CLP: An Archetypal ATP-Dependent Protease

描述（由申请人提供）：能量依赖性蛋白水解机可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26S蛋白酶体，Clp和Lon，在电子显微镜下观察时，它们具有共同的桶状结构和共同的机制。在Lon中，atp酶和蛋白水解活性编码在一个氨基酸序列中，使底物识别和蛋白水解成为一个高度耦合的过程。在Clp中，蛋白水解核心ClpP和atp酶组分CIpA、ClpX或某些细菌中的ClpC在两个独立的氨基酸序列上编码，是稳定的同源低聚物，可以独立纯化表征。HlsUV也是如此，它包含一个蛋白水解成分HslV，它与26S蛋白酶体的n型亚基同源，以及一个clp样atp酶HslU。因此，CIp和hslv系统作为解决机械问题的模型系统比Lon具有主要优势，弗拉纳根博士在他的提案中利用了这一特性。本研究主要对ClpX和HslU及其复合物及其各自的蛋白水解亚基和底物进行生化和x射线晶体学研究，以研究这些atp酶亚基如何结合、展开和将底物转运到蛋白水解亚基中进行降解。这些atp酶具有单独的伴侣活性，当与它们的蛋白水解组分形成复合物时，可转化为展开酶活性。因此，这类atp酶的特征可能在细胞蛋白质量控制中起关键作用。首席研究员还提出了实验，以了解如何同源寡聚蛋白水解亚基可以切割肽键，几乎没有明显的序列特异性；后一种特征是所有能量依赖性蛋白酶的特征。本文提出的实验将使我们对能量依赖性蛋白质水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，这是所有细胞中必不可少的过程。

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.