Mitochondrial Biogenesis in Health and Disease

健康和疾病中的线粒体生物发生

• 批准号: 9071590
• 负责人: Antoni Barrientos
• 金额: $ 49.96万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071590
• 关键词: Address; Applications Grants; Biochemistry; Biogenesis; Boxing; Cardiomyopathies; Catalytic Domain; Cell Nucleus; Cells; Complex; Cytoplasmic Granules; Disease; Enzymes; Gene Expression; Genes; Genetic; Goals; Health; Heme; Human; Human Cell Line; Individual; Mass Spectrum Analysis; Membrane; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Molecular; Molecular Chaperones; Nuclear; Oxidases; Oxidation-Reduction; Oxidative Phosphorylation; Pathology; Pathway interactions; Process; Protein Biosynthesis; Proteins; RNA; RNA Helicase; Recombinant Proteins; Reporting; Research; Resolution; Respiratory Chain; Ribosomes; Role; Structure; System; Techniques; Translational Regulation; Translations; Work; Yeasts; base; cytochrome c oxidase; innovation; insight; mitochondrial membrane; programs; public health relevance; transcription activator-like effector nucleases; yeast genetics

 DESCRIPTION (provided by applicant): Mitochondria allow our cells to use oxidative phosphorylation (OXPHOS) as a highly efficient way to generate ATP. The inner membrane-embedded OXPHOS system enzymes are multimeric complexes composed of proteins from two different genetic origins, namely the nuclear and the mitochondrial DNA. Nucleus-encoded proteins are synthesized in cytoplasmic ribosomes and imported into mitochondria. The mitochondrion-encoded proteins, usually catalytic core subunits of the complexes, are synthesized into distinct mitochondrial ribosomes. We have developed a scientific research program aiming at understanding the molecular mechanisms underlying the assembly of OXPHOS complexes and mitochondrial ribosomes. Our studies focus on the assembly of OXPHOS enzymes as individual complexes, with a focus on cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory chain. We have uncovered translational regulation, heme and redox sensing processes ruling COX biogenesis in yeast and/or human cells. Additionally, we aim to understand how OXPHOS complexes form higher order assemblies known as supercomplexes or respirasomes. We have already reported the first respirasome assembly pathway in human cells. Dedicated chaperone-like factors are required to assist and regulate complex and supercomplex assembly in mitochondria. While many have been already identified, their specific functions remain to be precisely characterized. In another aspect of our work we address the question of how mitochondrial ribosomes assemble into functional protein synthesis machineries. We have recently identified the first two DEAD-box RNA helicases acting on the assembly of the large mitoribosome subunit in yeast and human cells. Also, we have gained insight into the compartmentalization of the ribosome assembly process by identifying matrix RNA granules as the "mitochondriolus" (per equivalence to the nucleoulus). Despite high-resolution cryo-EM structures of yeast and human ribosomes have been recently reported, the pathway of mitoribosome biogenesis and the factors involved are poorly characterized. Studies outlined in this proposal will involve yeast genetics, gene disruption in human cells using new gene-editing techniques (TALENs and CRISPRs) and mechanistic biochemistry in yeast, human cell lines, isolated mitochondria and purified native and recombinant proteins to gain insight into the role/s of OXPHOS complex, supercomplex and mitoribosome assembly factors. To further fill the gaps, we are implementing innovative strategies to identify new assembly factors and to define the biosynthetic pathways under study, by applying quantitative mass spectrometry and structural approaches. The analysis of the principles of the biogenesis process and the activities of the assembly factors is of central importance for our understanding of the molecular basis of human mitochondrial disorders.

 描述（由申请人提供）：线粒体允许我们的细胞使用氧化磷酸化（OXPHOS）作为产生ATP的高效方式。嵌入内膜的OXPHOS系统酶是由来自两种不同遗传来源的蛋白质组成的多聚体复合物，即核和线粒体DNA。细胞核编码的蛋白质在细胞质核糖体中合成并输入线粒体。这些蛋白质通常是复合物的催化核心亚基，它们被合成到不同的线粒体核糖体中。我们已经开发了一个科学研究计划，旨在了解OXPHOS复合物和线粒体核糖体组装的分子机制。我们的研究集中在组装OXPHOS酶作为单独的复合物，重点是细胞色素c氧化酶，线粒体呼吸链的末端氧化酶。我们已经发现了翻译调节，血红素和氧化还原传感过程统治考克斯在酵母和/或人类细胞的生物合成。此外，我们的目标是了解OXPHOS复合物如何形成更高阶的组装体，称为超复合物或复合体。我们已经报道了人类细胞中的第一个蛋白酶体组装途径。需要专用的伴侣样因子来协助和调节线粒体中的复合物和超复合物组装。虽然已经确定了许多，但其具体功能仍有待准确描述。在我们的另一方面， 我们的工作解决了线粒体核糖体如何组装成功能性蛋白质合成机器的问题。我们最近已经确定了前两个死亡盒RNA解旋酶作用于酵母和人类细胞中的大的线粒体亚基的组装。此外，我们已经通过将基质RNA颗粒识别为“核仁”（相当于核仁），深入了解了核糖体组装过程的区室化。尽管最近已经报道了酵母和人类核糖体的高分辨率cryo-EM结构，但线粒体生物合成的途径和所涉及的因素的特征很差。该提案中概述的研究将涉及酵母遗传学，使用新的基因编辑技术（TALEN和CRISPR）在人类细胞中进行基因破坏，以及酵母，人类细胞系，分离的线粒体和纯化的天然和重组蛋白质中的机械生物化学，以深入了解OXPHOS复合物，超复合物和mitoribosome组装因子的作用。为了进一步填补空白，我们正在实施创新战略，通过应用定量质谱和结构方法，确定新的组装因子，并确定正在研究的生物合成途径。分析生物发生过程的原理和组装因子的活性对于我们理解人类线粒体疾病的分子基础至关重要。