Mechanisms of Chaperone-Mediated Control in the Assembly of the Proteasome Holoenzyme

蛋白酶体全酶组装中分子伴侣介导的控制机制

• 批准号: 9750305
• 负责人: Soyeon Park
• 金额: $ 31.8万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750305
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Aging; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cell physiology; Cells; Complex; Critical Pathways; Data; Dependence; Development; Disease; Escherichia coli; Event; Goals; Growth; Health; Holoenzymes; Human; In Vitro; Link; Malignant Neoplasms; Mediating; Mediator of activation protein; Medical; Modeling; Molecular Chaperones; Molecular Machines; Nerve Degeneration; Neurodegenerative Disorders; Nucleosome Core Particle; Nucleotides; Outcome; Pathologic; Pathway interactions; Phenotype; Polyubiquitination; Process; Proteins; Proteolysis; Public Health; Publishing; Quality Control; Regulation; Research; Role; Route; Saccharomycetales; Structure; Syndrome; System; Testing; Time; Ubiquitin; Ubiquitination; Variant; Work; Yeast Model System; Yeasts; base; cell growth; combat; human disease; in vivo; innovation; multicatalytic endopeptidase complex; mutant; new therapeutic target; prevent; protein degradation; reconstitution; therapeutic target; ubiquitin-protein ligase; yeast genetics

PROJECT SUMMARY The proteasome is a complex molecular machine and an essential mediator of ubiquitin-dependent protein degradation. Polyubiquitination of target proteins has been long considered the major regulator of proper protein degradation. However, little is known about a new and potent regulator of cellular protein degradation — mechanisms that control the rate and quality of proteasome assembly. It is well established that cells require multiple evolutionarily conserved chaperones to regulate proteasome assembly. A catalyzed assembly mechanism serves as an active regulator of proper proteasome assembly, and therefore of proteolysis itself. Under some pathological situations, cells are known to exploit these chaperones to alter the rate of proteasome- mediated proteolysis. Moreover, chaperone-mediated assembly of proteasomes has also been linked to major pathways for growth control. Therefore, understanding how chaperones control proteasome assembly is of wide- ranging biological and biomedical significance. The objective of this proposal is to elucidate how these chaperones function to regulate the rate and the quality of proteasome formation. Our central hypothesis is that the chaperones determine correct versus incorrect assembly of the proteasome by regulating both the ATP hydrolysis and the ubiquitination of the ATPase subunits en route to proteasome formation. Aim 1 will determine how chaperone-mediated inhibition of ATP hydrolysis by the proteasome subunits controls proteasome assembly events. Both in vitro and in vivo proteasome assembly assays will be used while the ATPase rates are directly modulated by specific yeast mutants. Aim 2 will determine how ubiquitinations of the proteasome subunits provide quality control during chaperone-mediated proteasome assembly. Biochemical approaches similar to Aim 1 will be used in combination with yeast genetics. The goal is to identify how the chaperones regulate ubiquitination of the proteasome subunits, and how ubiquitination affects assembly intermediates and the assembled proteasome holoenzyme. The expected outcome is the identification of the mechanisms by which the chaperones determine incorrect versus correct assembly events, and provide quality control, thereby enabling only the correct assembly events to proceed in forming the proteasome holoenzyme. The proposed research is innovative because it aims to identify mechanisms that regulate the number of functional proteasomes in the cell, which have been missing from the current paradigm of proteasome-mediated proteolysis. This contribution is significant for human health to help understand how this critical pathway is exploited to alter protein degradation in pathological conditions, including cancer, neurodegenerative disease and aging. These conditions are known to be impacted by the ubiquitin-proteasome system, a proven therapeutic target for altering protein degradation in disease. We anticipate that understanding chaperone-mediated regulation of the proteasome subunits will contribute to the development of new targeted drugs.

项目摘要 蛋白酶体是一个复杂的分子机器，是泛素依赖的蛋白质合成的重要介质。 蛋白质降解靶蛋白的多聚泛素化一直被认为是细胞内适当的蛋白质表达的主要调节因子。 蛋白质降解然而，人们对一种新的有效的细胞蛋白质降解调节剂知之甚少- 控制蛋白酶体组装速率和质量的机制。众所周知，细胞需要 多种进化上保守的分子伴侣来调节蛋白酶体组装。一种催化组装 该机制作为适当的蛋白酶体组装的主动调节器，因此也是蛋白水解本身的主动调节器。 在某些病理情况下，已知细胞利用这些分子伴侣来改变蛋白酶体的速率， 介导的蛋白水解。此外，分子伴侣介导的蛋白酶体组装也与主要的 控制生长的途径。因此，了解分子伴侣如何控制蛋白酶体组装具有广泛的意义。 包括生物学和生物医学意义。本提案的目的是阐明这些 伴侣蛋白的功能是调节蛋白酶体形成的速率和质量。我们的核心假设是， 分子伴侣通过调节ATP和蛋白酶体， 在蛋白酶体形成的过程中，ATP酶亚基的水解和泛素化。目标1将决定 蛋白酶体亚单位如何通过伴侣介导的ATP水解抑制来控制蛋白酶体 集会活动。将使用体外和体内蛋白酶体组装测定，同时ATP酶速率为 由特定的酵母突变体直接调节。目的2将确定蛋白酶体的泛素化 亚基在分子伴侣介导的蛋白酶体组装过程中提供质量控制。生物化学方法 类似于目标1的基因将与酵母遗传学组合使用。我们的目标是确定伴侣是如何 调节蛋白酶体亚基的遍在化，以及遍在化如何影响组装中间体和 组装的蛋白酶体全酶。预期的结果是确定了 所述分子伴侣确定不正确与正确的组装事件，并提供质量控制，从而 使得在形成蛋白酶体全酶时仅进行正确的装配事件。拟议 研究是创新的，因为它的目的是确定调节功能数量的机制， 蛋白酶体在细胞中，这已经从蛋白酶体介导的蛋白水解的当前范例失踪。 这一贡献对人类健康具有重要意义，有助于了解这一关键途径是如何被利用来改变 病理条件下的蛋白质降解，包括癌症、神经退行性疾病和衰老。这些 已知条件受到泛素-蛋白酶体系统的影响，泛素-蛋白酶体系统是一种经证实的用于改变 疾病中的蛋白质降解。我们预计，了解分子伴侣介导的调节， 蛋白酶体亚单位将有助于开发新的靶向药物。