BIOCHEMISTRY OF ENERGY DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

能量依赖性（细胞内）蛋白质降解的生物化学

• 批准号: 6100828
• 负责人: M R MAURIZI
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6100828
• 关键词: Escherichia coli; HeLa cells; active sites; adenosine triphosphate; adenosinetriphosphatase; bacterial proteins; bioenergetics; chemical stability; complementary DNA; electron microscopy; endopeptidases; enzyme activity; enzyme complex; enzyme structure; enzyme substrate; enzyme substrate complex; gel filtration chromatography; human genetic material tag; intermolecular interaction; molecular chaperones; molecular cloning; protein degradation; protein structure function

Our research is focused on the structure/function relationships of the ATP-dependent Clp and Lon proteases, which degrade important regulatory proteins as well as damaged and denatured proteins in E. coli and human cells. Wild-type Lon can be expressed using a Vaccinia expression system, and the protein is targeted to mitochondria and processed. Processing of human Lon appears to be autocatalytic, because a mutant Lon in which the active site serine residue has been altered is targeted to mitochondria but is not processed and co-expression of the mutant and the wild-type Lon proteases leads to processing of the mutant. Expression of wild-type Lon in HeLa cells causes the cells to round up and lose viability, whereas expression of the serine mutant has no deleterious effects. This system will be exploited to define functional changes in different Lon mutants and to identify physiological targets of human Lon protease. Stable transfectants of human Lon have been obtained using a construct with MDR1 as the dominant selectable marker. In this system also, high level expression of the inactive mutant but not of wild-type Lon has been possible. We have cloned and expressed E. coli ClpX, a member of the Clp family of ATPases. ClpX has ATP-dependent chaperone activity and is required for some specific ATP-dependent proteolytic activities dependent on ClpP. Gel filtration and electron microscopy show that ClpX subunits (Mr 46,000) associate to form a six- membered ring (Mr 280,000) that is stabilized by binding of ATP. In the presence of ATP, hexameric ClpX interacts with ClpP, a tetradecamer composed of superimposed seven-membered rings, to form a stable complex that can be isolated by gel filtration. In the complex, the rings of ClpP are flanked on each side by a single ring of ClpX. A symmetry mismatch thus exists between the seven-membered rings of ClpP and the six-membered ATPase rings for both ClpXP and ClpAP. Competition studies showed that ClpX and ClpA have nearly equal affinity for binding to ClpP, however no evidence for mixed complexes of ClpA, ClpX, and ClpP were observed suggesting that binding of a specific ATPase to one face of ClpP favors binding of a like ATPase to the opposite face of ClpP. The oligopeptide, FAPHMALVPV, is cleaved by ClpXP in the presence of non- hydrolyzable analogs of ATP with a turnover number of 10,000 min-1 (per tetradecamer of ClpP), indicating that ClpX, as does ClpA, allosterically activates affects ClpP to make the active site more accessible and to potentiate the catalytic efficiency of the proteolytic active site. Studies of subunit interactions in ClpP indicate that contacts between rings can be disrupted without breaking the subunit interactions within the heptameric rings. Treatment with high salt concentrations and low temperature lead to reversible separation of the ClpP rings, which can reassociate with complete restoration of enzymatic activity.

我们的研究主要集中在结构/功能关系