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Screening for Potential Inhibitors of the Pro‐angiogenic GTPase TCL Using Split‐Venus Reporter Proteins
Author(s) -
Florke Rebecca,
Hamann Michael
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.866.1
Subject(s) - gtpase , bimolecular fluorescence complementation , microbiology and biotechnology , cdc42 , chemistry , gtp' , fluorescence , small gtpase , green fluorescent protein , rab , biochemistry , biology , signal transduction , gene , physics , quantum mechanics , enzyme
TCL is a specific Rho GTPase involved in cytoskeletal signaling related to angiogenesis when it is activated and is implicated in the development of tumor vascularization. Therefore, TCL is a promising new target in anticancer treatments. In order to screen for inhibitors of TCL, a detection system was designed using the fluorescent protein Venus. The coding sequence for Venus was split to produce two non‐fluorescent fragments. TCL and the GTPase binding portion of PAK were then genetically fused to the two Venus sequences to detect GTP‐loaded TCL. Constitutively active (CA) and dominant negative (DN) mutants of TCL, as well as other Rho GTPases, were tested to establish the validity of this bimolecular fluorescence complementation (BiFC) method. In contrast to the DN GTPases, the CA GTPases yielded significantly higher fluorescence. To evaluate the ability of the BiFC assay to respond to inhibitors, a novel Cdc42‐specific inhibitor, ML141, was used with Cdc42 and resulted in decreased fluorescence. Initial screening of TCL with ML141 also produced a decrease in fluorescence, suggesting that ML141 may inhibit either the GTP loading of TCL or the interaction between GTP‐loaded TCL and PAK.