Abstract
One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.
Highlights
Elucidation of the biochemical and biological function of IFN-␥ induced mGBP2
Our results indicate that the intracellular localization in vesicle-like structures and the recruitment of mGBP2 to the PV membrane of T. gondii in IFN-␥-stimulated murine embryonic fibroblasts (MEFs) depend on multimerization and the GTPase activity
We have shown that the GTPase activity of mGBP2 increased in a concentrationdependent manner (Fig. 2), so we sought to determine whether mGBP2 forms oligomers as well
Summary
Elucidation of the biochemical and biological function of IFN-␥ induced mGBP2. Results: Mutations in the G-domain of mGBP2 inhibit GTPase activity and multimerization resulting in failure in the recruitment to T. gondii. Two families of GTPases, the 47- and 65-kDa guanylatebinding proteins (GBPs), account for a large percentage of the mRNA transcripts induced by IFN-␥, and the GBPs represent classical secondary response genes [3] Their implication in mammalian host defense and potential for pathogen specificity have been demonstrated (4 –7). The GBPs share unique biochemical properties such as low affinities for guanine nucleotides, in contrast to the small GTPases such as Ras, Rab, Rho, and ␣-subunits of heterotrimeric G proteins [12, 13] They are stable without guanine nucleotide, coordinate a chelated Mg2ϩ ion cofactor, and undergo nucleotide-dependent multimerization and a cooperative mechanism of GTP hydrolysis [14, 15]. Our results indicate that the intracellular localization in vesicle-like structures and the recruitment of mGBP2 to the PV membrane of T. gondii in IFN-␥-stimulated murine embryonic fibroblasts (MEFs) depend on multimerization and the GTPase activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
