Abstract
Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis, and migration. FGFs are essential for early embryonic development, organ formation, and angiogenesis. FGF1 also plays an important role in inflammation, wound healing, and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase fibroblast growth factor receptors in the presence of heparin sulfate proteoglycans and, therefore, require the release of the protein into the extracellular space. FGF1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex. The protein constituents of this complex include FGF1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). Because FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multiprotein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both complexes possibly formed during the non-classical pathway of FGF1 release.
Highlights
Otic cells is known as the classical ER/Golgi-dependent secretory pathway
Direct roles for synaptotagmin 1 (Syt1) and S100A13 in fibroblast growth factor 1 (FGF1) export have been demonstrated by both antisense RNA-mediated knockdown of Syt1 expression and the expression of a dominant negative S100A13 mutant that attenuates the export of FGF1 [12, 15]
When S100A13 is expressed in FGF1-free cells, it exhibits a spontaneous non-classical release both at 37 and 42 °C, but, when it is expressed in cells with FGF1, it is released only by heat shock [12]
Summary
Materials—Ingredients for Luria Broth were obtained from AMRESCO (USA). Aprotinin, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, Triton X-100, and -mercaptoethanol were obtained from Sigma. The S100A13 or C2A domain that was obtained was further purified by gel filtration on a Superdex-75 (Amersham Biosciences) column using fast protein liquid chromatography and 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl as the eluent. Assignments for the backbone 1H, 13C, and 15N resonances in the complexes containing FGF1, S100A13, and C2A were obtained through three-dimensional HNCA and HNCOCA experiments [26]. Docking Studies—HADDOCK (36 – 40) was applied to dock FGF1 and S100A13 for the FGF1-S100A13 tetrameric complex and to dock C2A and the FGF1-S100A13 tetrameric complex for the FGF1-S100A13-C2A heterohexameric complex using the previously determined structures established using ARIA and intermolecular NOEs. Intermolecular distance restraints were derived from a three-dimensional, 13C and 15N (F1)-filtered, 13C (F2)-edited, 12C (F3)-filtered NOESY experiment. The structures were analyzed with PROCHECK [35]
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