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 作为更大的前体合成的。我们的长期目标是阐明 多肽激素前体在细胞内调节中的作用 成熟蛋白的运输、翻译后加工和分泌 荷尔蒙。对于这些研究，PreproSRIF是一个有用的模型，因为它是 最简单的多肽激素前体。I.体外重建 激素原加工和分类。人们对这种分子知之甚少 内分泌细胞区分不同蛋白的机制 对于结构性和受调控的分泌途径。去调查 在Trans Golgi Network(TGN)中的激素前体处理和分类，我们将 使用来自表达proSRIF的GH3细胞的无细胞系统。我们 建议：(I)详细描述这一体外系统；(Ii) 研究ProSRIF在TGN中的切割及其伴随的包装 成熟的SRIF和内源性GH转化为新生的分泌颗粒； 从激素前体加工缺陷的细胞中制备体外系统 通过混合实验确定参与的细胞特定成分 激素原加工和分类。II.单基生物的识别- 残留物特异性激素原转换酶。在许多激素原中 生物活性多肽的两侧是一对碱性氨基酸，但在 几种前体激素序列在单一碱基上被裂解 残留物。我们在酵母细胞中发现了一种新的蛋白质分解活性 将ProSRIF-II异源切割在序列 编码该酶的基因；(Ii)利用酵母基因鉴定其 通过筛选胰岛c DNA文库获得哺乳动物同源基因； ProSRIF-II和ProSRIF-II在GH3细胞中的共表达 在细胞内被降解，以确定前激素是否随后被切割 至SRIF-28。我们的目标不仅仅是确定一种新的激素原加工方法 酶(S)还要了解细胞 区分作为蛋白质分解底物的前激素 从那些针对细胞内降解的物质进行处理。三. 肽类激素拓扑域的结构与功能研究 先驱物。SRIF前肽在细胞内调节中的作用 运输和正确的蛋白质分解处理。识别结构 影响前驱体分选和处理的特征，我们有过度- 在大肠杆菌中表达了多个proSRIF。我们将：(I)描述保守的 不同物种ProSRIF的结构域鉴定及其抗蛋白酶活性 和-前体的敏感区域；(Ii)纯化前SRIF以 均一性，制备纯化多肽的晶体并测定 它们的X射线晶体结构。这些研究将使我们能够 确定在细胞内起中介作用的结构“结构域” 运输。现在有证据表明胰岛素原加工过程中的缺陷 导致某些形式的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 糖尿病病因学的重要意义。

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.