Novel mechanisms of mitochondrial regulation by sirtuin deacetylases

描述(申请人提供)：线粒体是在真核细胞中执行许多重要功能的细胞质细胞器，其中大多数细胞ATP的生成。线粒体功能障碍与多种病理因素有关，如2型糖尿病、骨质疏松症、神经退行性变和癌症。尽管线粒体对人类健康至关重要，但线粒体功能的调控机制仍不完全清楚。这一应用的基本原理是，对这类机制的深入了解可能会使治疗方法的发展成为可能，以调节线粒体的功能，作为治疗各种人类疾病的方法。这项应用侧重于sirtuin蛋白在调节关键线粒体功能中的新角色。Sirtuins是一个脱乙酰酶家族，在无脊椎动物模型中促进寿命延长，并调节哺乳动物的不同过程。该应用基于两个新的观察结果。首先，线粒体sirtuin SIRT5在丙酮酸脱氢酶复合体(PDC)的脱乙酰化和抑制活性方面发挥了迄今尚未被描述的作用，PDC是一种线粒体全酶，在调节哺乳动物细胞中的葡萄糖氧化方面起着主要作用。PDC功能障碍与2型糖尿病、癌症和心脏缺血有关。刺激PDC活性的新方法--如抑制SIRT5--在这些和其他临床环境中将是有益的。其次，sirtuin SIRT6在刺激线粒体呼吸方面具有意想不到的作用。脂肪组织特异性SIRT6基因敲除(S6AKO)小鼠表现出明显的肥胖，部分原因可能是棕色脂肪组织(BAT)中的线粒体呼吸缺陷。这项应用的总体目标是阐明sirtuin蛋白调节线粒体的新机制，从而解决线粒体生物学中的一个关键知识缺口。这一应用的假设有两个。第一个假设是SIRT5通过减弱PDC活性来抑制葡萄糖氧化。第二个假设是，SIRT6促进线粒体呼吸，从而促进细胞和机体的动态平衡。这些假设将在两个具体目标上得到检验。首先，SIRT5在调控PDC中的作用将通过结合质谱学、突变、体内通量分析和高脂喂养在机制水平上得到阐明。第二，SIRT6在促进线粒体呼吸中的作用将从机械上定义。SIRT6抑制肥胖的功能将通过S6AKO小鼠的详细特征和BAT特异性SIRT6基因敲除的产生来阐明。这一应用具有创新性，因为它侧重于sirtuins在调节线粒体能量学方面的新功能。各种尖端技术将被用来检验这些假设。这一应用意义重大，因为它将在sirtuins和线粒体之间建立新的联系，可能为未来sirtuin指导的调节葡萄糖氧化和/或线粒体呼吸的疗法奠定基础。因此，这项工作属于NIGMS的总体任务。

DESCRIPTION (provided by applicant): Mitochondria are cytoplasmic organelles that perform many crucial functions in eukaryotic cells, among them generation of most cellular ATP. Mitochondrial dysfunction is implicated in diverse pathologies such as type 2 diabetes, sarcopenia, neurodegeneration, and cancer. Despite their central importance to human health, mechanisms by which mitochondrial functions are regulated remain incompletely understood. The rationale for this application is that improved insights into such mechanisms may permit development of therapeutics to modulate mitochondrial functions as treatments for a wide variety of human diseases. This application focuses on novel roles for sirtuin proteins in regulating key mitochondrial functions. Sirtuins are a family of deacetylases that promote increased longevity in invertebrate models and modulate diverse processes in mammals. The application is based on two novel observations. First, the mitochondrial sirtuin SIRT5 plays a hitherto undescribed role in deacetylating and suppressing activity of Pyruvate Dehydrogenase Complex (PDC), a mitochondrial holoenzyme with a major role in regulating glucose oxidation in mammalian cells. PDC dysfunction is implicated in type 2 diabetes, cancer, and cardiac ischemia. Novel means of stimulating PDC activity - as by SIRT5 inhibition - would be beneficial in these and other clinical settings. Second, the sirtuin SIRT6 has an unexpected role in stimulating mitochondrial respiration. Adipose tissue- specific SIRT6 knockout (S6AKO) mice show marked adiposity, potentially due in part to mitochondrial respiratory defects in brown adipose tissue (BAT). The overall objective of this application is to elucidate novel mechanisms of mitochondrial regulation by sirtuin proteins, thus addressing a key knowledge gap in mitochondrial biology. The hypotheses of this application are two-fold. The first hypothesis is that SIRT5 inhibits glucose oxidation by attenuating PDC activity. The second hypothesis is that SIRT6 promotes mitochondrial respiration to promote cellular and organismal homeostasis. These hypotheses will be tested in two specific aims. First, the roles of SIRT5 in regulating PDC will be elucidated at a mechanistic level through a combination of mass spectrometry, mutagenesis, in vivo flux analysis, and high fat feeding. Second, the role of SIRT6 in promoting mitochondrial respiration will be defined mechanistically. The function of SIRT6 in suppressing adiposity will be elucidated through detailed characterization of S6AKO mice, and through generation of BAT-specific SIRT6 knockouts. This application is innovative, since it focuses on novel functions for sirtuins in regulating mitochondrial energetics. A variety of cutting-edge techniques will be brought to bear to test these hypotheses. The application is significant, since it will establish novel links between sirtuins and mitochondria, potentially laying the groundwork for future sirtuin-directed therapies to modulate glucose oxidation and/or mitochondrial respiration. Hence this work falls within the overall mission of NIGMS.