Biophysical analyses of human resistin: oligomer formation suggests novel biological function.

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.

Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappaB-dependent pathway.

Resistin, a recently discovered 92 amino acid protein involved in the development of insulin resistance, has been associated with obesity and type 2 diabetes. The elevated serum resistin in human diabetes is often associated with a pro-inflammatory milieu. However, the role of resistin in the development of inflammation is not well understood. Addition of recombinant human resistin protein (hResistin) to macrophages (both murine and human) resulted in enhanced secretion of pro-inflammatory cytokines, TNF-alpha and IL-12, similar to that obtained using 5 microg/ml lipopolysaccharide. Both oligomeric and dimeric forms of hResistin were able to activate these cytokines suggesting that the inflammatory action of resistin is independent of its conformation. Heat denatured hResistin abrogated cytokine induction while treatment of recombinant resistin with polymyxin B agarose beads had no effect thereby ruling out the role of endotoxin in the recombinant hResistin mediated cytokine induction. The pro-inflammatory nature of hResistin was further evident from the ability of this protein to induce the nuclear translocation of NF-kappaB transcription factor as seen from electrophoretic mobility shift assays. Induction of TNF-alpha in U937 cells by hResistin was markedly reduced in the presence of either dominant negative IkappaBalpha plasmid or PDTC, a pharmacological inhibitor of NF-kappaB. A protein involved in conferring insulin resistance is also a pro-inflammatory molecule that has important implications.

Dimerization of human recombinant resistin involves covalent and noncovalent interactions.

Resistin, an adipocyte secreted cysteine rich hormone has been implicated as molecular link between obesity and type 2 diabetes in a murine model. Although, at the protein level mouse and human resistin show remarkable similarities with respect to conserved cysteine residues, the physiological role of human resistin is not yet clear. In the present study we describe the purification and refolding of human recombinant resistin using two different refolding processes. Gel filtration analysis of protein refolded by both the methods revealed that human recombinant resistin, like mouse resistin, has a tendency to form dimers. Interestingly, dimerization of resistin appears to be mediated by both covalent (disulfide bond mediated) and non-covalent interactions as seen on reducing and non-reducing SDS-PAGE. Circular dichroism spectral analysis revealed that human resistin peptide backbone is a mixture of alpha-helical and beta-sheet conformation with significant amounts of unordered structure, similar to the mouse resistin. It is likely that the first cysteine (Cyst22) of human resistin, which is equivalent to mouse Cyst26, may be involved in stabilizing the dimers through covalent interaction.

Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages.

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.

The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications.

The resistin gene is a potential candidate for the etiology of insulin resistance and type 2 diabetes and has been implicated as the molecular link between type 2 diabetes and obesity. Unlike the mouse resistin, expression of the human resistin appears to be regulated differently. We report comparative analyses of the mouse and human genomic fragments encoding the resistin gene. At the amino acid level the two proteins exhibit 59% identity. While at the mRNA level the human resistin shows 64.4% sequence identity with its mouse counterpart, the mouse resistin genomic sequence displays only 46.7% sequence identity with the human resistin and is almost three times bigger than the human resistin. The intronic sequences per se displayed the least identities (28.7%), however the intron boundaries were highly conserved between human and mouse. The mouse resistin carries a very large intron in the 3' UTR, which has a number of regulatory sequences possibly involved in differential gene expression. Of particular significance is the presence of a PPAR/RXR heterodimer binding site within intron X (IntX-PPRE) which may possibly confer TZD responsiveness. Oligonucleotides carrying the authentic PPAR/RXR binding element (Aco-PPRE) as well as IntX-PPRE specifically bound factors (PPAR/RXR heterodimers) present in differentiated 3T3-L1 adipocyte cells in an electrophoretic mobility shift assay. IntX-PPRE oligonucleotide modulated the expression of the luciferase reporter gene in transient transfection assays using 3T3-L1 cells.