体代谢的一种新的调节机制。该提案将使用SIRTS，4和5的敲除和转基因小鼠来进一步探索线粒体乙酰蛋白质组，即被SIRTS，4和5可逆脱乙酰化的蛋白质。将在各种器官中探索缺乏SIRTS，4和5的小鼠的代谢组，以确定由单个线粒体sirtuins调节的代谢途径。我们将进一步探索缺乏SIRTS的小鼠发生II型糖尿病的分子机制及其与脂质代谢紊乱的联系。最后，我们将研究线粒体sirtuins在不同生理条件下的表达调控，并筛选一个小分子库，以增强SIRTS表达的药物。这些药物对代谢综合征表现的影响将在小鼠中进行检查。我们预计，这些实验将增加我们对可逆的线粒体蛋白乙酰化及其酶在代谢调节和II型糖尿病中的作用的理解。 公共卫生相关性：线粒体是几种代谢疾病发病机制的关键参与者。最近鉴定了大量线粒体蛋白上可逆的赖氨酸乙酰化和鉴定了三种线粒体sirtuin（SIRTS，4和5），表明可逆的线粒体乙酰化在代谢紊乱的发病机制中起作用。了解SIRT 3、4、5在代谢综合征和其他代谢紊乱的发病机制中的作用可以产生新的治疗机会。

DESCRIPTION (provided by applicant): A large number of mitochondrial proteins are subject to reversible lysine acetylation, suggesting that this modification plays a significant role in mitochondrial biology. Three distinct NAD+dependent protein deacetylases, called SIRT3, 4 and 5, have been identified in mitochondria. The PIs of this proposal have extensively collaborated to characterize the phenotype of knockout mice lacking SIRT3. These mice show marked mitochondrial protein acetylation. SIRT3 expression increases during fasting and activates (3-oxidation of fatty acid in the liver. SIRTS directly deacetylates two key mitochondrial enzymes in the fatty acid oxidation pathway and increases their enzymatic activities. Metabolomic analysis of mice lacking SIRTS shows significantly greater levels of acylcarnitines during fasting than wild-type mice. SIRTS-/- mice also show reduced ATP levels and intolerance to cold exposure upon fasting consistent with their fatty acid oxidation defect. When SIRTS-/- mice are placed on a high fat diet, they show accelerated development of a syndrome that closely mimics the human metabolic syndrome: obesity, type II diabetes, lipid abnormalities, steatohepatltis and hepatocellular carcinoma. These findings identify acetylation as a novel regulatory mechanism for mitochondrial metabolism. This proposal will use knockout and transgenic mice for SIRTS, 4 and 5 to further explore the mitochondrial acetylproteome, i.e. the proteins that are reversibly deacetylated by SIRTS, 4 and 5. The metabolome of mice lacking SIRTS, 4 and 5 will be explored in a variety of organs to identify metabolic pathways that are regulated by individual mitochondrial sirtuins. We will further explore the molecular mechanism(s) responsible for the development of type II diabetes in mice lacking SIRTS and its link to disordered lipid metabolism. Finally, we will study the regulation of expression of mitochondrial sirtuins under different physiological conditions and screen a small molecule library for drugs that enhance SIRTS expression. The effect of these drugs on the manifestations ofthe metabolic syndrome will be examined in mice. We anticipate that these experiments will increase our understanding of the role of reversible mitochondrial protein acetylation and its enzymes in metabolic regulation and in type II diabetes. PUBLIC HEALTH RELEVANCE: Mitochondria are key players in the pathogenesis of several metabolic disorders. The recent identification of reversible lysine acetylation on a large number of mitochondrial proteins and the identification of three mitochondrial sirtuin (SIRTS, 4 and 5) suggest that reversible mitochondrial acetylation plays a role in the pathogenesis of metabolic disorders. Understanding the role of SIRT 3, 4, 5 in the pathogenesis of the metabolic syndrome and other metabolic disorders could yield novel therapeutic opportunities.