PROTEOSOME OF BACILLUS SUBTILIS IN SPORE DEVELOPMENT

枯草芽孢杆菌在孢子发育中的蛋白质体

• 批准号: 6469243
• 负责人: JAMES H HAGEMAN
• 金额: $ 6.87万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469243
• 关键词: Bacillus subtilis; bacterial genetics; bioenergetics; electron microscopy; endopeptidases; enzyme substrate; high performance liquid chromatography; membrane permeability; microorganism metabolism; protease inhibitor; proteasome; protein degradation; protein purification; proteolysis; sporogenesis; water channel

During the development of a bacterial spore, and during development in most biological systems, rapid and extensive degradation of pre-existing proteins occurs. The long term objective of the proposed work is to understand the molecular details of this process in the soil/marine bacterium Bacillus subtilis, including a subtilis cells contain a proteosome like particle of 1-2 MDa and genetic evidences indicates that a gene of B. subtilis (clpQ) codes for a protein homologous to the beta subunits of other known proteosomes. In higher cells the proteosome is responsible for the ATP-dependent degradation of most cellular proteins, is involved in inflammation responses, cell cycle control, antigen presentation and apoptosis, and it has thus become an important pharmacological target. It is proposed here to purify the B. subtilis particle to homogeneity and characterize it with respect to appearance through electromicroscopy, its subunit composition by detergent gel electrophoresis, its substrate specificity, its requirement for ATP and its sensitivity, its requirement for ATP and its sensitivity towards inhibitors such at alpha-lactacystin and peptide vinylsulfones. Specific inhibitors will be used to assess the possible role of the proteosome in the hydrolysis of bulk protein during sporulation. Known deletions in the clpQ-encoded beta subunit of the proteosome will also be used to assess what effect this lesion has on the structure and reactivity of the proteosome, and on the rate of intracellular proteolysis during spore formation. In the connection studies of rates of proteolysis in Bacillus species have indicated that degradation of intracellular protein can be completely cryptic due to a failure of amino acids to exchange freely across the membranes. A similar problem is likely to occur in many biological systems, but no way exists to test for this. An important aim if this proposal is to develop a new, simple method based on the free exchange of water across all biological membranes. It is proposed to use the shift in the infrared frequency of the Amide I band of the peptide carbonyl when 18O is present, introduced by hydrolysis in the peptide in the presence of [18O] water, instead of 16O. If the method is successfully developed, it will be used to analyze the role played by proteosome in proteolysis of bulk protein.

在细菌孢子的发育过程中，以及在大多数生物系统的发育过程中，预先存在的蛋白质发生快速和广泛的降解。本研究的长期目标是了解土壤/海洋细菌枯草芽孢杆菌（Bacillus subtilis）中这一过程的分子细节，包括枯草芽孢杆菌细胞中含有1-2 MDa的蛋白体样颗粒，遗传证据表明B的基因。枯草芽孢杆菌（clpQ）编码与其它已知蛋白体的β亚基同源的蛋白质。在高等细胞中，蛋白体负责大多数细胞蛋白的ATP依赖性降解，参与炎症反应、细胞周期控制、抗原呈递和凋亡，因此它已成为重要的药理学靶点。这里建议纯化B。将枯草杆菌颗粒均匀化，并通过电子显微镜表征其外观，通过去污剂凝胶电泳表征其亚基组成，其底物特异性，其对ATP的需求及其敏感性，其对ATP的需求及其对抑制剂如α-乳胞素和肽乙烯基砜的敏感性。将使用特异性抑制剂来评估蛋白体在孢子形成过程中大量蛋白质水解中的可能作用。蛋白体的clpQ编码的β亚基中的已知缺失也将用于评估这种损伤对蛋白体的结构和反应性以及对孢子形成期间细胞内蛋白水解速率的影响。关于芽孢杆菌属物种中蛋白水解速率的研究表明，由于氨基酸不能自由地跨膜交换，细胞内蛋白质的降解可能是完全隐蔽的。类似的问题很可能发生在许多生物系统中，但没有办法测试这一点。该提案的一个重要目标是开发一种新的、简单的方法，该方法基于水在所有生物膜上的自由交换。当存在18 O（通过在[18 O]水存在下水解肽而引入）而不是16 O时，建议使用肽羰基酰胺I带的红外频率偏移。如果该方法的成功开发，它将被用来分析蛋白酶体在蛋白质水解中所起的作用。