Abstract：
Metallothionein 3 (MT-3) is a small, cysteine-rich protein that binds to essential metals required for homeostasis, as well as to heavy metals that have the potential to exert toxic effects on cells. MT-3 is expressed by epithelial cells of the human kidney, including the cells of the proximal tubule. Our laboratory has previously shown that mortal cultures of human proximal tubular (HPT) cells express MT-3 and form domes in the cell monolayer, a morphological feature indicative of vectorial active transport, an essential function of the proximal tubule. However, an immortalized proximal tubular cell line HK-2 lacks the expression of MT-3 and fails to form domes in the monolayer. Transfection of HK-2 cells with the MT-3 gene restores dome formation in these cells suggesting that MT-3 is required for vectorial active transport. In order to determine how MT-3 imparts this essential feature to the proximal tubule, we sought to identify proteins that interact either directly or indirectly with MT-3. Using a combination of pulldowns, co-immunoprecipitations, and mass spectrometry analysis, putative protein interactants were identified and subsequently confirmed by Western analysis and confocal microscopy, following which proteins with direct physical interactions were investigated through molecular docking. Our data shows that MT-3 interacts with myosin-9, aldolase A, enolase 1, β-actin, and tropomyosin 3 and that these interactions are maximized at the periphery of the apical membrane of doming proximal tubule cells. Together these observations reveal that MT-3 interacts with proteins involved in cytoskeletal organization and energy metabolism, and these interactions at the apical membrane support vectorial active transport and cell differentiation in proximal tubule cultures.
摘要：
金属硫蛋白3(MT-3)是一种小的，富含半胱氨酸的蛋白质，与体内平衡所需的必需金属结合，以及可能对细胞产生毒性作用的重金属。MT-3由人类肾脏的上皮细胞表达，包括近端小管的细胞。我们的实验室先前已经表明，人类近端管状（HPT）细胞的凡人培养物表达MT-3并在细胞单层中形成圆顶，指示矢量主动运输的形态特征，近端小管的基本功能。然而,永生化的近端肾小管细胞系HK-2缺乏MT-3的表达，并且无法在单层中形成圆顶。用MT-3基因转染HK-2细胞可恢复这些细胞中的圆顶形成，表明MT-3是矢量主动运输所必需的。为了确定MT-3如何将这一基本特征赋予近端小管，我们试图鉴定与MT-3直接或间接相互作用的蛋白质.使用下拉的组合，共免疫沉淀，和质谱分析，鉴定了推定的蛋白质相互作用物，随后通过Western分析和共聚焦显微镜确认，随后通过分子对接研究了具有直接物理相互作用的蛋白质。我们的数据显示，MT-3与肌球蛋白-9，醛缩酶A，烯醇化酶1,β-肌动蛋白,和原肌球蛋白3，并且这些相互作用在圆顶近端小管细胞的顶端膜的外围最大化。这些观察结果共同表明，MT-3与参与细胞骨架组织和能量代谢的蛋白质相互作用，根尖膜上的这些相互作用支持近端小管培养物中的矢量主动运输和细胞分化。
