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: 6160928
• 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/6160928
• 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.

我们的研究重点是结构/功能关系 ATP 依赖性 Clp 和 Lon 蛋白酶，可降解重要的调节因子 大肠杆菌和人类中的蛋白质以及受损和变性的蛋白质 细胞。野生型 Lon 可以使用痘苗病毒表达 系统，蛋白质被靶向线粒体并被加工。 人类 Lon 的加工似乎是自催化的，因为突变体 靶向活性位点丝氨酸残基已改变的 Lon 线粒体但未被加工并且突变体和的共表达 野生型 Lon 蛋白酶导致突变体的加工。 HeLa 细胞中野生型 Lon 的表达导致细胞聚集 并失去活力，而丝氨酸突变体的表达没有 有害影响。该系统将被用来定义功能 不同 Lon 突变体的变化并确定生理目标 人类 Lon 蛋白酶。 Stable transfectants of human Lon have been 使用以 MDR1 作为显性选择标记的构建体获得。 在该系统中，失活突变体也高水平表达，但 not of wild-type Lon has been possible. We have cloned and expressed E. 大肠杆菌 ClpX，ATP 酶 Clp 家族的成员。 ClpX 具有 ATP 依赖性 伴侣活性，并且是某些特定 ATP 依赖性所必需的 proteolytic activities dependent on ClpP. Gel filtration and electron 显微镜显示 ClpX 亚基 (Mr 46,000) 结合形成六- 通过结合 ATP 来稳定的元环 (Mr 280,000)。在 ATP 存在时，六聚体 ClpX 与十四聚体 ClpP 相互作用 由七元环叠加而成，形成稳定的配合物 that can be isolated by gel filtration. In the complex, the rings of ClpP 的两侧各有一个 ClpX 环。对称性 因此 ClpP 的七元环与 ClpXP 和 ClpAP 的六元 ATP 酶环。竞争研究 表明 ClpX 和 ClpA 具有几乎相同的结合亲和力 ClpP，但没有证据表明存在 ClpA、ClpX 和 ClpP 的混合复合物 观察到表明特定 ATP 酶与一张脸的结合 ClpP 有利于类似 ATP 酶与 ClpP 的另一面结合。 寡肽 FAPHMALVPV 在存在以下物质的情况下被 ClpXP 裂解 ATP 的不可水解类似物，周转数为 10,000 min-1 （每个 ClpP 的十四角体），表明 ClpX 和 ClpA 一样， 变构激活影响 ClpP 使活性位点更多 易于使用并增强蛋白水解酶的催化效率 活跃站点。 ClpP 亚基相互作用的研究表明 可以在不破坏子单元的情况下破坏环之间的接触 interactions within the heptameric rings.高盐处理 浓度和低温导致可逆分离 ClpP环，可以重新结合并完全恢复酶促作用 活动。