Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules
Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules
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DOI：
10.1242/jeb.024646
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发表时间：
2009-02-01
影响因子：
2.8
通讯作者：
Zhang, Sheng
中科院分区：
文献类型：
--
作者：
Beyenbach, Klaus W.;Baumgart, Sabine;Zhang, Sheng
使用蛋白质组学方法，我们检查了当用利尿肽伊德皮宁-III（AK-III，10(-7) mol l(-1)）刺激分离的埃及伊蚊马氏小管1分钟时，胞浆蛋白的翻译后变化。从数千个马氏小管中提取对照 (C) 和伊皮素处理 (T) 肾小管的胞质溶胶，进行 2-D 电泳并对总蛋白和磷蛋白进行染色。通过凝胶图像分析来比较 C 和 T 凝胶的归一化点体积的变化。体积等于或超过 C/T 比率 +/-1.5 的斑点被机器人挑选用于胰蛋白酶的 ingel 消化，并提交通过 nanoLC/MS/MS 分析进行蛋白质鉴定。已鉴定的蛋白质涵盖了广泛的生物活性。由于已知激肽肽可快速刺激马氏小管跨上皮分泌电解质和水，因此我们重点关注那些可能介导跨上皮分泌增加的蛋白质。我们发现 AK-III 减少了 V 型 H+ ATPase 亚基 A 和 B、内质蛋白、钙网蛋白、膜联蛋白、蛋白激酶 A (PKA) II 型调节亚基和 rab GDP 解离抑制剂的胞质存在，并增加了内收蛋白、肌动蛋白、Ca2+ 结合蛋白调节钙蛋白/SMP30 和肌动蛋白解聚因子的胞质存在。 H89（PKA 抑制剂）对体液分泌的作用支持了 PKA 在 AK-III 诱导的 V 型 H+ ATP 酶激活中的假定作用。 H89 逆转 AK-III 对离体马氏小管跨上皮液体分泌的刺激作用。然而，AK-III 不会提高细胞内 cAMP（PKA 的常用激活剂）水平，表明 PKA 的激活不依赖于 cAMP，在 V 型 H+ ATP 酶的组装和激活过程中，可从细胞质中去除亚基 A 和 B。或者，蛋白激酶 C 也可以介导质子泵的激活。 Ca2+ 仍然是腺皮素的主要细胞内信使，明显通过蛋白激酶 C 向细胞旁复合物的重塑发出信号，从而增加跨上皮阴离子分泌。 AK-III 对主动跨细胞和被动旁细胞转运的影响即使不是协同作用，也是相加的，以带来快速利尿。
Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10(-7) mol l(-1)). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of +/-1.5 were robotically picked for ingel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK-III reduces the cytosolic presence of subunits A and B of the V-type H+ ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca2+-binding protein regucalcin/SMP30 and actin-depolymerizing factor. Supporting the putative role of PKA in the AK-III-induced activation of the V-type H+ ATPase is the effect of H89, an inhibitor of PKA, on fluid secretion. H89 reverses the stimulatory effect of AK-III on transepithelial fluid secretion in isolated Malpighian tubules. However, AK-III does not raise intracellular levels of cAMP, the usual activator of PKA, suggesting a cAMP-independent activation of PKA that removes subunits A and B from the cytoplasm in the assembly and activation of the V-type H+ ATPase. Alternatively, protein kinase C could also mediate the activation of the proton pump. Ca2+ remains the primary intracellular messenger of the aedeskinins that signals the remodeling of the paracellular complex apparently through protein kinase C, thereby increasing transepithelial anion secretion. The effects of AK-III on active transcellular and passive paracellular transport are additive, if not synergistic, to bring about the rapid diuresis.