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: 6289126
• 负责人: MICHAEL 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/6289126
• 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 mechanisms by which cellular proteins are selected for degradation and the structure/function relationships of the ATP-dependent proteases that degrade them. The ATP-dependent Lon and Clp proteases are found in all organisms, where they serve to modulate the levels of important regulatory proteins and contribute to protein quality control pathways by eliminating damaged proteins. The ATP-dependent proteases are high molecular weight complexes of a molecular chaperone tightly associated with a protease. Reconstruction of electron microscopic images of ClpAP has provided a structural model which helps explain its action and serves as a paradigm for other ATP- dependent proteases. ClpAP is composed 2 seven-membered rings of ClpP flanked on each side by a six-membered ring of ClpA. A large aqueous chamber containing the proteolytic active sites is enclosed by the rings of ClpP. The ClpA subunits enclose another aqueous chamber which may be the site where protein substrates are unfolded prior to translocation to the active site chamber in ClpP. ClpA has two structural distinct ATPase domains in each subunit. Studies with the ClpA mutant, K220V, have shown that the N-terminal ATPase domain (domain I) is required for chaperone activity. Binding of ClpP has allosteric effects on ClpA and can restore the ATPase and chaperone activities to some domain I mutants. In studies done in collaboration with Sue Wickner, NCI, we have shown that substrates bound to ClpAP can be degraded or released in a remodeled state. Thus, the same initial binding chamber is used for both refolding and for translocation to the protease. ClpA and ClpP exchange studies were conducted to measure the half-life of ClpAP complexes during catalysis. Our results show that the complex is stable during multiple rounds of substrate unfolding and degradation, thus showing that proteins interact with the assembled complex and can enter the unfolding and degradation chambers by translocation from external binding sites. Electron microscopic images confirm the model derived from kinetic studies. Studies are underway to dissect the functional domains of a distinct family of ATP-dependent proteases represented by E. coli Lon protease. The Lon ATPase and proteolytic functions lie within a single polypeptide chain. Molecular weight measurements made by ultracentrifugation and by scanning transmission electron microscopy indicate that Lon exists in hexameric and dodecameric forms, with the larger species stabilized by nucleotide binding. Limited proteolysis identified three distinct domains- an N- terminal domain, a central ATPase domain which is stabilized by nucleotide binding, and a C-terminal proteolytic domain which appears to have a limited peptidase activity on its own. Molecular weights of partial cleavage products suggest that the oligomerization domain of Lon lies within the central ATPase region. Electron micrographs of Lon indicate an elongated particle with a pseudo-two fold symmetry and micrographs of sub-oligomers reveal structures with a notched-ring-like appearance. Interactions between the functional domains of Lon may be analogous to those seen with the Clp proteases, suggesting that there is an underlying similarity in architecture for all ATP-dependent proteases. - ATPase, chaperone, post-translational regulation, proteolysis, Clp, protein folding, protein stability, - Neither Human Subjects nor Human Tissues

我们的研究重点是细胞蛋白被选择降解的机制以及降解它们的atp依赖性蛋白酶的结构/功能关系。atp依赖的Lon和Clp蛋白酶存在于所有生物体中，它们可以调节重要调节蛋白的水平，并通过消除受损蛋白来参与蛋白质质量控制途径。atp依赖性蛋白酶是与蛋白酶紧密相关的分子伴侣的高分子量复合物。ClpAP的电镜图像重建提供了一个结构模型，有助于解释其作用，并作为其他ATP依赖性蛋白酶的范例。ClpAP由两个七元环ClpP组成，每侧由一个六元环ClpA组成。含有蛋白水解活性位点的大水腔被ClpP环包围。ClpA亚基包裹着另一个水腔，该水腔可能是蛋白质底物在转运到ClpP活性位腔之前展开的位置。ClpA在每个亚基中有两个结构不同的atp酶结构域。对ClpA突变体K220V的研究表明，n端atp酶结构域（结构域I）是伴侣蛋白活性所必需的。ClpP结合对ClpA具有变构作用，可以恢复某些结构域I突变体的atp酶和伴侣活性。在与NCI的Sue Wickner合作进行的研究中，我们已经表明与ClpAP结合的底物可以降解或以重塑状态释放。因此，相同的初始结合腔用于重折叠和转运到蛋白酶。进行了ClpA和ClpP交换研究，以测量ClpAP配合物在催化过程中的半衰期。我们的研究结果表明，该复合物在多轮底物展开和降解过程中是稳定的，这表明蛋白质与组装的复合物相互作用，并可以通过从外部结合位点的易位进入展开和降解室。电子显微镜图像证实了动力学研究得出的模型。以大肠杆菌Lon蛋白酶为代表的一个独特的atp依赖性蛋白酶家族的功能域的研究正在进行中。长atp酶和蛋白水解功能位于一个多肽链内。通过超离心和扫描透射电子显微镜进行的分子量测量表明，Lon以六聚体和十二聚体形式存在，较大的种类通过核苷酸结合稳定。有限的蛋白水解鉴定出三个不同的结构域- N端结构域，一个通过核苷酸结合稳定的中心atp酶结构域，以及一个c端蛋白水解结构域，其本身似乎具有有限的肽酶活性。部分裂解产物的分子量表明，Lon的寡聚化结构域位于atp酶中心区域。电子显微图显示了一个具有伪双重对称的细长粒子，亚低聚物的显微图显示了具有缺口环状外观的结构。Lon功能域之间的相互作用可能类似于Clp蛋白酶之间的相互作用，这表明所有atp依赖性蛋白酶在结构上存在潜在的相似性。- atp酶，伴侣，翻译后调节，蛋白质水解，Clp，蛋白质折叠，蛋白质稳定性，-既不是人类受试者也不是人类组织