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，预先存在的蛋白质会发生迅速而广泛的降解。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）这一过程的分子细节，包括枯草芽孢杆菌细胞含有1-2 MDa的蛋白体样颗粒，遗传证据表明枯草芽孢杆菌（B. subtilis）的一个基因（clpQ）编码与其他已知蛋白体β亚基同源的蛋白质。在高等细胞中，该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，因此已成为重要的药理学靶点。本文拟将枯草芽孢杆菌颗粒纯化至均匀性，并通过电镜、洗涤凝胶电泳、底物特异性、对ATP的需求及其敏感性、对ATP的需求及其对α -乳糖蛋白酶和肽乙烯基砜等抑制剂的敏感性等方面对其进行表征。特异性抑制剂将用于评估在孢子形成过程中蛋白质体水解的可能作用。已知的clpq编码的蛋白体β亚基缺失也将用于评估这种损伤对蛋白体的结构和反应性的影响，以及对孢子形成过程中细胞内蛋白水解率的影响。有关芽孢杆菌中蛋白质水解速率的研究表明，由于氨基酸不能在细胞膜上自由交换，细胞内蛋白质的降解可能是完全隐式的。类似的问题很可能发生在许多生物系统中，但没有办法对此进行测试。该提案的一个重要目标是开发一种基于水在所有生物膜上自由交换的简单新方法。当肽基在[18O]水的存在下水解引入18O而不是16O时，我们建议利用肽基酰胺I波段红外频率的偏移。如果该方法开发成功，将用于分析蛋白体在体蛋白水解过程中所起的作用。

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.