FORMATION AND MAINTENANCE OF LYSOSOME STRUCTURE-FUNCTION

溶酶体结构功能的形成和维持

• 批准号: 3289084
• 负责人: Gregory E. Conner
• 金额: $ 9.96万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289084
• 关键词: autoradiography; biochemistry; cell transformation; chemical structure; complementary DNA; electron microscopy; endonuclease; flow cytometry; gel electrophoresis; gene complementation; genetic manipulation; immunoprecipitation; lysosomes; molecular cloning; pepsin; peptide chemical synthesis; proteolysis; radionuclide double label

The long term objective of my research is to describe the precise molecular structure and function of lysosomes. From biosynthesis to turnover, how do lysosomes assemble, organize and activate their constituent enzymes, while protecting the cell from autodigestion? Within this general framework, the proposed experiments will specifically ask: 1) what protein sequence determinants of cathepsin D, a major lysosomal protease, and pepsin, a secreted protease, are responsible for directing these two highly homologous digestive enzymes to different cellular compartments; ii) what are the general cellular effects of a loss of cathepsin D activity in lysosomes; iii) what is the physical disposition of cathepsin D in the various cellular compartments including lysosomes, i.e. is it membrane bound or soluble; and iv) what parameters define cathepsin D turnover or degradation? To answer these questions it will be necessary to isolate functionally expressible recombinant DNA molecules coding for cathepsin D and pepsin and isolate cultured cells conditionally defective in cathepsin D activity. The recombinant clones will be used to construct deletion and fusion proteins which will, in turn, be assayed for complementation of the cathepsin D deficient cells. This approach will allow rapid screening of randomly constructed (i.e. shotgun) recombinant molecules and thus, a complete search of cathepsin D and pepsin primary sequence for putative sorting signals. To evaluate the effect of a loss of cathepsin D activity, the conditionally defective cells will be characterized in terms of their biochemical defect, changes in protein synthesis and turnover, morphology, and growth characteristics. Pulse-chase cell fractionation procedures will be used to study the turnover of cathepsin D as it relates to lysosome structure and function in normal cultured cells. The studies of lysosome physiology, described here, are basic in nature but will be valuable to our understanding of lysosome related pathologies at the cellular and molecular level.

我研究的长期目标是描述精确的分子 溶酶体的结构和功能。 从生物合成到周转，如何进行 溶酶体组装、组织和激活其组成酶，而 保护细胞免遭自身消化？ 在这个总体框架内， 建议的实验将具体询问：1）什么蛋白质序列 组织蛋白酶 D（一种主要的溶酶体蛋白酶）和胃蛋白酶（一种 分泌的蛋白酶，负责指导这两个高度 不同细胞区室的同源消化酶； ii) 什么 是组织蛋白酶 D 活性丧失的一般细胞效应 溶酶体； iii) 组织蛋白酶 D 在体内的物理性质是什么？ 各种细胞区室，包括溶酶体，即它是膜吗 结合或可溶； iv) 哪些参数定义了组织蛋白酶 D 的周转率或 降解？ 为了回答这些问题，需要进行功能隔离 编码组织蛋白酶 D 和胃蛋白酶的可表达重组 DNA 分子， 分离组织蛋白酶 D 活性有条件缺陷的培养细胞。 重组克隆将用于构建缺失和融合 反过来，这些蛋白质将被检测以进行互补 组织蛋白酶 D 缺陷细胞。 这种方法将允许快速筛选 随机构建（即鸟枪法）重组分子，因此， 完整搜索组织蛋白酶 D 和胃蛋白酶一级序列以寻找假定的 排序信号。 为了评估组织蛋白酶 D 活性丧失的影响， 有条件缺陷的细胞将根据其特征进行表征 生化缺陷、蛋白质合成和周转、形态的变化， 和生长特征。 脉冲追踪细胞分级分离程序将 用于研究组织蛋白酶 D 的周转，因为它与溶酶体相关 正常培养细胞的结构和功能。 这里描述的溶酶体生理学研究本质上是基础性的，但是 对于我们理解溶酶体相关的病理学非常有价值 细胞和分子水平。