PHYSIOLOGY OF PROTEASOMES IN HALOFERAX VOLCANII

HALOFERAX VOLCANII 中蛋白酶体的生理学

• 批准号: 6386886
• 负责人: JULIE A MAUPIN-FURLOW
• 金额: $ 17.87万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386886
• 关键词: Archaea; RNase protection assay; biological signal transduction; gene mutation; messenger RNA; microorganism metabolism; molecular cloning; nucleic acid sequence; posttranscriptional RNA processing; proteasome; protein metabolism

DESCRIPTION: (Adapted from the Investigator's Abstract) Our long term goal is to obtain a better understanding of energy-dependent proteolysis in the cell, especially as it relates to the metabolically diverse Archaea which play a major role in global carbon mineralization and the production of greenhouse gases. Surprisingly, little is known about regulated protein turnover in the Archaea which are closely related to the Eucarya. Energy-dependent proteolysis is now known to be central to the regulation of cell division, metabolism, transcription, and other essential functions directly related to human health. Proteases which mediate energy-dependent hydrolysis comprise a small group of structurally related proteins. These proteases form a compartment with narrow openings which isolate the proteolytic active-sites away from the cytoplasmic constituents, thus, avoiding, non-specific protein degradation. An energy-dependent component of the protease is needed for recognition and/or unfolding of substrates which are then fed into the proteolytic chamber. This energy-dependence provides a proofreading step to insure that the proper substrate has been selected for destruction. Although energy-dependent proteases are predicted based on archaeal genome sequences, the only component which has been purified is the 20S proteasome. These 20S proteasomes only act on unfolded proteins in vitro and, thus, have not facilitated the identification of native substrates in the archaeal cell. The main objective of this study is to examine proteasome-mediated degradation in the archaeon Haloferax volcanii using genetic methods to determine in vivo proteasome functions. To achieve these goals, the 20S proteasome mRNA transcripts and protein levels will be analyzed under a variety of culture conditions which will provide information of the regulation, promoters, and operons of the proteasomes. The levels of active proteasome proteins will also be modified through chromosomal mutagenesis as well as the homologous express of epitope-tagged proteasome genes from plasmids. These approaches will enable us to link the in vivo level of the various proteasome proteins with cell phenotype(s) and, thus, provide an understanding of the role of these proteins in archaeal physiology. Furthermore, expression of the epitope-tagged proteasome subunits in H. volcanii will enable us to determine the arrangement of the alpha1, alpha2 and beta subunits in the 20S proteasomes. Several genetic methods are also presented which will facilitate the identification of foreign and native proteins which are degraded by the proteasome in the cell. This study will, therefore, unveil pathways which are regulated by the proteasomes and lay a foundation for understanding the biology of proteolysis as a mechanism of post-transcriptional regulation in this unusual group of organisms.

描述：（改编自研究者摘要）我们的长期目标是 为了更好地了解细胞中能量依赖性蛋白水解作用， 特别是因为它与代谢多样化的古细菌有关，这些古细菌发挥着重要作用 在全球碳矿化和温室气体产生中发挥着重要作用 气体。令人惊讶的是，人们对蛋白质周转的调节知之甚少。 古菌与真核菌密切相关。能量依赖性蛋白水解 现在已知它对于细胞分裂、新陈代谢的调节至关重要， 转录以及其他与人类健康直接相关的重要功能。 介导能量依赖性水解的蛋白酶包括一小群 结构相关的蛋白质。这些蛋白酶形成一个狭窄的隔室 将蛋白水解活性位点与细胞质隔离的开口 成分，从而避免非特异性蛋白质降解。一个 识别和/或需要蛋白酶的能量依赖性成分 底物展开，然后送入蛋白水解室。这 能量依赖性提供了一个校对步骤，以确保正确的 已选择要销毁的基材。虽然依赖能源 蛋白酶是根据古菌基因组序列预测的，古菌基因组序列是唯一的组成部分 纯化后的就是20S蛋白酶体。这些 20S 蛋白酶体只起作用 体外未折叠的蛋白质，因此没有促进 古细菌细胞中天然底物的鉴定。主要目标 这项研究的目的是检查古细菌中蛋白酶体介导的降解 Haloferax volcanii 使用遗传方法确定体内蛋白酶体 功能。为了实现这些目标，20S 蛋白酶体 mRNA 转录本和 将在多种培养条件下分析蛋白质水平 将提供调节、启动子和操纵子的信息 蛋白酶体。活性蛋白酶体蛋白的水平也将被改变 通过染色体突变以及同源表达 来自质粒的表位标记的蛋白酶体基因。这些方法将使我们能够 将各种蛋白酶体蛋白的体内水平与细胞联系起来 表型，从而提供对这些蛋白质作用的理解 在古菌生理学中。此外，表位标记的表达 H. volcanii 中的蛋白酶体亚基将使我们能够确定排列 20S 蛋白酶体中的 alpha1、alpha2 和 beta 亚基。几个遗传 还提出了有助于识别外国人的方法 以及被细胞中的蛋白酶体降解的天然蛋白质。这 因此，研究将揭示蛋白酶体调节的途径 并为理解蛋白水解生物学奠定基础 这一不寻常的群体的转录后调控机制 有机体。