CLP: An Archetypal ATP-Dependent Protease

CLP：一种原型 ATP 依赖性蛋白酶

• 批准号: 6908083
• 负责人: John M Flanagan
• 金额: $ 32万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6908083
• 关键词: X ray crystallography; adenosine triphosphate; binding sites; bioenergetics; enzyme activity; enzyme complex; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; molecular chaperones; proteasome; protein degradation; protein structure function; proteolysis; serine proteinases; structural biology

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.

描述（由申请人提供）：能量依赖性的蛋白水解机降解异常蛋白质和许多短寿命的调节蛋白。这些机器分为三个原型组，称为26S蛋白酶体，CLP和LON，当在电子显微镜中观看时，共享一个共同的桶形结构和一种共同的机制。在LON中，ATPase和蛋白水解活性在单个氨基酸序列中编码，从而使底物识别和蛋白水解成为高度耦合的过程。在CLP中，将蛋白水解核心，CLPP和ATPase成分，CIPA，CLPX或某些细菌CLPC中的蛋白水解核心组成，在两个单独的氨基酸序列上编码，并且是稳定的同性恋者，可以独立纯化。对于包含蛋白水解成分的HLSUV也是如此，HSLV与26S蛋白酶体的N型亚基同源，以及类似CLP的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蛋白水解亚基降解。这些ATPases具有伴侣活性，该活性在与其蛋白水解成分的复合物中转换为进食酶活性。因此，这类ATPase的特征可能在细胞蛋白质质量控​​制中具有关键作用。主要研究者还提出了实验，以了解均具有明显序列特异性的同型蛋白水解亚基如何裂解肽键。后一个特征是所有依赖能量蛋白酶的标志。本文提出的实验将允许对依赖能量依赖性蛋白水解的机理有所了解，因此人们可以期望对蛋白质质量控​​制的过程有更深入的了解，这在所有细胞中都是必不可少的过程。