Septate junctional proteins: Proteomic identification and role in diuresis

隔膜连接蛋白：蛋白质组学鉴定及其在利尿中的作用

• 批准号: 7178177
• 负责人: Klaus W Beyenbach
• 金额: $ 20.68万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7178177
• 关键词: Active Biological Transport; Aedes; Affect; Anopheles Genus; Antibodies; Attention; Binding; Biological Assay; Blood; Cell membrane; Cells; Cloning; Culicidae; Databases; Depth; Diuresis; Diuretics; Drosophila genus; Enzymes; Epithelial; Epithelial Cells; Epithelium; Event; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gel; Genome; Genomics; Goals; Homologous Gene; Hormones; Ingestion; Insect Proteins; Insecta; Intestines; Invertebrates; JAM protein; Knowledge; Localized; Malpighian Tubules; Mediating; Membrane Proteins; Methods; Molecular; Molecular Biology; Molecular Cloning; Neurotransmitters; Pancreas; Pathway interactions; Peptides; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Plasma; Post-Translational Protein Processing; Protein Dephosphorylation; Protein Phosphatase G; Proteins; Proteomics; RNA; Race; Rate; Regulation; Renal tubule structure; Research Personnel; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Salivary; Salivary Glands; Sampling; Septate; Signal Pathway; Signal Transduction; Speed; Standards of Weights and Measures; Stomach; Students; Tight Junctions; Time; Vertebrates; Water; Work; base; basolateral membrane; design; feeding; intestinal epithelium; junctional adhesion molecule; occludin; programs; receptor binding; research study; response; voltage

DESCRIPTION (provided by applicant): In the past, studies of epithelial transport have focused largely on the active transport pathway through epithelial cells. The transport pathway between cells, the paracellular pathway, is now receiving increased attention. But no epithelium displays the dynamic regulation of paracellular transport as well as Malpighian (renal) tubules of the blood-feeding mosquito Aedes aegypti. Here, the diuretic hormone leucokinin triggers a 10-fold increase in paracellular Cl conductance that helps rid the mosquito of the unwanted plasma fraction of the blood meal. The effects of leucokinin are reversible, and switch like on/off changes suggest the post- translational modification of paracellular proteins. At the level of Malpighian tubules, leucokinin binds to a G protein-coupled receptor that leads to Ca entry into the cell via Ca channels activated by Ca store depletion. How Ca goes on to increase the Cl conductance of the septate junction (functional equivalent of the vertebrate tight junction) is unknown. Furthermore, the proteins that define the conductance and the permselectivity of septate junctions are largely unknown. Accordingly, one goal is to identify the septate junctional proteins in Malpighian tubules of A. aegypti by the methods of proteomics. The proteomic analysis will focus on plasma membrane proteins that may extend into the paracellular space to define paracellular conductance and permselectivity. Physiological/pharmacological studies in intact Malpighian tubules seek to uncover the Ca-activated kinases, phosphatases, and/or G-proteins that mediate the rapid paracellular conductance changes, and proteomic analyses will attempt to identify those septate junctional proteins undergoing posttranslational modifications with the rapid on/off effects of leucokinin. Time permitting, the goal of molecular cloning is to confirm the proteomic identification of septate junctional proteins, and newly produced antibodies seek to examine whether identified septate junctional proteins are indeed located at the septate junction of Malpighian tubules. While identifying new proteins of the invertebrate septate junction, the results may reveal how kinases and other enzymes regulate paracellular transport, thereby integrating transcellular and paracellular transport pathways with additive if not synergistic effect. A similar integration may well take place in vertebrate epithelia where hormones and neurotransmitters are known to trigger quick and potent responses in secretory epithelia such as salivary glands, stomach, pancreas and intestine.

描述(申请人提供)：过去，对上皮细胞转运的研究主要集中在通过上皮细胞的主动转运途径上。细胞之间的运输途径，即细胞旁途径，现在正受到越来越多的关注。但没有上皮细胞表现出埃及伊蚊的细胞旁运输和马氏管的动态调节。在这里，利尿激素亮氨酸激动素触发细胞旁氯电导增加10倍，这有助于清除蚊子血液中不需要的血浆部分。Leucokinin的作用是可逆的，开关类似开/关的变化提示细胞旁蛋白的翻译后修饰。在马氏管水平，亮氨酸激肽与G蛋白偶联受体结合，导致钙通过钙储备库耗尽激活的钙通道进入细胞。钙如何继续增加隔状连接(脊椎动物紧密连接的功能等价物)的氯电导尚不清楚。此外，定义隔膜连接的电导和渗透选择性的蛋白质在很大程度上是未知的。因此，一个目标是用蛋白质组学的方法鉴定埃及伊蚊马氏管中的分离连接蛋白。蛋白质组学分析将重点放在可能延伸到细胞旁空间的质膜蛋白上，以确定细胞旁的电导和渗透选择性。在完整的马氏管中的生理学/药理学研究试图揭示介导细胞旁快速电导变化的钙激活的激酶、磷酸酶和/或G-蛋白，而蛋白质组学分析将试图识别那些经历翻译后修饰的隔膜连接蛋白，以及亮激肽的快速开/关效应。在时间允许的情况下，分子克隆的目标是确认间隔连接蛋白的蛋白质组学鉴定，新产生的抗体试图检查已识别的间隔连接蛋白是否确实位于马氏管的间隔连接。在识别无脊椎动物隔区连接的新蛋白质时，结果可能揭示激酶和其他酶如何调节细胞旁运输，从而整合跨细胞和细胞旁运输途径，如果不是协同作用的话是相加的。类似的整合也可能发生在脊椎动物的上皮细胞中，众所周知，激素和神经递质可以在唾液腺、胃、胰腺和肠道等分泌上皮细胞中触发快速而有效的反应。