Regulation of Heme Synthesis and Mitochondrial Physiology by the ClpX Unfoldase

描述（由申请人提供）：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，但这些适应的控制机制知之甚少。蛋白展开酶ClpX是原核生物中一种重要的调控元件，通过选择底物介导细胞状态的变化和对胁迫条件的反应。在真核生物中，ClpX是线粒体中广泛保守的成分，但尚未发现线粒体ClpX底物，ClpX对线粒体生理的具体贡献尚不清楚。本提案旨在定义线粒体ClpX如何调节线粒体生理。酿酒酵母大规模遗传互作图谱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。代谢分析将用于确定Mcx1对这一步的贡献，并缩小一组候选底物。Mcx1调节血红素生物合成的底物相互作用的特性和机制将通过Mcx1处理底物的互补体内和体外实验来确定。这些努力将定义一种新的机制，通过这种机制调节一种必需辅因子的生物合成。酿酒葡萄球菌和秀丽隐杆线虫的表型数据，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。通过结合质谱法的基于活性的蛋白质捕获策略，将对Mcx1的潜在更广泛的生理底物进行采样；这一策略可能表明Mcx1调节的其他线粒体过程，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理解其作为必要的辅助因子血红素生物合成中的控制元件的作用，并通过一种无偏倚的方法来对Mcx1对线粒体生理学的更广泛贡献进行采样，从而定义线粒体调节因子ClpX同源物Mcx1的功能库，以及其活性被控制的机制。这些研究可以为血红素生物合成异常引起的贫血以及卟啉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。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.