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

基本信息

批准号：
3233566
负责人：
PHILIPPE A HALBAN
金额：
$ 9.76万
依托单位：
UNIVERSITY OF GENEVA
依托单位国家：
美国
项目类别：
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

项目摘要

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细胞产生的胰岛素由一系列后翻译事件：将前硫蛋白引入管腔 ;前硫蛋白转化率；运输促进高尔基络合物并包装成未成熟的蛋白酶，网格蛋白涂层颗粒；促胰岛素转化为胰岛素。颗粒酸化和网状蛋白不涂层；颗粒释放，存储或降解。该项目的最终目的是表征每个步骤都有分子细节。只有这样才有可能定义负责有缺陷胰岛素产生的病变糖尿病。实验取决于天然胰岛（大鼠或小腿）或大鼠胰岛素瘤细胞。这些方法结合了分析生物化学，细胞生物学，形态和重组DNA 技术。具体目的和实验方法是： 1。将前硫蛋白引入RER：信号（领导者）序列带有负责的结构信息穿透膜穿透。这个地区胰岛素基因将被突变（位点定向大鼠的诱变胰岛素II基因）和转染的突变基因（垂体细胞系）。胰岛素及其前体的产生将将与天然基因转染的ATT20细胞进行比较。合成，转化和细胞内的相对速率将检查运输/定位/包装。 2。靶向/包装促颗粒蛋白到颗粒中，并将其靶向随后转化为胰岛素：将使用类似的方法研究有关第三纪和区域结构信息负责高尔基体识别的促硫蛋白分子复合物和通过转化酶（S）。 3。颗粒成熟（网格蛋白不涂层），杂志酸化和促硫蛋白转化：分离的完整颗粒将使用。负责去除网格蛋白的机制将研究未成熟的颗粒。实验会还解决了脱衣，酸化和是否是否 conversion依只是同时或实际上相互依存的事件。 4。新合成胰岛素的预释放：新胰岛素的优先释放的机制将是使用天然大鼠胰岛进行研究。亲密的假设新形成的颗粒与细胞骨架的关联是负责通过检查代理的效果来评估负责已知会干扰微管聚合。 5。颗粒含量的细胞内降解：融合后预计淋巴细胞（crinophagy）胰岛素的颗粒被预测为降解的速度速度较差，而不是C肽/促硫磺蛋白（因为它是以晶体形式稳定）。胰岛素和C肽的速率降解将在大鼠胰岛B细胞中测量。颗粒溶酶体接触，保险丝尚不清楚。这假设细胞骨架是作为框架 Crinophagy将通过实验测试。