BIOCHEMISTRY OF ENERGY-DEPENDENT (INTRACELLULAR) PROTEIN DEGRADATION

我们的研究集中在选择细胞蛋白进行降解的机制以及降解它们的依赖于ATP的蛋白酶的结构/功能关系。依赖于ATP的Lon和CLP酶存在于所有生物体中，它们调节重要的调节蛋白的水平，并通过清除受损的蛋白质来参与蛋白质质量控制途径。ATP依赖的蛋白水解酶是与蛋白水解酶紧密结合的分子伴侣的高相对分子质量复合体。重建ClpAP的电子显微镜图像提供了一个结构模型，有助于解释其作用，并为其他依赖ATP的蛋白酶提供了一个范例。ClpAP由2个7元的ClpP环组成，两侧各有一个6元的ClpA环。一个包含蛋白分解活性部位的大水室被ClpP的环所包围。ClpA亚基包裹着另一个水室，该水室可能是蛋白质底物在转位到ClpP中的活性部位之前展开的位置。ClpA在每个亚基中有两个结构不同的ATPase结构域。对ClpA突变体K220V的研究表明，N端ATPase结构域(结构域I)是伴侣活性所必需的。ClpP的结合对ClpA具有变构作用，可以恢复某些结构域I突变体的ATPase和伴侣活性。在与NCI的Sue Wickner合作进行的研究中，我们已经证明了与ClpAP结合的底物可以在重塑状态下被降解或释放。因此，相同的初始结合室用于复性和转位到蛋白水解酶。用ClpA和ClpP交换研究了ClpAP络合物在催化过程中的半衰期。我们的结果表明，该复合体在多轮底物展开和降解过程中是稳定的，从而表明蛋白质与组装的复合体相互作用，并可以从外部结合部位移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。目前正在研究以大肠杆菌Lon蛋白水解酶为代表的一组不同的ATP依赖蛋白水解酶的功能结构域。Lon-ATPase和蛋白分解功能位于单一的多肽链中。超速离心法和扫描电子显微镜的分子量测量表明，Lon以六聚体和十二聚体的形式存在，较大的物种通过核苷酸结合而稳定。限制性蛋白水解法确定了三个不同的结构域--N-末端结构域、由核苷酸结合稳定的中央ATPase结构域和C-末端蛋白水解结构域，该结构域本身似乎具有有限的肽酶活性。部分裂解产物的相对分子质量表明，LON的寡聚区位于ATPase中心区域。Lon的电子显微镜照片显示了具有伪两重对称性的细长粒子，而亚低聚物的显微照片显示了具有缺口环状外观的结构。Lon功能结构域之间的相互作用可能类似于CLP蛋白酶的相互作用，这表明所有依赖于ATP的蛋白酶在结构上都有一个潜在的相似性。-ATPase，伴侣，翻译后调节，蛋白质分解，CLP，蛋白质折叠，蛋白质稳定性，-非人类受试者或人类组织

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