Vasopressin Cytotoxicity in Inherited Diabetes Insipidus

遗传性尿崩症中加压素的细胞毒性

• 批准号: 6769432
• 负责人: DAVID R COOL
• 金额: $ 24.31万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769432
• 关键词: Golgi apparatus; apoptosis; chimeric proteins; confocal scanning microscopy; cytotoxicity; diabetes insipidus; endoplasmic reticulum; fluorescence microscopy; gene mutation; genetic disorder; green fluorescent proteins; immunoelectron microscopy; immunoprecipitation; molecular chaperones; molecular pathology; necrosis; neurophysins; peptide hormone biosynthesis; proteasome; protein localization; protein transport; secretory protein; terminal nick end labeling; tissue /cell culture; vasopressins

The overall goal of this study is to investigate the molecular basis for the neuroendocrine disease familial neurohypophyseal diabetes insipidus (FNDI). FNDI is an inherited disorder caused by specific mutations in the gene that encodes pre-provasopressin. In this disease, mutant pro-vasopressin is misfolded and accumulates in the ER, is missorted to the constitutive secretory pathway and is not processed correctly. The hypothesis is that accumulation of pro-vasopressin mutations in the ER or Golgi due to improper folding or sorting causes cytotoxic effects that destroy the vasopressin producing cells. In order to test this hypothesis, a chimeric protein, pro- vasopressin-green fluorescent protein (VP-GFP) has been created that allows the mutants to be followed by fluorescent microscopy and immunoprecipitation. In Specific Aim 1 the intracellular fate of FNDI mutations to provasopressin will be studied. Ten FNDI-mutants will be expressed in Neuro-2a and AtT20 cells and their retention, processing and secretion analyzed by confocal and immuno-electron microscopy and pulse-chase experiments. Specific Aim 2 involves investigation of other proteins affected by the mutations, i.e., ER chaperones, proteasome enzymes, secretory granule proteins. In Specific Aim 3, the hypothesis that accumulation of the FNDI-mutations causes cytotoxicity will be tested. Two of the FNDI-mutations will be stably expressed and the cells evaluated to determine if the mutants are cytotoxic and the mechanism by which they produce the cytotoxicity, either necrosis or apoptosis or both. The fate of the mutant VP-GFP in all three specific aims will be followed by confocal microscopy, immunoelectron microscopy and pulse-chase secretion immunoprecipitation experiments either directly or with antibodies to GFP, AVP or neurophysin. Necrosis and apoptosis will be determined using specific assays, e.g., TUNEL, Comet, LDH and PS. The results from these experiments will contribute to understanding the molecular and cellular mechanisms that cause degeneration of vasopressin secreting cells in FNDI and possibly other neurodegenerative and ER storage diseases.

这项研究的总体目的是研究神经内分泌疾病家族性神经植物学糖尿病（FNDI）的分子基础。 FNDI是由编码预胃蛋白酶的基因特异性突变引起的遗传疾病。 在这种疾病中，突变的促抗抑制蛋白被错误折叠并积聚在ER中，被错过了本构分泌途径，并且未正确处理。假设是，由于不当折叠或分类导致细胞毒性作用破坏了加压素产生的细胞，因此在ER或高尔基体中促抗抑制蛋白突变的积累。为了检验这一假设，已经创建了一种嵌合蛋白，加压素绿色荧光蛋白（VP-GFP），该蛋白（VP-GFP）允许突变体进行荧光显微镜和免疫沉淀。 在特定的目标1中，将研究FNDI突变对前封闭素的细胞内命运。 十种FNDI突变剂将在Neuro-2a和Att20细胞中表达，并通过共焦和免疫电子显微镜和脉冲练习实验分析其保留，加工和分泌。具体目标2涉及研究受突变影响的其他蛋白质，即ER伴侣，蛋白酶体酶，分泌颗粒蛋白。 在特定的目标3中，将测试FNDI突变导致细胞毒性的假设。 将稳定表达FNDI突变，并评估细胞以确定突变体是否是细胞毒性的，以及产生细胞毒性的机制，即坏死或凋亡。 在所有三个特定目标中，突变体VP-GFP的命运将直接直接或直接使用抗GFP，AVP或神经磷酸剂的抗体的共聚焦显微镜，免疫电子显微镜和脉冲练习的免疫沉淀实验。 坏死和凋亡将使用特定测定法（例如Tunel，Comet，LDH和PS）确定。 这些实验的结果将有助于理解引起FNDI以及可能其他神经退行性和ER储存疾病的加压素分泌细胞变性的分子和细胞机制。