The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.
Interferon γ (IFN-γ) has recently been implicated in cancer immunosurveillance. Among the most abundant proteins induced by IFN-γ are guanylate binding proteins (GBPs), which belong to the superfamily of large GTPases and are widely expressed in various species. Here, we investigated whether the well-known human GBP-1 (hGBP-1), which has been shown to exert antiangiogenic activities and was described as a prognostic marker in colorectal carcinomas, may contribute to an IFN-γ-mediated tumor defense. To this end, an IFN-independent, inducible hGBP-1 expression system was established in murine mammary carcinoma (TS/A) cells, which were then transplanted into syngeneic immune-competent Balb/c mice. Animals carrying TS/A cells that had been given doxycycline for induction of hGBP-1 expression revealed a significantly reduced tumor growth compared with mock-treated mice. Immunohistochemical analysis of the respective tumors demonstrated a tightly regulated, high-level expression of hGBP-1. No signs of an enhanced immunosurveillance were observed by investigating the number of infiltrating B and T cells. However, hemoglobin levels as well as the number of proliferating tumor cells were shown to be significantly reduced in hGBP-1-expressing tumors. This finding corresponded to reduced amounts of vascular endothelial growth factor A (VEGF-A) released by hGBP-1-expressing TS/A cells in vitro and reduced VEGF-A protein levels in the corresponding mammary tumors in vivo. The results suggest that hGBP-1 may contribute to IFN-γ-mediated antitumorigenic activities by inhibiting paracrine effects of tumor cells on angiogenesis. Consequently, owing to these activities GBPs might be considered as potent members in an innate, IFN-γ-induced antitumoral defense system.
Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic “hairpin” structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.
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