| Resistin is a novel cytokine that is specifically secreted from adipocytes. It has recently been shown to induce resistance to the effects of insulin, a hallmark of type II diabetes. Resistin secretion is dysregulated in obese humans and several animal models of obesity. Thus, it may provide a molecular link between obesity and diabetes. To begin to investigate the molecular functions of resistin, we have determined the high resolution crystal structure of resistin and resistin like molecule beta (RELM-beta).; Resistin is a disulfide-rich secreted protein; consequently, it is unlikely to fold properly when produced in E.coli over-expression systems. To achieve the high production levels of natively folded resistin necessary for structural studies, we developed a general system for the high level expression of eukaryotic genes in mammalian cell lines. This system utilizes an internal ribosome entry site (IRES) to produce a bicistronic message composed of the gene of interest and green fluorescent protein (GFP) from the same mRNA. Fluorescence activated cell sorting is then used to select cells with the most intense fluorescence, which by analogy, express the gene of interest at high levels. This system was used to express the mouse resistin and RELM-beta proteins.; We determined crystal structures of resistin in two separate crystal forms (C2221 and C2) by single wavelength anomalous diffraction and molecular replacement, respectively. The RELM-beta structure was solved by molecular replacement using the C2 crystal form of resistin as a search model. These structures show that the resistin protein family adopts a novel hexameric structure. Each protomer in the hexamer is composed of a disulfide-rich beta-sandwich head, and an amino-terminal alpha-helical tail segment. Three protomers associate by their alpha-helical segments to form a trimer that is composed of a three-stranded coiled-coil. Two trimers associate by three solvent-exposed disulfide bonds at their amino terminus to form a hexamer. The high solvent exposure of these disulfides led us to postulate that there are potentially two forms of the molecule in vivo, trimers and hexamers. This was confirmed by size exclusion chromatography of mouse serum under both reducing and non-reducing conditions, and subsequent western blot analysis.; Adiponectin is another adipocyte specific hormone that has effects opposite that of resistin, namely it is an insulin sensitizer. The disulfide dependent multimeric assembly of these two hormones has intriguing parallels, despite their physiologically opposite effects on glucose homeostasis, and suggests the possibility of a general mechanism of disulfide regulation for modulating the activity of adipocyte secreted hormones. |
