Functional Mapping of ABC Transporters Protein Interaction Networks

人类基因组编码48种ABC蛋白，分为7个亚家族（ABC A - G）。几个实验室的研究表明，这些ABC蛋白运输各种内源性底物，包括氯离子、谷胱甘肽、胆固醇、LTC4、血红素、甾醇和胆汁盐；然而，人们对调节其功能的机制知之甚少。我们相信这种调控机制可以通过识别ABC转运蛋白的相互作用蛋白来揭示。使用肽扫描方法鉴定相互作用蛋白，我们已经证明ABCB1和ABCC1 （ABCB1/C1）连接域（L1）中的序列结合细胞提取物中的可溶性α/ß-微管蛋白。最近，在与张博士实验室的合作中，我们还展示了ABCC1-ATP合成酶-α的相互作用。通过L1磷酸化，ATP合酶-α与ABCC1连接域的结合显著增加，而与微管蛋白的结合则被消除。我们推测这些相互作用可能调节ABCB1/C1亚细胞定位或提示ABCC1调节细胞外ATP水平的新功能。该项目的长期目标是绘制ABC转运体的蛋白质相互作用网络，以努力了解调节其功能的机制。我们将首先关注所有相互作用蛋白的鉴定和功能表征，以两种表征良好的ABC转运蛋白（即ABCB1/C1）。然而，同样的方法将扩展到其他ABC蛋白。这个应用程序的具体目标是：使用各种蛋白质相互作用方法，从肽扫描方法和质谱法开始，鉴定所有与ABCB1/C1相互作用的蛋白质。使用全长和gst融合构建的下拉分析确认这种相互作用。比较ABCB1/C1蛋白在不同组织提取物中的相互作用。ABCB1/C1蛋白相互作用的体内验证。使用ABCB1/C1及其各自相互作用蛋白的抗体，使用化学/光交联和共聚焦显微镜，在体内使用ABCB1/C1及其各自相互作用蛋白的野生型和突变型ABCB1/C1相互作用域验证蛋白质相互作用。评估蛋白质相互作用对ABCB1/C1转运功能、亚细胞定位、蛋白质半衰期、磷酸化和侧枝敏感性表型的影响。鉴于ABC转运体在植物、酵母和寄生虫生物学中的作用，上述目标的完成将对不同领域的研究产生影响。我们认识到，使用非计算方法绘制人类ABC转运蛋白相互作用组是一项雄心勃勃的任务，然而使用高通量平行肽扫描合成与质谱应该促进这一任务。此外，从这个项目中获得的知识应该增强计算方法的预测能力。

The human genome encodes for 48 ABC proteins that are classified into seven subfamilies (ABC A - G). Research from several laboratories has shown these ABC proteins to transport various endogenous substrates, including chloride ions, glutathione, cholesterol, LTC4, hemin, sterols and bile salts; however little is known about the mechanisms that regulate their functions. We believe that such regulatory mechanisms can be unmasked by identifying ABC transporters' interacting proteins. Using peptide-scanning approach to identify interacting proteins, we have shown that sequences in ABCB1 and ABCC1 (ABCB1/C1) linker domains (L1) bind soluble α/ß-tubulin in cell extracts. More recently, in collaboration with Dr. Zhang's lab, we also demonstrated ABCC1-ATP synthase-α interactions. The binding of ATP synthase-α to ABCC1 linker domain was dramatically increased by L1 phosphorylation, while that to tubulin was abolished. We speculate that these interactions may modulate ABCB1/C1 subcellular localization or suggest a novel function for ABCC1 in regulating extracellular ATP level. The long-term objective of this program is to map the protein interaction networks of ABC transporters in an effort to understand the mechanisms that regulate their functions. We will begin by focusing on the identification and functional characterization of all interacting proteins to two well-characterized ABC transporters (i.e., ABCB1/C1). However, the same approach will be expanded to other ABC proteins. The specific aims of this application are to:***I. Identify all interacting proteins with ABCB1/C1 using various protein-interaction methods, beginning with peptide-scanning approach and mass-spectrometry. Confirm such interactions with pull-down assays using full-length and GST-fusion constructs. Compare ABCB1/C1 protein interactions using different tissue extracts.***II. In vivo validation of ABCB1/C1 protein interactions. Protein interactions will be validated using wild type and mutant variants of ABCB1/C1 interacting domains in vivo, using chemical/photo-crosslinking and confocal microscopy with antibodies to ABCB1/C1 and their respective interacting protein.***III. Assess the effects of protein interactions on ABCB1/C1 transport functions, subcellular localization, proteins half-lives, phosphorylation and the collateral sensitivity phenotype.***The completion of the above objectives should have an impact on diverse areas of studies given the role of ABC transporters in plant, yeast and parasite biology. We recognize that mapping the human ABC transporters interactome using non-computational approaches is an ambitious undertaking, however the use of high-throughput parallel peptide scanning synthesis with mass-spectrometry should facilitate this undertaking. In addition, knowledge gained from this program should enhance the predictive power of computational approaches.