Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal
Jacob O Agola1, Daniel Sivalingam, Daniel F Cimino
1Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 25, 2015
Summary
We developed a novel flow cytometry assay to measure Rab7-RILP protein interactions. This assay quantifies small molecule effects on GTPase activity, aiding drug discovery for vesicular transport disorders.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rab7 is crucial for vesicular transport, mediating endosome-to-lysosome delivery.
- Rab7 function depends on effector proteins like Rab-interacting lysosomal protein (RILP), which links Rab7 to motor proteins for cytoskeletal transport.
Purpose of the Study:
- To develop and validate a novel bead-based flow cytometry assay for quantifying Rab7-RILP protein interactions.
- To assess the utility of this assay for studying drug-target interactions and small molecule effects on GTPase activity.
Main Methods:
- A bead-based flow cytometry assay was developed to measure the binding of GTP-bound Rab7 to RILP.
- Binding kinetics, including dose-dependence, saturation, and temperature effects, were characterized.
- A small molecule inhibitor (CID 1067700/ML282) was used to test the assay's ability to identify modulators of Rab7-RILP interaction.
Main Results:
- The assay successfully demonstrated specific, dose-dependent, and saturable binding between GTP-bound Rab7 and RILP.
- Binding was found to be rapid and temperature-dependent.
- The small molecule inhibitor ML282 was shown to inhibit the Rab7-RILP interaction by stabilizing Rab7 in an inactive conformation.
Conclusions:
- The developed flow cytometry assay provides a quantitative method for characterizing small GTPase protein-protein interactions.
- This assay is adaptable for high-throughput screening and can be used to evaluate small molecule inhibitors targeting these interactions, relevant for drug discovery.
