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: 2463651
• 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/2463651
• 关键词: Escherichia coli; 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; human genetic material tag; human tissue; intermolecular interaction; molecular chaperones; protein degradation

ATP-dependent proteases degrade important regulatory proteins and help dispose of damaged and denatured proteins in the cell. Our research is focused on the specificity and mechanism of action of the Clp and Lon proteases of E. coli and human cells. Sequence analysis of a genomic clone of human LON, which produces an ATP-dependent protease targeted to mitochondria, has located the positions of 16 introns. This information will help in determining structurally distinct regions of Lon and suggests fusion joints for the construction of chimeric Lon proteases. To date, human LON cDNA clones, as well as several chimeras of human and E. coli lon, have proven to be unstable in E. coli, and the protein is poorly expressed and unstable. Other expression systems are being used (Vaccinia) or are under construction (yeast) to provide model systems to test the effects of mutational changes on Lon activity in vivo. In studies in which purified E. coli Lon protease was used to degrade purified CcdA, ATP hydrolysis was shown to be required for disruption of the secondary structure of CcdA, presumably to allow greater ease of entry to the proteolytic active sites. Conditions that stabilize secondary structure, such as functional interaction with other proteins, protect CcdA from degradation. The sequence of E. coli ClpA suggested, and electron micrographs confirmed, that the protein has two ATPase domains, and mutational studies indicated that the C-terminal domain was important for the activation of ClpP. In protease protection assays, addition of ClpP protected the C-terminal but not the N-terminal domain of ClpA, indicating that the C-terminal ATPase domain interacts with ClpP. A novel member of the Clp protease family, ClpYQ, was cloned and the proteins purified. ClpY and ClpQ were isolated separately and could be combined to form a high molecular weight complex with ATP-dependent protein degrading activity. Electron micrographs show that subunits of ClpQ are arranged in the form of a hexagonal ring. In this regard, ClpQ differs strikingly from the heptagonal 20 S proteasome, despite the significant degree of homology between these proteins. Binding of ATP stabilizes an oligomeric form of ClpY which also has a hexagonal structure. Because earlier studies had shown that ClpA is a hexamer and ClpP is a heptamer, it was postulated that asymmetric interactions between subunits may be important during the catalytic cycle. However, the identical six-fold symmetries of ClpY and ClpQ in the ClpYQ complex indicate that a symmetry mismatch is not essential for coupling activity of the ATP-dependent chaperone to that of the proteolytic component.

ATP依赖性蛋白酶降解重要的调节蛋白， 处理细胞中受损和变性的蛋白质。 我们的研究 是集中在特异性和作用机制的CLP和 大肠杆菌的Lon蛋白酶。大肠杆菌和人类细胞。 序列分析 人LON的基因组克隆，其产生ATP依赖性蛋白酶 针对线粒体，定位了16个内含子的位置。 这些信息将有助于确定结构上不同的区域 并建议融合关节的嵌合Lon的建设 蛋白酶 迄今为止，人LON cDNA克隆以及几种嵌合体 人和E. coli lon，已证明在E.杆菌和 该蛋白质表达差且不稳定。 其它表达系统 正在使用（牛痘）或正在建设（酵母），以提供 测试突变变化对Lon活性的影响的模型系统 in vivo. 在研究中，纯化的E. coli Lon蛋白酶 为了降解纯化的CcdA，ATP水解被证明是必需的。 破坏CcdA的二级结构，可能是为了 更容易进入蛋白水解活性位点。 的条件 稳定二级结构，例如与 其他蛋白质，保护CcdA免受降解。 对E.杆菌 ClpA表明，电子显微镜证实， 具有两个ATP酶结构域，突变研究表明， C端结构域对ClpP的激活起重要作用。 在 蛋白酶保护测定，ClpP的加入保护了C-末端 而不是ClpA的N-末端结构域，表明ClpA的C-末端结构域 ATP酶结构域与ClpP相互作用。 Clp蛋白酶的新成员 克隆了ClpYQ家族，并纯化了蛋白质。 ClpY和ClpQ 分别分离得到，并可结合形成高分子 具有ATP依赖性蛋白质降解活性的重量复合物。 电子 显微照片显示，ClpQ的亚基以α-淀粉样蛋白的形式排列， 六角环 在这方面，ClpQ与 七角形20 S蛋白酶体，尽管有显着程度的同源性 这些蛋白质之间。 ATP的结合稳定了寡聚体形式 的ClpY，其也具有六方结构。 因为早期的研究 已经表明ClpA是六聚体，ClpP是七聚体， 假设亚基之间的不对称相互作用可能是 在催化循环中起重要作用。 然而，同样的六重 ClpYQ复合物中ClpY和ClpQ的对称性表明， 对称性错配对于偶联活性不是必需的， ATP依赖性分子伴侣与蛋白水解组分的分子伴侣。