Septate junctional proteins: Proteomic identification and role in diuresis

描述（由申请人提供）：过去，上皮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，即细胞旁途径，现在正受到越来越多的关注。但没有上皮显示动态调节的细胞旁运输以及马氏管（肾）的吸血蚊子埃及伊蚊。在这里，利尿激素白细胞激肽触发细胞旁氯电导增加10倍，有助于清除蚊子不需要的血浆部分的血餐。白细胞激肽的作用是可逆的，开关样的开/关变化表明细胞旁蛋白的翻译后修饰。在马氏管水平，白细胞激肽与G蛋白偶联受体结合，导致Ca通过Ca库耗竭激活的Ca通道进入细胞。钙是如何增加隔膜连接（脊椎动物紧密连接的功能等同物）的Cl电导的尚不清楚。此外，蛋白质定义的电导和隔连接的选择透过性在很大程度上是未知的。因此，一个目标是鉴定A.蛋白质组学方法对埃及伊蚊进行了研究。蛋白质组学分析将集中在质膜蛋白，可能会延伸到细胞旁空间，以确定细胞旁电导和渗透选择性。在完整的马氏管的生理/药理学研究试图揭示钙激活的激酶，磷酸酶，和/或G-蛋白介导的快速细胞旁电导的变化，和蛋白质组学分析将试图确定那些分隔连接蛋白进行翻译后修饰的快速开/关白细胞激肽的影响。如果时间允许，分子克隆的目标是确认分隔连接蛋白的蛋白质组学鉴定，新产生的抗体试图检查鉴定的分隔连接蛋白是否确实位于马氏管的分隔连接处。在确定新的蛋白质的无脊椎动物隔膜连接，结果可能会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的添加剂，如果没有协同效应。类似的整合也可能发生在脊椎动物上皮细胞中，已知激素和神经递质在分泌上皮细胞如唾液腺、胃、胰腺和肠中触发快速和有效的反应。

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.