Molecular architecture of protein-RNA recognition sites

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein–protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein–protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein–protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein–protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2′ position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein–protein interfaces is insignificant.     

Induction of oxidative stress and histopathological changes by sub-chronic doses of triazophos

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with aconcomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histoarchitectural alterations at sub-chronic doses of triazophos in rats.     

Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.     

The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity

Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations. Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA. Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes. Thus, increasing the ssDNA tail length increases the unwinding efficiency. The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules. The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site. The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity. The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated. The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.     

Role of the N-terminal epidermal growth factor-like domain of factor X/Xa

The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with approximately 3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with approximately 12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a one-stage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.