The effect of mitochondrial CoA degradation on glucose and fatty acid metabolism

项目摘要/摘要 线粒体的代谢依赖于辅酶A(CoA)来支持像脂肪酸这样的代谢过程 氧化、酮生和糖异生。辅酶A的积累会导致脂肪酸氧化增加， 导致糖尿病患者糖异生。骨骼肌中异常高的CoA水平使线粒体变钝 通过降低电子传递链的络合物I的活性来合成ATP。尽管有这样的证据 将CoA增加与代谢失调联系起来，线粒体内CoA水平的调节机制 目前还不得而知。Nudt8是Nudex水解酶超家族中一个未知的成员。这种蛋白质 已被注释为潜在的CoA降解酶，因为它与Nudt7，a 以前的特征CoA二磷酸水解酶，驻留在过氧化物酶体中。我们的初步数据显示 Nudt8特别降解游离CoA和CoA硫代酯。此外，Nudt8定位于线粒体 在心脏、肾脏、肝脏、棕色脂肪组织和肌肉等高度氧化的组织中表达。 Nudt8的CoA降解活性，再加上它对线粒体的定位，表明了它在其中的作用 酶在调节线粒体CoA水平，从而调节新陈代谢。拟议的研究将利用 腺相关病毒实现Nudt8在肝脏中的过表达，接下来分析其对 线粒体辅酶A池的大小和组成，以及对线粒体脂肪酸和葡萄糖代谢的影响。 此外，还将阐明调控Nudt8表达和活性的机制。 拟议的研究将在现有的优秀研究设施的支持下完成 申请人并与韦斯特生物化学系内外的其他调查人员合作 弗吉尼亚大学。高度协作的环境与个性化的培训计划相结合，将使 申请者需要掌握广泛的技术技能和发展批判性思维能力，这两者都 对于学员成长为成功的科学家来说是必不可少的。申请者将参加严格的课程 工作和口头报告；这些练习将提供广泛的新陈代谢和 增强申请者交流科学思想的能力。总体而言，拟议的组合 研究和培训计划将培养出一位全面发展的科学家，以及对监管的宝贵见解 线粒体辅酶A池和靶向Nudt8以纠正线粒体失调的可能性 导致各种代谢性疾病的代谢。

PROJECT SUMMARY/ABSTRACT Mitochondrial metabolism is dependent on coenzyme A (CoA) to support metabolic processes like fatty acid oxidation, ketogenesis, and gluconeogenesis. Accumulation of CoA results in increased fatty acid oxidation, which drives gluconeogenesis in diabetes. Abnormally high CoA levels in skeletal muscle blunt mitochondrial ATP synthesis by decreasing the activity of complex I of the electron transport chain. Despite this evidence linking increased CoA to metabolic dysregulation, the mechanisms regulating CoA levels within the mitochondria are currently unknown. Nudt8 is an uncharacterized member of the Nudix hydrolase superfamily. This protein has been annotated as a potential CoA-degrading enzyme due to its high sequence homology to Nudt7, a previously characterized CoA diphosphohydrolase that resides in the peroxisomes. Our preliminary data show that Nudt8 specifically degrades free CoA and CoA thioesters. Additionally, Nudt8 localizes to the mitochondria and is expressed in highly oxidative tissues such as the heart, kidney, liver, brown adipose tissue, and muscle. The CoA-degrading activity of Nudt8, coupled with its localization to the mitochondria, suggests a role for this enzyme in the regulation of mitochondrial CoA levels and thus, metabolism. The proposed research will utilize adeno-associated virus to achieve over-expression of Nudt8 in the liver, followed by analysis of its effects on mitochondrial CoA pool size and composition, and on the mitochondrial metabolism of fatty acids and glucose. Additionally, mechanisms regulating Nudt8 expression and activity will be elucidated. The proposed research will be completed with the support of the excellent research facilities available to the applicant and in collaboration with other investigators within and outside the Department of Biochemistry at West Virginia University. The highly collaborative environment, combined with a personalized training plan, will allow the applicant to acquire skills in a broad range of techniques and to develop critical thinking skills, both of which are essential for a trainee to develop into a successful scientist. The applicant will participate in rigorous course work and oral presentations; these practices will provide an extensive knowledge base in metabolism and enhance the ability of the applicant to communicate his scientific ideas. Overall, the proposed combination of research and training plans will produce a well-rounded scientist as well as valuable insight into the regulation of the mitochondrial CoA pool and potential of targeting Nudt8 to correct the dysregulated mitochondrial metabolism that underlies a variety of metabolic diseases.