CELL BIOLOGY OF BIOACTIVE PEPTIDE SECRETION

多肽与许多生理过程有关。以及 促肾上腺皮质激素、旁分泌和自分泌等多肽的内分泌作用 已经被提出了。多肽作为神经调节剂和神经递质 在许多突触上。复杂的后翻译中的某些步骤 从失活的前激素原到生物活性的加工途径 多肽是以组织特有的方式进行的。建议数 研究的重点是了解多肽前体是如何在 组织特异性方式和多肽翻译后处理的方式 酶在发现酶的不同细胞类型中发挥作用。1] 控制潜在的内蛋白水解酶切割位点是否为 利用将通过比较前神经肽的加工来确定 含有突变体的Y(ProNPY)和前ACTH/β-内啡肽(PAE)分子 一元、二元、三元和四元加工区。以下为稳定或 瞬时将cDNA导入AtT-20小鼠皮质激素细胞， 产品多肽将通过生物合成标记和多肽进行鉴定 分析。N-和O-连接糖基化位点改变的突变关闭 将研究切割部位和更远的切割部位的影响 关于路由和处理。2]细胞类型特异性 翻译后加工将通过表达野生型和 突变的proNPY和PAE在AtT-20细胞、GH3大鼠促体腺激素细胞、 原代大鼠垂体促黑素细胞和原发房 肌细胞。将比较内源性和外源性多肽的定位。 通过免疫荧光和免疫电子显微镜观察。3]确定 控制多肽-甘氨酸α-酰胺化单加氧酶的途径 (PAM；EC 1.14.17.3)到正确的亚细胞细胞器 通过在成纤维细胞中表达6种自然存在的PAM形式和 MDCK细胞。在含有大量内源性物质的细胞中进行路由 将PAM的催化结构域用一种 NPY或大鼠血清白蛋白等报告。文献中的数据会 支持PAM跨膜形式的细胞质结构域的作用 在布线中。4]如果转染数据表明细胞质的作用 在PAM结构域的路由中，将使用细胞质结构域亲和性柱 提纯细胞质结合(组装)蛋白；任何可能的组装 蛋白质将被提纯。5]将引入针对PAM的反义cDNA 分为几种细胞类型，以确定缺乏PAM的后果。 候选内切酶将通过以下方式在哺乳动物细胞中表达 转染法和将一种新的蛋白水解酶导入 反义c DNA阻断蛋白水解酶表达或分泌途径的研究 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.