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: 7365144
• 负责人: Klaus W Beyenbach
• 金额: $ 18.35万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365144
• 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（由申请人提供）：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。细胞间的运输途径，即细胞旁通路，现在受到越来越多的关注。但没有上皮显示出血媒埃及伊蚊的细胞旁运输和马尔比氏（肾）小管的动态调节。在这里，利尿激素白细胞介素触发细胞旁Cl电导增加10倍，帮助蚊子摆脱不需要的血浆部分的血粉。白细胞介素的作用是可逆的，并且开关式的开/关变化提示细胞旁蛋白的翻译后修饰。在马氏小管水平，白细胞介素与G蛋白偶联受体结合，通过钙储存耗尽激活的钙通道导致钙进入细胞。钙如何继续增加间隔连接（与脊椎动物紧密连接的功能等效）的氯离子电导尚不清楚。此外，定义电导和分离连接的许可选择性的蛋白质在很大程度上是未知的。因此，利用蛋白质组学方法鉴定埃及伊蚊马尔匹氏小管中的分离连接蛋白是一个目标。蛋白质组学分析将集中在可能延伸到细胞旁空间的质膜蛋白上，以定义细胞旁电导和许可选择性。完整马尔比氏小管的生理/药理学研究试图揭示介导细胞旁电导快速变化的钙活化激酶、磷酸酶和/或g蛋白，蛋白质组学分析将试图识别那些经过翻译后修饰的分离连接蛋白，这些连接蛋白具有白细胞介素的快速开/关作用。在时间允许的情况下，分子克隆的目标是确认分离连接蛋白的蛋白质组学鉴定，而新产生的抗体则试图检验鉴定的分离连接蛋白是否确实位于马尔皮管的分离连接处。在鉴定无脊椎动物分离连接的新蛋白时，结果可能揭示激酶和其他酶如何调节细胞旁运输，从而将跨细胞和细胞旁运输途径整合在一起，即使不是协同作用，也是相加的。类似的整合很可能发生在脊椎动物上皮中，激素和神经递质在分泌上皮如唾液腺、胃、胰腺和肠中引发快速而有效的反应。

项目成果

Ca2+ and cAMP signaling pathways interact to increase the diuretic effect of serotonin in Malpighian tubules of the kissing bug. Focus on "Serotonin triggers cAMP- and PKA-1-mediated intracellular calcium waves in Malpighian tubules of Rhodnius prolixus".

Ca2 和 cAMP 信号通路相互作用，增加接吻虫马氏小管中血清素的利尿作用。

• DOI: 10.1152/ajpregu.00336.2014
• 发表时间: 2014
• 期刊: American journal of physiology. Regulatory, integrative and comparative physiology
• 作者: Pannabecker,ThomasL;Beyenbach,KlausW
• 通讯作者: Beyenbach,KlausW

Transcellular and paracellular pathways of transepithelial fluid secretion in Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti.

黄热病蚊子埃及埃及的马皮亚（肾脏）小管中跨细胞流体分泌的跨细胞和细胞细胞途径。

• DOI: 10.1111/j.1748-1716.2010.02195.x
• 发表时间: 2011-07
• 期刊: Acta physiologica (Oxford, England)
• 作者: Beyenbach KW;Piermarini PM
• 通讯作者: Piermarini PM