Determination of the Rab27-Effector Binding Affinity Using a High-Throughput FRET-Based Assay.

Thus far, two Rab27 isoforms (Rab27a and Rab27b) have been identified that interact with their eleven downstream effectors proteins, preferentially in their GTP-bound state. In recent years, a number of studies has suggested roles for Rab27-effector protein interactions in the development of cancer cell invasion and metastasis, and immune and inflammatory responses. Here we develop an in vitro fluorescence resonance energy transfer (FRET)-based protein-protein interaction assay to report Rab27 protein interactions with their effectors. We particularly focus on determining the interaction of mouse (m) Synaptotagmin-like protein (Slp)1 and mSlp2 effector proteins with human (h)Rab27. Green fluorescent protein (GFP)-N-terminus Rab27 binding domains (m-Slp1 and m-Slp2) recombinant proteins were used as donor fluorophores, whereas mCherry-hRab27a/b recombinant proteins were used as acceptor fluorophores. The conditions of this assay were validated and optimized, and the specificity of the assay was confirmed. Accordingly, this assay can be used to assess and identify key determinants and/or candidate inhibitors of Rab27-effector interactions.

In Vitro Fluorescence Resonance Energy Transfer-Based Assay Used to Determine the Rab27-Effector-Binding Affinity.

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.