The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full‐length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain–domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild‐type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full‐length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an “extended” catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.     

Fractalkine and apoptotic/anti-apoptotic markers in granulosa cells of women with polycystic ovarian syndrome

Molecular Biology Reports - Owing to the role of fractalkine in regulating cellular apoptosis/proliferation, we investigated fractalkine effects on apoptosis/proliferation signaling of granulosa...     

The effects of chronic long-term intermittent hypobaric hypoxia on blood rheology parameters

The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on blood rheology is not completely investigated. We designed this study to determine the effect of CLTIHH on blood rheology parameters. Present study was performed in 16 male Spraque-Dawley rats that divided into CLTIHH and Control groups. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mmHg; 5 hours/day, 5 days/week, 5 weeks). The control rats stayed in the same environment as the CLTIHH rats but they breathed room air. In the blood samples aspirated from the heart, hematocrit, whole blood viscosity, plasma viscosity, plasma fibrinogen concentration, erythrocyte rigidity index and oxygen delivery index were determined. The whole blood viscosity, plasma viscosity, hematocrit and fibrinogen concentration values in the CLTIHH group were found to be higher than those of the control group. However, no significant difference was found in erythrocyte rigidity index and oxygen delivery index between the groups. Our results suggested that CLTIHH elevated whole blood viscosity by increasing plasma viscosity, fibrinogen concentration and hematocrit value without effecting the erythrocyte deformability. Hence, CLTIHH that may occur in intermittent high altitude exposure and some severe obstructive sleep apnea (OSA) patients may be responsible for hemorheologic changes in those subjects.     

The 8-oxoguanine DNA N-glycosylase 1 (hOGG1) Ser326Cys variant affects the susceptibility to Graves' disease

Oxidative DNA damage, caused by either endogenous or exogenous sources of reactive oxygen species (ROS), has been linked several diseases including Graves' disease (GD). 7,8‐Dihydro‐8‐oxoguanine (8‐oxoG) is a major lesion produced by ROS and is considered a key biomarker of oxidative DNA damage. In humans, 8‐oxoG is mainly repaired by 8‐oxoguanine DNA N‐glycosylase‐1 (hOGG1), which is an essential component of the base excision repair (BER) pathway. The functional studies showed that hOGG1 Ser326Cys polymorphism is associated with the reduced DNA repair activity and increased risk for some oxidative stress‐related diseases. In this study, we firstly investigated hOGG1 Ser326Cys polymorphism in GD. According to our results, Cys/Cys genotype frequency in the GD patients (23.4%) was significantly higher than the controls (9.2%). Cys/Cys genotype had an 3.5‐fold [95% CI (confidence interval): 2.10–6.01, p < 0.001] the Cys allele had 1.83‐fold (95% CI: 1.43–2.34, p < 0.001) increase in the risk for developing GD. Our results suggest that Ser326Cys polymorphism of the hOGG1 gene is associated with GD risk. Copyright © 2011 John Wiley & Sons, Ltd.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

An extracellular--pepstatin insensitive acid protease produced by Thermoplasma volcanium

In this study, some parameters for the production and caseinolytic activity of an extracellular thermostable acid protease from a thermoacidophilic archaeon Thermoplasma volcanium were determined. The highest level of growth and enzyme production were detected at pH 3.0 over an incubation period of 192 h at 60 degrees C. The pH optimum for the acid protease activity was 3.0 and the enzyme was fairly stable over a broad pH range (pH 3.0-8.0). The temperature for maximum activity of the enzyme was 55 degrees C and activity remained stable between 50 degrees C and 70 degrees C. These features could be of relevance for various biotechnological applications of this enzyme. Serine-(PMSF), cysteine-(DTT), metallo-(EDTA) and aspartate-(pepstatin) protease inhibitors did not inhibit the caseinolytic activity of the enzyme. Therefore, Tp. volcanium acid protease could be a member of the pepstatin-insensitive carboxyl proteinases.