Global proteomic analysis of insulin receptor interactors in glomerular podocytes.
Global proteomic analysis of insulin receptor interactors in glomerular podocytes.
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DOI：
10.12688/wellcomeopenres.16072.1
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发表时间：
2020
影响因子：
--
通讯作者：
Lennon R
中科院分区：
文献类型：
--
作者：
Hosawi SB;Humphries JD;Coward RJ;Knight D;Humphries MJ;Lennon R
背景：胰岛素信号传导参与多种细胞活动，包括蛋白质合成、增殖和细胞存活。胰岛素抵抗描述了细胞不能有效激活胰岛素信号通路;导致包括肾脏在内的多个器官系统的病理效应。在糖尿病肾病中，存在进行性肾小球功能障碍，并且最近的研究已经证明肾脏足细胞是胰岛素作用的直接靶点。在这项研究中，我们定义了基于文献的胰岛素受体（INSR）相互作用组，并利用无偏倚的蛋白质组学方法来检查足细胞中的INSR相互作用。 方法：在基础和胰岛素抵抗条件下表征表达INSR的人足细胞。用100 nM胰岛素刺激2、7和15分钟后，通过全细胞免疫沉淀分离INSR。通过无标记质谱法（MS）分析所得INSR复合物以检测蛋白质相互作用物。 结果：我们确定了27个已知的，直接的INSR相互作用，除了新的相互作用，包括含双皮质素结构域的蛋白2（DCDC 2）。通过免疫沉淀和免疫荧光证实了DCDC 2与INSR的相互作用，并且在胰岛素抵抗条件下，DCDC 2与INSR的关联增加。在足细胞中siRNA敲低DCDC 2导致细胞形态学改变和改变的INSR定位。 结论：本研究提供了深入了解足细胞中INSR相互作用的复杂性，并强调DCDC 2作为一种新的INSR结合蛋白。这种新的相互作用在胰岛素信号和足细胞生物学的参与可能解释胰岛素抵抗如何改变肾小球滤过屏障的形态和完整性。
Background: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively; leading to pathological effects in multiple organ systems including the kidney. In diabetic kidney disease, there is progressive glomerular dysfunction and recent studies have demonstrated that the kidney podocyte is a direct target for insulin action. In this study we defined the literature-based insulin receptor (INSR) interactome and utilised an unbiased proteomic approach to examine INSR interactors in podocytes. Methods: Human podocytes expressing the INSR were characterised under basal and insulin resistant conditions. The INSR was isolated by whole cell immunoprecipitation following a time course stimulation of 2, 7, and 15 minutes with of 100nM insulin. The resulting INSR complexes were analysed by label-free mass spectrometry (MS) to detect protein interactors. Results: We identified 27 known, direct INSR interactors in addition to novel interactors including doublecortin domain-containing protein 2 (DCDC2). The interaction of DCDC2 with the INSR was confirmed by immunoprecipitation and immunofluorescence, and under insulin resistant conditions, DCDC2 had increased association with the INSR. siRNA knockdown of DCDC2 in podocytes resulted in cell morphological change and altered INSR localisation. Conclusion: This study provides insight into the complexity of INSR interactors in podocytes and highlights DCDC2 as a novel INSR binding protein. Involvement of this novel interactor in insulin signalling and podocyte biology may explain how insulin resistance alters morphology and integrity of the glomerular filtration barrier.