PROTEOSOME OF BACILLUS SUBTILIS IN SPORE DEVELOPMENT

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，先前存在的蛋白质会发生快速和广泛的降解。这项拟议工作的长期目标是了解土壤/海洋细菌枯草芽孢杆菌中这一过程的分子细节，包括枯草杆菌细胞含有1-2个丙二醛的蛋白体样颗粒，遗传证据表明枯草杆菌的一个基因(ClpQ)编码与其他已知蛋白酶体的β亚基同源的蛋白质。在高等细胞中，蛋白小体负责大多数细胞蛋白的ATP依赖的降解，参与炎症反应、细胞周期控制、抗原提呈和细胞凋亡，因此成为一个重要的药理靶点。本文建议将枯草杆菌颗粒纯化成均一种，并从形态、洗涤剂凝胶电泳法的亚基组成、底物专一性、对ATP的要求和敏感性、对ATP的要求以及对α-lactacystin和多肽乙烯基砜等抑制剂的敏感性等方面对其进行表征。特定的抑制剂将被用来评估蛋白小体在孢子形成过程中对大宗蛋白的水解可能起到的作用。蛋白小体的clpQ编码的β亚基的已知缺失也将被用来评估这种损伤对蛋白小体的结构和反应能力以及孢子形成过程中细胞内蛋白分解速度的影响。在这方面，对芽孢杆菌中蛋白质分解速率的研究表明，由于氨基酸不能在膜上自由交换，细胞内蛋白质的降解可能完全是隐蔽的。类似的问题可能会出现在许多生物系统中，但没有方法来测试这一点。这一提议的一个重要目标是开发一种新的、简单的方法，其基础是跨所有生物膜的水的自由交换。建议利用当存在18O时，通过在有[18O]水存在的情况下在多肽中进行水解而引入的多肽羰基的酰胺I带的红外频率的移动，而不是16O。如果该方法开发成功，将被用来分析蛋白质小体在散装蛋白质的蛋白质分解中所起的作用。

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.