The Rab27 subfamily consists of Rab27a/b isoforms that have similar but not identical functions. Those functions include the regulation of trafficking, docking, and fusion of various lysosome-related organelles and secretory granules; such as melanosomes in melanocytes and lytic granules in cytotoxic T lymphocytes. Rab27a/b exert their specific and versatile functions by interacting with 11 effector proteins, preferentially in their GTP-bound state. In recent years, a number of studies have identified roles for Rab27 proteins and their effectors in cancer cell invasion and metastasis, immune response, inflammation, and allergic responses. These findings suggest that Rab27–effector protein interaction inhibitors could contribute to the development of effective strategies to treat these diseases. To facilitate inhibitor identification, in this study we developed a fluorescence resonance energy transfer-based protein–protein interaction assay that reports Rab27–effector interactions. Green fluorescent protein (GFP)-mouse (m) synaptotagmin-like protein (Slp)1 and GFP-mSlp2 (N-terminus Rab27-binding domains) recombinant proteins were used as donor fluorophores, whereas mCherry-human (h) Rab27a/b recombinant proteins were used as acceptor fluorophores. The in vitro binding affinity of mSlp2 to Rab27 was found to be higher compared with mSlp1 and was evidenced by the effective concentration 50 value differences (mSlp2-hRab27b = 0.15 μM < mSlp2-hRab27a = 0.2 μM < mSlp1-hRab27a = 0.32 μM < mSlp1-hRab27b = 0.33 μM). The specificity of the assay was assessed using unlabeled rat (r) Rab27a and hRab27b recombinant proteins as typical competitive inhibitors for Rab27–effector interactions and was evidenced by the inhibitory concentration 50 value differences. Accordingly, this in vitro assay can be employed in identification of candidate inhibitors of Rab27–effector interactions.

中文翻译：

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用于确定Rab27-效应子结合亲和力的基于体外荧光共振能量转移的分析方法。

Rab27亚家族由功能相似但不相同的Rab27a / b亚型组成。这些功能包括调节各种溶酶体相关细胞器和分泌颗粒的运输，对接和融合；例如黑素细胞中的黑素体和细胞毒性T淋巴细胞中的溶解颗粒。Rab27a / b通过与11种效应蛋白相互作用而发挥其特定的通用功能，这些蛋白优先处于其GTP结合状态。近年来，许多研究已经确定Rab27蛋白及其效应物在癌细胞侵袭和转移，免疫反应，炎症和过敏反应中的作用。这些发现表明，Rab27-效应子蛋白相互作用抑制剂可以促进治疗这些疾病的有效策略的发展。为方便识别抑制剂，在这项研究中，我们开发了一种基于荧光共振能量转移的蛋白质-蛋白质相互作用测定方法，该方法报告了Rab27-效应子的相互作用。绿色荧光蛋白（GFP）-小鼠（m）突触素样蛋白（Slp）1和GFP-mSlp2（N端Rab27结合域）重​​组蛋白用作供体荧光团，而mCherry-人类（h）Rab27a / b重组蛋白被用作受体荧光团。的发现mSlp2对Rab27的体外结合亲和力高于mSlp1，并通过有效浓度50值差异来证明（mSlp2-hRab27b = 0.15μM<mSlp2-hRab27a = 0.2μM<mSlp1-hRab27a = 0.32μM<mSlp1- hRab27b = 0.33μM）。使用未标记的大鼠（r）Rab27a和hRab27b重组蛋白作为Rab27-效应子相互作用的典型竞争性抑制剂来评估测定的特异性，并通过抑制浓度50值的差异来证明。因此，该体外测定可用于鉴定Rab27-效应子相互作用的候选抑制剂。