Structure and phospholipase function of peroxiredoxin 6: identification of the catalytic triad and its role in phospholipid substrate binding

Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 alpha-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10(6) M(-1) s(-1) and a dissociation constant of 0.36 microM. Both binding and PLA(2) activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA(2) activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.     

Increased yield of high purity recombinant human interferon-gamma utilizing reversed phase column chromatography

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-gamma), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30 matrix in the purification of rhIFN-gamma from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-gamma monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50 and 100 microg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg/g cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 x 10(7) to 4 x 10(7)IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-gamma in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.     

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.     

Decreased Prevalence of Lymphatic Filariasis among Diabetic Subjects Associated with a Diminished Pro-Inflammatory Cytokine Response (CURES 83)

Epidemiological studies have shown an inverse correlation between the incidence of lymphatic filariasis (LF) and the incidence of allergies and autoimmunity. However, the interrelationship between LF and type-2 diabetes is not known and hence, a cross sectional study to assess the baseline prevalence and the correlates of sero-positivity of LF among diabetic subjects was carried out (n = 1416) as part of the CURES study. There was a significant decrease in the prevalence of LF among diabetic subjects (both newly diagnosed [5.7%] and those under treatment [4.3%]) compared to pre-diabetic subjects [9.1%] (p = 0.0095) and non-diabetic subjects [10.4%] (p = 0.0463). A significant decrease in filarial antigen load (p = 0.04) was also seen among diabetic subjects. Serum cytokine levels of the pro-inflammatory cytokines—IL-6 and GM-CSF—were significantly lower in diabetic subjects who were LF positive, compared to those who were LF negative. There were, however, no significant differences in the levels of anti-inflammatory cytokines—IL-10, IL-13 and TGF-β—between the two groups. Although a direct causal link has yet to be shown, there appears to be a striking inverse relationship between the prevalence of LF and diabetes, which is reflected by a diminished pro-inflammatory cytokine response in Asian Indians with diabetes and concomitant LF.