STRUCTURE AND FUNCTION OF THE MITOCHONDRIAL LON PROTEASE

描述（改编自申请人的摘要）：提出的调查路线 在这项研究计划是基于假设， ATP依赖性Lon蛋白酶监测蛋白质的完整性和表达。 线粒体基因组以及新生的 多肽。本提案的目标是分析结构， 酵母Lon的功能，并鉴定哺乳动物和酵母蛋白， 功能上与Lon重叠。分子生物学，遗传学， 生物化学和结构生物学将用于实现以下目标： 分析Lon对线粒体DNA-蛋白质相互作用的调控作用。的 从细菌到人类，Lon的DNA结合功能是保守的，然而， 这种活性的生理重要性尚不清楚。研究者 将鉴定酵母Lon结合的内源mtDNA序列，分析 Lon在线粒体DNA-蛋白质复合物（称为类核蛋白）中的作用，并确定 负责增加双链DNA结合的蛋白质 缺乏Lon的线粒体 为了鉴定和表征哺乳动物和酵母蛋白， 功能与Lon一个温敏基因的遗传互补 将使用设计用于在酵母中表达的哺乳动物文库的突变体， 贯彻鉴定的哺乳动物蛋白质将在以下方面进行表征： 哺乳动物细胞系统。此外，转录水平的全基因组调查 将使用以下方法对缺失Lon基因的酵母进行改变： 寡核苷酸探针微阵列。 阐明同源七聚体的结构动力学和功能 利用圆二色性和高分辨电子 显微镜 线粒体功能障碍与多种退行性疾病有关， 疾病、衰老和癌症。最典型的基因突变导致了 线粒体功能丧失的原因是缺失、点突变和碱基突变。 线粒体DNA的替换。细胞对压力的反应 氧化损伤和衰老可能涉及ATP依赖性的作用， 选择性降解变性、聚集和未组装的蛋白酶 proteins.了解ATP依赖性蛋白酶 通过监测蛋白质的生物合成以及 线粒体基因组的完整性和表达，将提供深入了解 有助于防止线粒体功能障碍的机制。

DESCRIPTION (adapted from applicant's abstract): The line of inquiry proposed in this research program is based on the hypothesis that the mitochondrial ATP-dependent Lon protease monitors both the integrity and expression of the mitochondrial genome as well as the folding and assembly of nascent polypeptides. The goals of this proposal are to analyze the structure and function of yeast Lon and to identify mammalian and yeast proteins that functionally overlap with Lon. A combination of molecular biology, genetics, biochemistry and structural biology will be used to address the following aims: To analyze the role of Lon in regulating the mtDNA-protein interactions. The DNA-binding function of Lon is conserved from bacteria to humans, however the physiological importance of this activity is not understood. The investigator will identify the endogenous mtDNA sequences bound by yeast Lon, analyze the role of Lon in mtDNA-protein complexes called nucleoids and identify the protein(s) responsible for increased double-stranded DNA-binding in mitochondria that lack Lon. To identify and characterize mammalian and yeast proteins that have a shared function with Lon. Genetic complementation of a temperature-sensitive Lon mutant using mammalian libraries designed for expression in yeast will be carried out. The mammalian proteins identified will be characterized in mammalian cell systems. In addition, a genome-wide survey of transcript level changes in yeast deleted for the Lon gene will be conducted using oligonucleotide probe microarrays. To elucidate the structural dynamics and function of the homo-heptameric ring-shaped Lon protease using circular dichroism and high-resolution electron microscopy. Mitochondrial dysfunction has been implicated in a wide variety of degenerative disorders, aging and cancer. The best-characterized genetic mutations that lead to a loss of mitochondrial function are deletions, point mutations and base substitutions in mitochondrial DNA. Cellular responses to stress caused by oxidative damage and aging are likely to involve the action of ATP-dependent proteases which selectively degrade denatured, aggregated and unassembled proteins. Understanding the molecular details of how ATP-dependent proteases ensure cellular homeostasis by monitoring protein biogenesis as well as the integrity and expression of the mitochondrial genome, will provide insight into mechanisms that help to prevent mitochondrial dysfunction.