REVERSIBLE MITOCHONDRIAL PROTEIN ACETYLATION AND METABOLIC REGULATION

描述（由申请人提供）：大量线粒体蛋白可发生可逆赖氨酸乙酰化，表明这种修饰在线粒体生物学中起着重要作用。已经在线粒体中发现了三种不同的NAD+依赖蛋白去乙酰化酶，称为SIRT3, 4和5。该提案的pi广泛合作表征缺乏SIRT3的敲除小鼠的表型。这些小鼠显示出明显的线粒体蛋白乙酰化。禁食期间SIRT3表达增加，激活肝脏脂肪酸的(3)氧化。SIRTS直接使脂肪酸氧化途径中的两个关键线粒体酶去乙酰化，并增加它们的酶活性。代谢组学分析显示，缺乏SIRTS的小鼠在禁食期间的酰基肉碱水平明显高于野生型小鼠。SIRTS-/-小鼠在禁食时也表现出ATP水平降低和对冷暴露的不耐受，这与它们的脂肪酸氧化缺陷一致。当SIRTS-/-小鼠被喂食高脂肪食物时，它们表现出一种与人类代谢综合征非常相似的综合征的加速发展：肥胖、II型糖尿病、脂质异常、脂肪性肝炎和肝细胞癌。这些发现表明乙酰化是线粒体代谢的一种新的调节机制。本提案将使用SIRTS， 4和5基因敲除和转基因小鼠进一步探索线粒体乙酰蛋白组，即被SIRTS， 4和5可逆去乙酰化的蛋白质。缺乏SIRTS， 4和5的小鼠的代谢组将在各种器官中进行探索，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SIRTS缺失小鼠II型糖尿病发生的分子机制及其与脂质代谢紊乱的联系。最后，我们将研究不同生理条件下线粒体sirtuins的表达调控，筛选增强SIRTS表达的药物小分子文库。这些药物对代谢综合征表现的影响将在小鼠中进行研究。我们预计这些实验将增加我们对可逆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。

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.