Regulation of mitochondrial metabolism by lysine acylation

项目摘要 脂肪酸氧化(FAO)是心脏、肌肉和肝脏等的一种关键的能量产生途径 器官。粮农组织途径基因的先天错误与这些器官的功能障碍和高 死亡率。此外，粮农组织的中断也见于多基因疾病，如肥胖、糖尿病和 癌症。随着血液代谢物的质谱学分析的进步，粮农组织的缺陷很容易出现 诊断出来了。然而，尽管进行了30年的密集研究，调节粮农组织在人类中的治疗方案 患者仍然有限，效果不佳。关于粮农组织酶的监管的知识差距和 粮农组织途径在线粒体能量代谢大格局中的功能组织 限制了新疗法的发展。在这笔赠款的前一个资助期，我们建立了 作为粮农组织调节剂的可逆赖氨酸翻译后修饰(乙酰化、琥珀酸化)。我们展示了 这种sirtuin酶对目标赖氨酸进行脱酰化，并将其恢复到天然状态，是重要的参与者。 最大限度地发挥粮农组织途径的作用。在本提案中，我们假设赖氨酸酰化 调节FAO酶的活性，定位到线粒体膜内层，以及更高级的- 订购粮农组织蛋白质和呼吸链之间的代谢复合体。在具体目标1中，我们将采用 我们在前一个资助期开创的体外方法，以确定sirtuin靶向的赖氨酸 粮农组织膜相关酶肉碱棕榈酰转移酶-2(CPT2)，线粒体三功能 蛋白质(TFP)和酰辅酶A脱氢酶-9(ACAD9)。我们将进行突变研究，以确定 每一个sirtuin靶向赖氨酸残基的功能作用。在具体目标2中，我们将调查物理 三种线粒体sirtuin(SIRT3、SIRT4和SIRT5)与内部的功能相互作用 线粒体膜。我们假设sirtuins负责管理线粒体膜内层，以便 促进高级代谢复合体的组装和操作，如粮农组织之间形成的代谢复合体 以及电子传输链。最后，特殊目标3将评估赖氨酸酰化对这些的影响。 利用小鼠模型结合高阶复合体和体外组装的蛋白质复合体。 了解sirtuins在调节粮农组织和代谢超复合体中的作用将奠定基础 致力于开发操纵人类先天缺陷患者线粒体功能的新疗法 新陈代谢疾病，以及患有肥胖、糖尿病和癌症等慢性病的人。

PROJECT ABSTRACT Fatty acid oxidation (FAO) is a critical energy producing pathway in heart, muscle, and liver, among other organs. Inborn errors in genes of the FAO pathway are associated with dysfunction in these organs and a high rate of mortality. Additionally, disruptions in FAO are seen in polygenic diseases such as obesity, diabetes, and cancer. With advances in mass spectrometry profiling of blood metabolites, FAO defects can be readily diagnosed. However, despite 30 years of intensive study, treatment options for modulating FAO in human patients remain limited and ineffective. Knowledge gaps regarding the regulation of FAO enzymes and the functional organization of the FAO pathway within the greater landscape of mitochondrial energy metabolism have limited the development of new therapies. In the previous funding period of this grant, we established reversible lysine post-translational modifications (acetylation, succinylation) as regulators of FAO. We showed that sirtuin enzymes, which deacylate target lysines and restore them to the native state, are important players in maximizing function of the FAO pathway. In the present proposal we hypothesize that lysine acylation regulates FAO enzyme activity, localization to the inner mitochondrial membrane, and the assembly of higher- order metabolic complexes between FAO proteins and the respiratory chain. In Specific Aim 1, we will employ in vitro methods that we pioneered in the previous funding period to identify sirtuin-targeted lysines on the membrane-associated FAO enzymes carnitine palmitoyltransferase-2 (CPT2), mitochondrial trifunctional protein (TFP), and acyl-CoA dehydrogenase-9 (ACAD9). We will perform mutagenesis studies to determine the functional role of each of the sirtuin-targeted lysine residues. In Specific Aim 2 we will investigate physical and functional interactions between the three mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5) and the inner mitochondrial membrane. We hypothesize that the sirtuins police the inner mitochondrial membrane in order to facilitate assembly and operation of higher-order metabolic complexes such as those formed between FAO and the electron transport chain. Finally, Specific Aim 3 will evaluate the effects of lysine acylation on these higher-order complexes using a combination of mouse models and protein complexes assembled in vitro. Understanding the role of the sirtuins in regulating FAO and metabolic supercomplexes will lay the ground work for developing new therapies that manipulate mitochondrial function in human patients with inborn errors of metabolism, as well as those with chronic diseases such as obesity, diabetes, and cancer.