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