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。细胞间的运输途径，即细胞旁途径，现在受到越来越多的关注。但没有上皮细胞像吸血蚊子埃及伊蚊的马尔皮基（肾）小管那样表现出细胞旁运输的动态调节。在这里，利尿激素白激肽触发细胞旁 Cl 电导增加 10 倍，帮助蚊子清除血粉中不需要的血浆部分。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。在马氏小管水平，白细胞激肽与 G 蛋白偶联受体结合，导致 Ca 通过由 Ca 储存耗尽激活的 Ca 通道进入细胞。 Ca 如何继续增加隔膜连接（相当于脊椎动物紧密连接的功能）的 Cl 电导尚不清楚。此外，定义隔膜连接的电导和选择性通透性的蛋白质在很大程度上是未知的。因此，一个目标是通过蛋白质组学方法鉴定埃及伊蚊马氏小管中的隔膜连接蛋白。蛋白质组分析将重点关注可能延伸到细胞旁空间的质膜蛋白，以定义细胞旁电导和选择性通透性。对完整马氏小管的生理/药理学研究试图揭示介导快速旁细胞电导变化的 Ca 激活激酶、磷酸酶和/或 G 蛋白，蛋白质组学分析将尝试识别那些经历翻译后修饰、白细胞激肽快速开/关效应的分隔连接蛋白。如果时间允许，分子克隆的目标是确认隔膜连接蛋白的蛋白质组学鉴定，并且新产生的抗体试图检查所识别的隔膜连接蛋白是否确实位于马氏小管的隔膜连接处。在鉴定无脊椎动物隔膜连接的新蛋白质时，结果可能揭示激酶和其他酶如何调节细胞旁转运，从而将跨细胞和细胞旁转运途径整合为相加效应（如果不是协同效应）。类似的整合很可能发生在脊椎动物上皮细胞中，已知激素和神经递质会在唾液腺、胃、胰腺和肠等分泌性上皮细胞中引发快速而有效的反应。

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.