Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins.
in (partial) fulfillment of the requirements for obtaining the degree Dr. rer. nat. by Victoria Langer. Supporting grants were from the German Research Foundation (DFG) (KFO 257 [subproject 4 to M Stürzl and MJW], FOR 2438 [subproject 2 to EN and M Stürzl], SFB/TRR241 [subproject A06 to M Stürzl and NBL], and BR 5196/2-1 [to NBL]); the Interdisciplinary Center for Clinical Research (IZKF) of the Clinical Center Erlangen (D28 to EN and M Stürzl); the W. Lutz Stiftung (to M Stürzl); and the Forschungsstiftung Medizin am Universitätsklinikum Erlangen (to M Stürzl).
The human guanylate-binding protein 1 (GBP-1) is among the proteins the most highly induced by interferon-γ (IFN-γ) in every cell type investigated as yet. In vivo, GBP-1 expression is associated with the presence of inflammation and has been observed in autoimmune diseases, inflammatory bowel diseases (IBD) and cancer. In colorectal carcinoma (CRC), the expression of GBP-1 in the desmoplastic stroma has been previously reported to correlate with the presence of an IFN-γ-dominated T helper type 1 (Th1) micromilieu and with an increased cancer-related 5-year survival. In the present study, the analysis of GBP-1 expression in a series of 185 CRCs by immunohistochemistry confirmed that GBP-1 is expressed in stroma cells of CRCs and revealed a significantly less frequent expression in tumor cells, which was contradictory with the broad inducibility of GBP-1. Furthermore, three of six CRC cell lines treated with IFN-γ were unable to express GBP-1 indicating that colorectal tumor cells tend to downregulate GBP-1. On the contrary, non-transformed colon epithelial cells strongly expressed GBP-1 in vitro in presence of IFN-γ and in vivo in inflammatory bowel diseases. Reconstitution of GBP-1 expression in a negative CRC cell line inhibited cell proliferation, migration and invasion. Using RNA interference, we showed that GBP-1 mediates the antitumorigenic effects of IFN-γ in CRC cells. In addition, GBP-1 was able to inhibit tumor growth in vivo. Altogether, these results suggested that GBP-1 acts directly as a tumor suppressor in CRC and the loss of GBP-1 expression might indicate tumor evasion from the IFN-γ-dominated Th1 immune response.
g Gamma interferon (IFN-␥) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-␥ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-␥-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-␥-dependent defense strategies.
The dissection of the pleiotropic activities of IFN-γ in IBD provides new insights to the pathological functions of this cytokine and may be of high relevance for the optimization of combination therapy approaches.
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