PROCESSING OF PROINSULIN/INSULIN BY B-CELL ORGANELLES

B 细胞细胞器对胰岛素原/胰岛素的加工

• 批准号: 3233574
• 负责人: PHILIPPE A HALBAN
• 金额: $ 10.21万
• 依托单位: UNIVERSITY OF GENEVA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233574
• 关键词: Golgi apparatus; clathrin; diabetes mellitus; endoplasmic reticulum; granule; high performance liquid chromatography; hormone biosynthesis; hormone metabolism; insulin; intracellular transport; laboratory rat; lysosomes; molecular pathology; neoplastic cell culture for noncancer research; pancreatic islet function; phagocytosis; point mutation; proinsulin; protein signal sequence; protein transport; proteolysis; radioimmunoassay; radiotracer; secretion; transfection

Insulin production by the B-cell consists of a series of post- translational events: introduction of preproinsulin into the lumen of the RER; prepro- to proinsulin conversion; transport of proinsulin to the Golgi complex and packaging into immature, clathrin coated granules; proinsulin to insulin conversion. granule acidification and clathrin uncoating; granule release, storage or degradation. The ultimate aim of this Project is to characterize each step in molecular detail. Only then will it be possible to define the lesions responsible for defective insulin production in diabetes. The experiments depend upon native islets (rat or calf) or rat insulinoma cells. The methods combine analytical biochemistry, cell biology, morphology and recombinant DNA techniques. The specific aims and experimental approaches are: 1. Introduction of preproinsulin into the RER: The signal (leader) sequence carries structural information responsible for penetration through the RER membrane. This region of the insulin gene will be mutated (site directed mutagenesis of rat insulin II gene) and the mutated gene transfected into AtT20 cells (pituitary cell line). Production of insulin and its precursors will be compared with AtT20 cells transfected with the native gene. Relative rates of synthesis, conversion and intracellular transport/targeting/packaging will be examined. 2. Targeting/packaging of proinsulin into granules, and subsequent conversion to insulin: A similar approach will be used to study the tertiary and quarternary structural information on the proinsulin molecule responsible for recognition in the Golgi complex and by the converting enzyme(s). 3. Granule maturation (clathrin uncoating), intragranular acidification and proinsulin conversion: Isolated, intact granules will be used. The mechanism responsible for removal of clathrin from immature granules will be studied. The experiments will also address the issue of whether uncoating, acidification and conversion are merely contemporaneous or actually interdependent events. 4. The preperential release of newly synthesized insulin: The mechanism underlying preferential release of new insulin will be studied using native rat islets. The hypothesis that intimate association of newly formed granules with the cytoskeleton is responsible will be evaluated by examining the effect of agents known to disturb microtubule polymerization. 5. Intracellular degradation of granule contents: After fusion of granules with lysosomes (crinophagy) insulin is predicted to be degraded less rapidly than C-peptide/proinsulin (since it is stabilized in the crystal form). The rates of insulin and C-peptide degradation will be measured in rat islet B-cells. How granules and lysosomes come in contact and fuse is unknown. The hypothesis that the cytoskeleton serves as the framework for crinophagy will be tested experimentally.

B细胞产生胰岛素的过程包括一系列的后 翻译事件：将前胰岛素原引入管腔 胰岛素前体转化为胰岛素原; 胰岛素原到高尔基复合体并包装成未成熟的， 网格蛋白包被颗粒;胰岛素原向胰岛素的转化。 颗粒 酸化和网格蛋白脱壳;颗粒释放、储存或 降解 该项目的最终目的是描述 每一步的分子细节。 只有这样， 定义导致胰岛素产生缺陷的病变， 糖尿病 实验依赖于天然胰岛（大鼠或小牛） 或大鼠胰岛素瘤细胞。 这些方法联合收割机结合了分析 生物化学、细胞生物学、形态学和重组DNA 技术. 具体目标和实验方法是： 1. 将前胰岛素原引入RER：信号（先导） 序列携带结构信息， 穿过RER膜。 的这个区域 胰岛素基因将被突变（大鼠的定点诱变 胰岛素II基因）和转染到AtT 20细胞中的突变基因 （垂体细胞系）。 胰岛素及其前体的生产将 与用天然基因转染的AtT 20细胞进行比较。 合成、转化和细胞内 将审查运输/目标/包装问题。 2. 将胰岛素原靶向/包装成颗粒，和 随后转换为胰岛素：将使用类似的方法 研究其第三、第四纪构造信息， 胰岛素原分子负责识别高尔基体 复合物和转化酶。 3. 颗粒成熟（网格蛋白未包衣），颗粒内 酸化和胰岛素原转化：分离的完整颗粒 将用于 清除网格蛋白的机制 未成熟的颗粒将被研究。 实验将 还解决了是否脱涂层，酸化和 转换仅仅是同时发生的，或者实际上 相互依存的事件。 4. 新合成胰岛素的预分泌： 新胰岛素优先释放的潜在机制将是 研究使用的是原生鼠胰岛。 亲密关系的假设 新形成的颗粒与细胞骨架的结合是 将通过检查代理人的效果来评估责任人 已知干扰微管聚合。 5. 颗粒内容物的细胞内降解： 颗粒与溶酶体（分泌吞噬）胰岛素预计是 降解速度低于C肽/胰岛素原（因为它是 以晶体形式稳定）。 胰岛素和C肽的比率 将在大鼠胰岛B细胞中测量降解。 如何颗粒 和溶酶体接触融合的情况尚不清楚。 的 假设细胞骨架作为框架， 将通过实验来测试食毛癖。