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: 5200937
• 负责人: M R MAURIZI
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 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/5200937
• 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 mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; human genetic material tag; intermolecular interaction; mutant; protein degradation

Rapid proteolytic degradation is a major mechanism for modulating the intracellular concentrations of regulatory proteins and affects developmental pathways, stress responses, metabolic adaptation, and essential cell cycle control processes. In vivo, most protein degradation is energy-dependent. Our research is focused on the ATP-dependent Clp and Lon proteases of E. coli and their homologs in mitochondria of human cells. The gene for a human Lon protease has been mapped to chromosome 19, region 13.3, and a cosmid clone of the genomic DNA has been isolated. A human cDNA encoding a homolog of ClpP has been isolated. Human ClpP is more than 50 percent identical to the bacterial protease at the amino acid level. Human ClpP expressed in E. coli and purified appears to have an oligomeric structure similar to the double-donut formed by E. coli ClpP but is not activated by E. coli ClpA. Electron microscopic studies show E. coli ClpAP has a structure analogous to that of the eukaryotic 26S proteasome. ClpP, the proteolytic core of the enzyme, is composed of superimposed seven-membered rings, whereas ClpA, the ATP-dependent component with protein unfolding activity, is a bi-lobed ring of only six subunits. In the ClpAP complex, a hexamer of ClpA is bound to each heptameric face of ClpP. Changes in these asymmetric interactions between subunits during the catalytic cycle may be important for unfolding or translocation of substrates during ATP-dependent proteolysis. A ClpA mutant with a lysine to glutamine substitution in the ATP-binding site of the first domain forms mixed hexamers with wild-type ClpA. Exchange studies with the mutant ClpA indicate that the half-time for subunit dissociation of ClpA under assay conditions is more than 8 min. Since the turnover number for peptide bond cleavage during proteolysis is at least 15 per min, multiple rounds of degradation occur without dissociation of ClpAP. Purified E. coli Lon protease requires ATP hydrolysis to degrade purified native CcdA, a physiological substrate for Lon, whereas degradation of a truncated form of CcdA by Lon is activated by nucleotide binding alone. The same peptide bonds are cleaved in each protein. Thus, ATP hydrolysis does not affect the specificity of the interaction between substrates and the proteolytic active site, but is required for unfolding or conformational alteration of the protein. Interaction with CcdB completely protects CcdA from degradation by Lon, demonstrating the importance of macromolecular interactions in determining the stability of regulatory proteins in vivo.

蛋白质的快速降解是调节细胞周期的主要机制 调节蛋白在细胞内的浓度及其影响 发育途径、应激反应、代谢适应和 基本的细胞周期控制过程。在体内，大多数蛋白质 降解是依赖能源的。我们的研究主要集中在 大肠杆菌及其同系物依赖于ATP的CLP和Lon蛋白水解酶 人类细胞的线粒体。人类Lon蛋白水解酶的基因 被定位到19号染色体13.3区，以及一个粘粒克隆 基因组DNA已经被分离出来。编码一个同源基因的人的cDNA 已分离出ClpP。人类ClpP基因的同源性超过50% 在氨基酸水平上对细菌蛋白水解酶的影响。人类ClpP 在大肠杆菌中表达和纯化的似乎有一个寡聚体 与E.ColiClpP形成的双甜甜圈相似的结构，但 不被大肠杆菌ClpA激活。电子显微镜研究表明，E。 ColiClpAP具有与真核26S相似的结构 蛋白酶体。ClpP是该酶的蛋白分解核心，由以下组成 叠加的七元环，而ClpA，依赖于ATP 具有蛋白质展开活性的组分，是仅有的一个双叶环 六个亚基。在ClpAP复合体中，ClpA的六角体结合到 ClpP的每个七聚体表面。这些不对称的变化 催化循环期间亚基之间的相互作用可能是 对于在以下过程中底物的展开或移动很重要 依赖于三磷酸腺苷的蛋白分解。赖氨酸为谷氨酰胺的ClpA突变体 第一结构域形式混合的ATP结合位点上的取代 带有野生型ClpA的六聚体。与突变体ClpA的交流研究 表明ClpA亚基解离的半衰期 测定条件为8min以上。由于成交额为 蛋白质分解过程中的肽键断裂速度至少为每分钟15次， 在没有解离ClpAP的情况下，发生了多轮降解。 纯化的大肠杆菌Lon蛋白酶需要ATP水解酶才能降解 纯化的天然CCDA，Lon的生理底物，而 Lon对截断形式的CCDA的降解通过以下方式激活 单独的核苷酸结合。每一个都裂解了相同的多肽键 蛋白。因此，三磷酸腺苷的水解不影响其特异性。 底物与蛋白降解活性部位之间的相互作用，但 蛋白质的展开或构象改变所必需的。 与Ccdb的交互通过以下方式完全保护CCDA不会降级 Lon，展示了大分子相互作用在 测定体内调节蛋白的稳定性。