BIOSYNTHESIS AND PROCESSING OF PEPTIDE HORMONE PRECURSOR

胰岛激素生长抑素（SRIF）、胰岛素和胰高血糖素是 合成为较大的前体。 我们的长期目标是阐明 多肽激素前体在介导细胞内 成熟蛋白的转运、翻译后加工和分泌 激素. PreproSRIF是这些研究的有用模型，因为它是 最简单的肽激素前体。 I. 体外复溶 激素原加工和分选。 知之甚少的分子 内分泌细胞区分蛋白质的机制 组成和调节分泌途径。 探讨 激素原的加工和排序在跨高尔基体网络（TGN），我们将 使用来源于表达proSRIF的GH 3细胞的无细胞系统。 我们 建议：（i）详细描述该体外系统;（ii） 研究TGN中的proSRIF裂解和伴随包装 成熟SRIF和内源性GH转化为新生分泌颗粒;（iii） 从缺乏激素原加工的细胞制备体外系统， 通过混合实验确定细胞特异性成分参与 激素原处理和分选。 二. 识别一个单一的对象- 残留特异性激素前体转化酶。 在许多激素原中， 生物活性肽的侧翼是成对的碱性氨基酸，然而， 几个前体的激素序列被切割在单一的碱基 残基 我们在酵母细胞中鉴定了一种新的蛋白水解活性， 正确切割异源表达的proSRIF-II， （ii）利用酵母基因来鉴定其 通过筛选胰岛cDNA文库的哺乳动物等同物;（iii） 在GH 3细胞中共表达蛋白酶cDNA和proSRIF-II，其中proSRIF-II 在细胞内降解，以确定激素原是否随后被切割 SRIF-28。 我们的目标不仅是确定一种新的激素原加工 酶，但也要了解细胞的分子基础， 区分作为蛋白水解底物的激素原 从那些靶向细胞内降解的物质中分离出来。 三. 肽激素拓扑结构域的结构与功能研究 前兆。 SRIF前肽在介导细胞内 运输和正确的蛋白水解加工。 识别结构 影响前体分类和处理的功能，我们有超过- 在大肠杆菌中表达了几种proSRIF。 我们将：（一）描述保守的 通过鉴定蛋白酶抗性， （ii）纯化proSRIF， 制备纯化多肽的晶体并测定其均一性。 它们的X射线晶体结构。 这些研究将使我们能够 鉴定在介导细胞内 运输 现在有证据表明胰岛素原加工过程中的缺陷 导致某些形式家族性高胰岛素血症。 因此 了解胰岛激素的生物合成和分类 关于糖尿病病因的重要意义。

The pancreatic islet hormones somatostatin (SRIF), insulin and glucagon are synthesized as larger precursors. Our long term goal is to elucidate the function of polypeptide hormone precursors in mediating intracellular transport, post-translational processing and secretion of the mature hormone. PreproSRIF is a useful model for these studies since it is one of the simplest peptide hormone precursors. I. In vitro Reconstitution of Prohormone Processing and Sorting. Little is known about the molecular mechanisms whereby endocrine cells discriminate between proteins destined for the constitutive and regulated secretory pathways. To investigate prohormone processing and sorting in the Trans Golgi Network (TGN), we will use a cell-free system derived from GH3 cells expressing proSRIF. We propose to: (i) Characterize this in vitro system in detail; (ii) Investigate proSRIF cleavage in the TGN and the concomitant packaging of mature SRIF and endogenous GH into nascent secretory granules; (iii) Prepare in vitro systems from cells deficient in prohormone processing and by mixing experiments identify cell-specific components involved in prohormone processing and sorting. II. Identification of a Monobasic- residue Specific Prohormone Converting Enzyme. In many prohormones the bioactive peptide is flanked by pairs of basic amino acids, however in several precursors the hormone sequence is cleaved at single basic residues. We identified a novel proteolytic activity in yeast cells which cleaves heterologously expressed proSRIF-II correctly at a sequence the gene encoding this enzyme; (ii) Exploit the yeast gene to identify its mammalian equivalent by screening a pancreatic islet cDNA library; (iii) Co-express the protease cDNA and proSRIF-II in GH3 cells, where proSRIF-II is degraded intracellularly, to determine if the prohormone is then cleaved to SRIF-28. Our goal is not only to identify a novel prohormone processing enzyme(s) but also to understand the molecular basis whereby cells discriminate between prohormones which are substrates for proteolytic processing from those targeted for intracellular degradation. III. Structure-Function Studies on Topogenic Domains in Peptide Hormone Precursors. The SRIF propeptide functions in mediating intracellular transport and correct proteolytic processing. To identify structural features that effect precursor sorting and processing, we have over- expressed several proSRIFs in E.coli. We will: (i) Characterize conserved domains in different species of proSRIF by identifying protease-resistant and -sensitive regions of the precursors; (ii) Purify the proSRIFs to homogeneity, prepare crystals of the purified polypeptides and determine their X-ray crystallographic structure. These studies will enable us to identify structural "domains" which function in mediating intracellular transport. There is now evidence that defects in proinsulin processing lead to certain forms of familial hyperinsulinemia. Therefore understanding the biosynthesis and sorting of the islet hormones has important implications concerning the etiology of diabetes.