CELL BIOLOGY OF BIOACTIVE PEPTIDE SECRETION

肽参与许多生理过程。 沿着 促肾上腺皮质激素、旁分泌和自分泌等肽类的内分泌作用 已提出 肽作为神经调质和神经递质 在许多突触上。 复杂的翻译后过程中的某些步骤 从非活性前激素原到生物活性前激素原的加工途径 肽以组织特异性方式进行。 拟议 研究的重点是了解肽前体是如何在一个 组织特异性方式和肽如何翻译后加工 酶在它们所存在的不同细胞类型中发挥作用。 决定潜在的内切蛋白水解切割位点是否 将通过比较前神经肽的加工来确定所利用的 含有Y（proNPY）和pro-ACTH/β-内啡肽（PAE）分子的突变体 一元、二元、三元和四元加工场所。 在稳定或 将cDNA瞬时转染入AtT-20小鼠促肾上腺皮质激素细胞， 产物肽将通过生物合成标记和肽 分析。 改变N-和O-连接糖基化位点的突变关闭 将研究切割位点的影响 路由和处理。 2]细胞类型特异性 将通过表达野生型和 AtT-20细胞，GH 3大鼠体乳细胞， 原代大鼠中间垂体促黑素细胞和原代心房肌 肌细胞 将比较内源性肽和外源性肽的定位 通过免疫荧光和免疫电子显微镜。 3]确定 控制肽基-甘氨酸α-酰胺化单加氧酶的路线 (PAM; EC 1.14.17.3）与正确的亚细胞器连接 通过在成纤维细胞中表达PAM的6种天然存在形式， MDCK细胞。 在含有大量内源性 PAM将通过将PAM的催化结构域替换为 报告基因如NPY或大鼠血清白蛋白。 文献中的数据 支持跨膜形式PAM的胞质结构域的作用 in routing路由. 4]如果转染数据表明细胞质中 在路由中，将使用胞质结构域亲和柱 纯化细胞质结合（组装）蛋白;任何潜在的组装 蛋白质将被纯化。 5]将引入PAM的反义cDNA 几种细胞类型，以确定缺乏PAM的后果。 候选内切蛋白酶将在哺乳动物细胞中表达， 转染和将新的蛋白酶引入细胞的结果 分泌途径或用反义cDNA阻断蛋白酶表达将 接受检查。

Peptides are implicated in many physiological processes. Along with the endocrine actions of peptides such as ACTH, paracrine and autocrine actions have been proposed. Peptides act as neuromodulators and neurotransmitters at many synapses. Certain steps in the complex post-translational processing pathway leading from inactive preprohormones to bioactive peptides are carried out in a tissue specific fashion. The proposed studies focus on understanding how peptide precursors are processed in a tissue specific manner and how peptide post-translational processing enzymes function in the diverse cell types in which they are found. 1] The properties governing whether potential endoproteolytic cleavage sites are utilized will be determined by comparing the processing of pro-neuropeptide Y (proNPY) and pro-ACTH/beta-endorphin (PAE) molecules containing mutant mono-, di-, tri- and tetrabasic processing sites. Following stable or transient transfection of cDNAs into AtT-20 mouse corticotrope cells, product peptides will be identified by biosynthetic labeling and peptide analyses. Mutations altering sites for N- and O-linked glycosylation close to and further away from the cleavage site will be investigated for effects on routing and processing. 2] The cell-type specificity of posttranslational processing will be compared by expressing wild-type and mutant proNPY and PAE in AtT-20 cells, GH3 rat somatomammotrope cells, primary rat intermediate pituitary melanotropes and primary atrial myocytes. Localization of endogenous and foreign peptides will be compared by immunofluorescence and immunoelectron microscopy. 3] Determining governing the routing of peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) to the correct subcellular organelles will be examined by expressing the 6 naturally occurring forms of PAM in fibroblasts and MDCK cells. Routing in cells containing substantial levels of endogenous PAM will be examined by replacing the catalytic domain of PAM with a reporter such as NPY or rat serum albumin. Data in the literature would support a role for the cytoplasmic domain of the transmembrane forms of PAM in routing. 4] If transfection data suggest a role for the cytoplasmic domain of PAM in routing, a cytoplasmic domain affinity column will be used to purify a cytoplasmic binding (assembly) protein; any potential assembly proteins will be purified. 5] Antisense cDNAs to PAM will be introduced into several cell types to determine the consequences of lack of PAM. Candidate endoproteases will be expressed in mammalian cells by transfection and the consequence of introducing a novel protease into the secretory pathway or blocking protease expression with antisense cDNA will be examined.