PROCESSING OF PROINSULIN/INSULIN BY B-CELL ORGANELLES

由B细胞产生的胰岛素包括一系列的后 翻译事件：将前胰岛素原引入管腔 前胰岛素原转化为胰岛素原；转运 胰岛素原到高尔基复合体并包装成未成熟的， 笼状蛋白包衣颗粒；胰岛素原转化为胰岛素。颗粒剂 酸化和网状蛋白去涂层；颗粒释放、储存或 退化。该项目的最终目标是 分子细节中的每一步。只有这样，才有可能 确定导致胰岛素分泌缺陷的病变 糖尿病。实验依赖于本地的胰岛(大鼠或小牛)。 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、形态与重组DNA 技巧。具体目标和实验方法如下： 1.将前胰岛素原引入内质网：信号(引导者) 序列携带结构信息，负责 穿透粗面内质网膜。这一地区的 胰岛素基因将发生突变(大鼠的定点突变 胰岛素II基因)和突变基因导入AtT20细胞 (垂体细胞系)。胰岛素及其前体的生产将 与转导天然基因的AtT20细胞进行比较。 合成、转化率和细胞内的相对速率 将对运输/目标/包装进行检查。 2.将胰岛素原靶向/包装成颗粒，以及 随后转换为胰岛素：将使用类似的方法 研究三系和四系的结构信息 高尔基体中负责识别的胰岛素原分子 复合体和转化酶(S)。 3.颗粒成熟(去膜蛋白)，颗粒内 酸化和胰岛素原转化：分离的、完整的颗粒 将会被使用。脱除网壳蛋白的机理 从未成熟的颗粒中提取将被研究。这些实验将会 还解决了脱膜、酸化和 皈依仅仅是同时代的或者实际上 相互依存的事件。 4.新合成胰岛素的预释放： 新胰岛素优先释放的机制将是 使用本地大鼠胰岛进行研究。关于亲密关系的假设 新形成的颗粒与细胞骨架的联系 责任人将通过检查代理人的效果进行评估 已知会干扰微管聚合。 5.颗粒内容物的细胞内降解：融合后 含有溶酶体(吞噬细胞)的胰岛素颗粒被预测为 降解速度不如C-肽/胰岛素原快(因为它 以晶体形式稳定)。胰岛素和C-肽的比率 将在大鼠胰岛B细胞中测量降解情况。How颗粒 和溶酶体接触，融合未知。这个 假设细胞骨架作为 将在实验中测试吞噬小鸡的能力。

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.