Regulation of Heme Synthesis and Mitochondrial Physiology by the ClpX Unfoldase

ClpX 解折叠酶对血红素合成和线粒体生理学的调节

• 批准号: 8310476
• 负责人: Julia R. Kardon
• 金额: $ 5.39万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-16 至 2015-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8310476
• 关键词: 5-Aminolevulinate synthase; Address; Affect; Aminolevulinic Acid; Anabolism; Anemia; Biological Assay; Caenorhabditis elegans; Cells; Coupled; Cytoplasm; Data; Defect; Development; Diabetes Mellitus; Disease; Ensure; Eukaryota; Failure; Genetic; Genome; Health; Heme; Homologous Gene; Human; Indium; Maps; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Mitochondria; Mitochondrial Proteins; Molecular; Pathology; Pathway interactions; Physiological; Physiology; Porphyrias; Prevention therapy; Process; Production; Prokaryotic Cells; Proteins; Publishing; Regulation; Regulatory Element; Research; Role; Saccharomyces cerevisiae; Sampling; Stress; Substrate Interaction; Surveys; Work; Yeasts; base; biological adaptation to stress; cofactor; defined contribution; heme biosynthesis; human disease; in vitro Assay; in vivo; insight; novel; research study; respiratory; response; unfoldase

DESCRIPTION (provided by applicant): The biosynthetic and respiratory capacities of mitochondria must adapt to cellular demand and environmental conditions to ensure organismal health, but control mechanisms for these adaptations are little understood. The protein unfoldase ClpX is an important regulatory element in prokaryotes, mediating changes in cell state and responses to stress conditions through its selection of substrates. In eukaryotes, ClpX is a widely conserved component of the mitochondrion, but no mitochondrial ClpX substrates have yet been discovered, and the specific contributions of ClpX to mitochondrial physiology are not known. This proposal seeks to define how mitochondrial ClpX modulates mitochondrial physiology. Analysis of large-scale genetic interaction maps in S. cerevisiae and phenotypic assays indicated that the yeast mitochondrial ClpX homolog, Mcx1, promotes the first step in the biosynthesis of heme. Metabolic profiling will be used to define the contribution of Mcx1 to this step and to narrow a set of candidate substrates. The identity and mechanism of the substrate interaction through which Mcx1 modulates heme biosynthesis will be determined through complementary in vivo and in vitro assays for substrate processing by Mcx1. These efforts will define a novel mechanism by which the biosynthesis of an essential cofactor is regulated. Phenotypic data in S. cerevisiae and C. elegans, as well as analogy with its prokaryotic homologs, indicate that mitochondrial ClpX may regulate other mitochondrial processes as well. Through an activity-based protein trapping strategy combined with mass spectrometry, the potentially broader repertoire of physiological substrates of Mcx1 will be sampled; this strategy may indicate other mitochondrial processes that Mcx1 regulates, and will allow common motifs that target mitochondrial proteins to Mcx1 to be defined. These studies seek to define the functional repertoire of a mitochondrial regulator, the ClpX homolog Mcx1, and the mechanisms by which its activity is controlled, through a targeted approach toward understanding its role as a control element in the biosynthesis of the essential cofactor heme, and through an unbiased approach that will sample the broader contribution of Mcx1 to mitochondrial physiology. These studies could inform the development of new targets for therapy in anemias resulting from aberrant heme biosynthesis as well as porphyrias. In addition, mitochondrial maladaptation underlies many other diseases in humans, including metabolic diseases and various degenerative ailments resulting from diabetes. Defining molecular mechanisms by which mitochondrial respond and adapt to cellular demand and to environmental stress will provide a new framework for understanding how failures in these responses contribute to disease. PUBLIC HEALTH RELEVANCE: The failure of mitochondria to respond appropriately to cellular demand and environmental condition underlies a wide spectrum of human disease, including inborn and acquired metabolic diseases, diabetes, and some types of anemia. The research proposed here will address how a mitochondrial regulator, the protein unfoldase ClpX, influences mitochondrial activities, in particular the production of the essential cofactor heme, i response to physiological need. This work will give insight into mitochondrial regulatory mechanisms that may be disrupted in human disease, or that could be targeted in therapy and prevention.

描述（申请人提供）：线粒体的生物合成和呼吸能力必须适应细胞需求和环境条件，以确保生物体健康，但对这些适应的控制机制知之甚少。蛋白质解折叠酶ClpX是原核生物中的重要调控元件，通过其对底物的选择介导细胞状态的变化和对应激条件的响应。在真核生物中，ClpX是线粒体中广泛保守的组分，但尚未发现线粒体ClpX底物，ClpX对线粒体生理学的具体贡献尚不清楚。该提案旨在定义线粒体ClpX如何调节线粒体生理学。大规模遗传互作图谱分析。酿酒酵母和表型测定表明，酵母线粒体ClpX同源物，Mcx 1，促进血红素生物合成的第一步。代谢谱将用于确定Mcx 1对该步骤的贡献，并缩小一组候选底物。通过Mcx 1调节血红素生物合成的底物相互作用的身份和机制将通过Mcx 1底物处理的互补体内和体外测定来确定。这些努力将确定一种新的机制，通过这种机制来调节一种必需辅因子的生物合成。S.酿酒酵母和C. elegans以及与其原核同源物的类比表明，线粒体ClpX也可以调节其他线粒体过程。通过基于活性的蛋白质捕获策略结合质谱分析，将对Mcx 1的潜在更广泛的生理底物库进行采样;该策略可能表明Mcx 1调节的其他线粒体过程，并将允许定义靶向Mcx 1的线粒体蛋白的共同基序。 这些研究旨在定义线粒体调节因子ClpX同源物Mcx 1的功能库，以及控制其活性的机制，通过有针对性的方法来理解其作为必需辅因子血红素生物合成中的控制元件的作用，并通过公正的方法来采样Mcx 1对线粒体生理学的更广泛贡献。这些研究可以为血红素生物合成异常和卟啉症引起的贫血的治疗提供新的靶点。此外，线粒体适应不良是人类许多其他疾病的基础，包括代谢疾病和糖尿病引起的各种退行性疾病。定义线粒体响应和适应细胞需求和环境压力的分子机制将为了解这些反应的失败如何导致疾病提供新的框架。 公共卫生关系：线粒体不能对细胞需求和环境条件做出适当反应是人类疾病的基础，包括先天性和获得性代谢疾病、糖尿病和某些类型的贫血。这里提出的研究将解决线粒体调节剂，蛋白质解折叠酶ClpX，如何影响线粒体活动，特别是生产的必要辅因子血红素，i响应生理需要。这项工作将深入了解线粒体调控机制，这些机制可能在人类疾病中被破坏，或者可能在治疗和预防中被靶向。