Regulation of Heme Synthesis and Mitochondrial Physiology by the ClpX Unfoldase

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，以确保机体健康，但对这些适应的控制机制知之甚少。蛋白质解折叠酶ClpX是原核生物中一个重要的调控元件，通过选择底物来调节细胞状态的变化和对胁迫条件的反应。在真核生物中，ClpX是线粒体中一个广泛保守的成分，但目前还没有发现线粒体的ClpX底物，也不知道ClpX对线粒体生理的具体贡献。这项提议试图定义线粒体ClpX如何调节线粒体生理。对酿酒酵母大规模遗传互作图谱的分析和表型分析表明，酵母线粒体ClpX同源物Mcx1促进了血红素生物合成的第一步。代谢图谱将被用来确定Mcx1对这一步骤的贡献，并缩小一组候选底物的范围。Mcx1调节血红素生物合成的底物相互作用的同一性和机制将通过Mcx1对底物处理的体内和体外互补试验来确定。这些努力将定义一种新的机制，通过这种机制来调节基本辅因子的生物合成。酿酒酵母和线虫的表型数据以及与其原核同源物的相似性表明，线粒体ClpX也可能调节其他线粒体过程。通过基于活性的蛋白质捕获策略与质谱学相结合，将采样Mcx1潜在的更广泛的生理底物；这一策略可能表明Mcx1调控的其他线粒体过程，并将允许定义针对Mcx1的线粒体蛋白的共同基序。这些研究试图通过一种有针对性的方法来了解其作为必要辅因子血红素生物合成中的控制元件的作用，并通过一种公正的方法来采样Mcx1对线粒体生理学的更广泛贡献，以确定线粒体调节因子ClpX同系物Mcx1的功能及其控制其活性的机制。这些研究可以为血红素生物合成异常引起的贫血和门静脉症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，包括代谢性疾病和由糖尿病引起的各种退行性疾病。确定线粒体对细胞需求和环境压力做出反应和适应的分子机制，将为理解这些反应的失败如何导致疾病提供一个新的框架。

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.