Agola Jacob O, Sivalingam Daniel, Cimino Daniel F, Simons Peter C, Buranda Tione, Sklar Larry A, Wandinger-Ness Angela
Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA.
Methods Mol Biol. 2015;1298:331-54. doi: 10.1007/978-1-4939-2569-8_28.
Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7-RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead 'locks' the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein-protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein-protein interactions, which can be relevant to drug discovery and development.
Rab7促进囊泡从早期内体向晚期内体的运输和传递，以及从晚期内体向溶酶体的运输和传递。Rab7在囊泡运输中的作用取决于其与效应蛋白的相互作用，其中包括Rab相互作用溶酶体蛋白（RILP），它有助于将活性Rab7（结合GTP）募集到动力蛋白-动力蛋白激活蛋白运动复合体上，以促进晚期内体在细胞骨架上的运输。在此，我们详细介绍一种基于磁珠的新型流式细胞术检测方法，用于测量Rab7与Rab相互作用溶酶体蛋白（RILP）效应蛋白的相互作用，并证明其在定量评估和研究药物-靶点相互作用方面的实用性。结合GTP的Rab7与RILP的特异性结合很容易得到证明，且呈剂量依赖性和饱和性，能够测定解离常数（Kd）和最大结合量（Bmax）。此外，结合几乎是瞬时的且依赖温度。在该检测方法的一项新应用中，一种Rab7核苷酸结合的竞争性小分子抑制剂（CID 1067700或ML282）被证明可抑制Rab7-RILP相互作用。因此，该检测方法能够区分该小分子并非诱导活性构象，而是将GTP酶“锁定”在非活性构象。总之，这项工作证明了使用流式细胞术检测方法定量表征涉及小GTP酶的蛋白质-蛋白质相互作用的实用性，并且该方法已适用于高通量筛选。此外，该方法提供了一个测试小分子对蛋白质-蛋白质相互作用影响的平台，这可能与药物发现和开发相关。
