Recombinant expression of the FMN binding protein Flavoredoxin from the Bacterium Desulfovibrio gigas

Abstract

The glomerular mesangial cells (GMC) play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC. Therefore, we studied pattern secretion of matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-3, MMP-9 and their biological tissue inhibitor of matrix metalloproteinases (TIMPs), TIMP-1 and TIMP-2 by cultured human GMC. We also measured MMP-l/TIMP-1 complex level in the cell culture supernatants. For this purpose, the GMC were incubated under serum-free conditions with medium (RPMI-1640) alone or in combination with TNF-alpha (30 ng/ml) or PMA (50 ng/ml) for 24,48 and 72 h. The metalloproteinases and their inhibitors were assayed by established ELISA techniques. Our results showed that the lowest and largest secretions were related to MMP-9 and MMP-2, respectively. The results indicated that the MMPs and TIMPs secretion were increased by TNF-alpha (MMP-1, MMP-2, TIMP-1 and TIMP-2) and PMA (MMP-2, TIMP-1 and TIMP-2), significantly (P < 0.05). These results suggest that GMC can synthesis and release various MMPs and their inhibitors (TIMPs) that, in part, control turnover of extracellular matrix proteins.An increased burden of oxidants and depletion of antioxidant defenses may contribute to the age-related decline in adrenal steroidogenesis. I n this study, we sought to determine whether aging influences the sensitivity of the adrenal to LPSstress related activation of NF-kB, a redox-sensitive transcription factor. Young mature (5 mo) and old (24-26 mo) rats were injected without or with LPS and nuclear extracts studied using electrophoretic mobility shift assay (EMSA) andWestern blotting. The adrenals from old animals showed -50% reduction in activation of NF-kB D N A binding activity as determined by EMSA, when treated with LPS as compared to values from the adrenals of young animals. Supershift analysis confirmed the presense of p65 and p50 proteins in D N A complexes. Aging decreased both total and nuclear levels of p65, but had no effect o n IkB inhibitory proteins. These findings suggest that aging-induced increases in oxidative stress might be related to alterations in NF-kB signaling, which could contribute to the adverse affects of aging on adrenal steroidogenesis. Desulfovibrio gigus is a sulphate reducing bacterium, very rich in several proteins involved in oxido-reduction reactions. These proteins show a great variety of prosthetic groups, playing an important role in the biochemical understanding of this bacterium. Some of these proteins are very well characterized, however, the function of many remains unclear. The recombinant overexpressed proteins help us to get information on its function and structure. One of these important proteins is the flavoredoxin. Flavoredoxin is a FMN binding protein that participates in the reduction of bisulphite from molecular hydrogen. According to the significant homolo...