Regulation of nuclear translocation of the Myb1 transcription factor by Tv Cyclophilin 1 in the protozoan parasite, Trichomonas vaginalis [Gene Regulation]

May 15th, 2014 by Hsu, H.-M., Chu, C.-H., Wang, Y.-T., Lee, Y., Wei, S.-Y., Liu, H.-W., Ong, S.-J., Chen, C., Tai, J.-H.

In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 (rTvCyP1) exhibited typical peptidyl-prolyl isomerase activity, with kcat/KM of ~7.1 μM-1s-1. In a pull-down assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of rTvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of R63 in the catalytic motif, or inhibited by cyclosporine A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1, but a much lower level of Myb1 associated with the vesicles, than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected to a much higher level in the protein complex of HA-TvCyP1 than that of HA-TvCyP1(R63A) immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, 105YGPKWNK111, was identified in Myb1, in which G106 and P107 were essential for its binding to TvCyP1. Mutation of G106 or P107 in HA-Myb1 respectively resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.
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