CLP: An Archetypal ATP-Dependent Protease

描述（由申请人提供）：能量依赖性蛋白水解机器降解异常蛋白和许多短寿命调节蛋白。这些机器分为三个原型组，称为26 S蛋白酶体，Clp和Lon，在电子显微镜下观察时具有共同的桶状结构和共同的机制。在Lon中，ATP酶和蛋白水解活性在单个氨基酸序列内编码，使得底物识别和蛋白水解成为高度耦合的过程。在Clp中，蛋白水解核心ClpP和ATP酶组分ClpA、ClpX或在一些细菌中的ClpC在两个单独的氨基酸序列上编码，并且是可以独立地纯化表征的稳定的同源寡聚体。对于HlsUV也是如此，其含有与26 S蛋白酶体的n型亚基同源的蛋白水解组分HslV和Clp样ATP酶HslU。因此，Clp和HslUV系统作为解决机制问题的模型系统比Lon具有主要优势，并且Flanagan博士在他的提案中利用了这一性质。该提案集中于ClpX和HslU及其与其各自的蛋白水解亚基和底物的复合物的生物化学和X射线晶体学研究，以检查这些ATP酶亚基如何结合，解折叠底物并将其转移到蛋白水解亚基中进行降解。这些ATP酶单独具有伴侣活性，当与其蛋白水解组分形成复合物时，其转化为解折叠酶活性。因此，这类ATP酶的特征可能在细胞蛋白质质量控制中起关键作用。主要研究者还提出了实验，以了解同源寡聚蛋白水解亚基如何在几乎没有明显序列特异性的情况下切割肽键;后者的特征是所有能量依赖性蛋白酶的标志。本文提出的实验将允许能量依赖性蛋白质水解的机制的全面理解，因此，人们可以期望更深入地了解蛋白质质量控制的过程，这是所有细胞中的一个基本过程。

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.