CLP: An Archetypal ATP-Dependent Protease

描述(申请人提供)：依赖能量的蛋白质水解机降解异常蛋白质和许多短暂的调节蛋白质。这些机器分为三个原型组，分别称为26S蛋白酶体、CLP和LON，在电子显微镜下观察时，它们共享一个共同的桶状结构和一个共同的机制。在LON中，ATPase和蛋白降解活性被编码在一个单一的氨基酸序列中，使得底物识别和蛋白降解是一个高度耦合的过程。在CLP中，蛋白分解核心ClpP和ATPase成分CIPA、ClpX或在一些细菌中ClpC编码在两个独立的氨基酸序列上，是可以独立纯化的稳定的同源低聚物。HlsUV也是如此，它包含一个蛋白水解组分，HslV，它与26S蛋白酶体的n型亚基同源，以及一个类似CLP的ATPase，HslU。因此，与Lon相比，CIP和HslUV系统作为解决机械问题的模型系统具有重大优势，Flanagan博士在他的提案中利用了这一特性。该提案集中于ClpX和HslU及其与它们各自的蛋白水解亚单位和底物的络合物的生化和X射线结晶学研究，以考察这些ATPase亚基如何结合、展开和转移底物到蛋白水解亚单位进行降解。这些ATPase单独具有伴侣活性，当与其蛋白水解组分形成复合体时，该活性转化为未折叠酶活性。因此，这类ATPase的功能很可能在细胞蛋白质质量控制中发挥关键作用。首席研究人员还提议进行实验，以了解同源寡聚蛋白水解亚单位如何在几乎没有明显的序列特异性的情况下裂解多肽键；后一特征是所有能量依赖的蛋白酶的标志。本文提出的实验将使人们对依赖能量的蛋白质分解的机制有一个全面的了解，从而可以更深入地了解蛋白质质量控制的过程，这是所有细胞的一个必不可少的过程。

DESCRIPTION (provided by applicant): Energy-dependent proteolytic machines degrade abnormal proteins and many short-lived regulatory proteins. These machines, which fall into three archetypal groups called the 26S proteasome, Clp and Lon, share a common barrel like architecture when viewed in the electron microscope and a common mechanism. In Lon, the ATPase and proteolytic activities are coded within a single amino acid sequence, making substrate recognition and proteolysis a highly coupled process. In Clp the proteolytic core, ClpP, and ATPase component, CIpA, ClpX, or in some bacteria ClpC, are coded for on two separate amino acid sequences and are stable homo-oligomers that can be purified characterized independently. The same is true for HlsUV, which contains a proteolytic component, HslV that is homologous to the n-type subunits of the 26S proteasome, and a Clp-like ATPase, HslU. Thus, CIp and HslUV systems have a major advantage over the Lon as model systems for addressing mechanistic questions, and Dr. Flanagan has exploited this property in his proposal.This proposal is focused on biochemical and x-ray crystallographic studies of ClpX and HslU and their complexes with their respective proteolytic subunits and substrates to examine how these ATPase subunits bind, unfold and translocate substrates into the proteolytic subunits for degradation. These ATPases have a chaperone activity alone that converts to an unfoldase activity when in a complex with their proteolytic component. Thus features of this class of ATPase are likely to have a key role in cellular protein quality control. The principal investigator has additionally proposed experiments to understand how the homo-oligomeric proteolytic subunits can cleave peptide bonds with little apparent sequence specificity; this latter feature is a hallmark of all of the energy-dependent proteases. The experiments proposed herein will allow an overall understanding of the mechanism of energy dependent proteolysis and thus one can expect a deeper understanding of the process of protein quality control which is a essential process in all cells.