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：（根据调查员的摘要进行了改编）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， 尤其是与代谢多样的古细菌有关的 在全球碳矿化和温室生产中的主要作用 气体。令人惊讶的是，关于调节的蛋白质更新知之甚少 古细菌与桉树密切相关。能量依赖性蛋白水解 现在已知是细胞分裂的调节，代谢的核心， 转录和其他与人类健康直接相关的基本功能。 介导能量依赖水解的蛋白酶包括一小部分 结构相关的蛋白质。这些蛋白酶形成一个狭窄的隔室 分离蛋白水解活性位点的开口远离细胞质 因此，成分避免了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 然后馈入蛋白水解室的底物的展开。这 能量依赖性提供了一个校对步骤，以确保适当 已经选择了底物进行破坏。虽然依赖能量 蛋白酶是基于古细胞基因组序列预测的，这是唯一的成分 纯化的是20s蛋白酶体。这些20s蛋白酶体仅起作用 在体外展开的蛋白质上，因此没有促进 鉴定古细胞中天然底物。主要目标 这项研究是检查蛋白酶体介导的古老降解 使用遗传学方法来确定体内蛋白酶体 功能。为了实现这些目标，20年代的蛋白酶体mRNA转录本和 将在多种培养条件下分析蛋白质水平 将提供法规，发起人和操纵子的信息 蛋白酶体。活性蛋白酶体蛋白的水平也将被修改 通过染色体诱变以及 来自质粒的表位标记的蛋白酶体基因。这些方法将使我们 将各种蛋白酶体蛋白的体内水平与细胞联系起来 表型（S），因此提供了对这些蛋白质的作用的理解 在古细菌生理学中。此外，表达式标签的表达 H. dovanii中的蛋白酶体亚基将使我们确定布置 20S蛋白酶体中的alpha1，alpha2和beta亚基的。几个遗传 还提出了将有助于识别外国的方法 和天然蛋白质被细胞中的蛋白酶体降解。这 因此，研究将揭开受蛋白酶体调节的途径 并为理解蛋白水解的生物学作为一种基础 在这一不寻常的组中，转录后调节的机制 有机体。