NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.     

Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.     

Orthogonal chemical genetic approaches for unraveling signaling pathways

While chemical genetic approach uses small molecules to probe protein functions in cells or organisms, orthogonal chemical genetics refers to strategies that utilize reengineered protein-small molecule interfaces, to alter specificities, in order to probe their functions. The advantage of orthogonal chemical genetics is that the changes at the interfaces are generally so minute that it goes undetected by natural processes, and thus depicts a true physiological picture of biological phenomenon. This review highlights the recent advances in the area of orthogonal chemical genetics, especially those designed to probe signaling processes. Dynamic protein-protein and enzyme-substrate interactions following stimuli form the foundation of signal transduction. These processes not only break spatial and temporal boundaries between interacting proteins, but also impart distinct regulatory properties by creating functional diversity at the interfaces. Functional and temporal modulation of these dynamic interactions by specific chemical probes provides extremely powerful tools to initiate, ablate, decouple and deconvolute different components of a signaling pathway at multiple stages. Not surprisingly, multiple receptor-ligand reengineering approaches have been developed in the last decade to selectively manipulate these transient interactions with the aim of unraveling signaling events. However, given the diversity of protein-protein interactions and novel chemical genetic probes developed to perturb these processes, a short review cannot do adequate justice to all aspects of signaling. For this reason, this review focuses on some orthogonal chemical-genetic strategies that are developed to study signaling processes involving enzyme-substrate interactions.     

Quantitative assessment of FGF regulation by cell surface heparan sulfates

Heparin/heparan sulfate-like glycosaminoglycans (HSGAGs) modulate the activity of the fibroblast growth factor (FGF) family of proteins. Through interactions with both FGFs and FGF receptors (FGFRs), HSGAGs mediate FGF-FGFR binding and oligomerization leading to FGFR phosphorylation and initiation of intracellular signaling cascades. We describe a methodology to examine the impact of heparan sulfate fine structure and source on FGF-mediated signaling. Mitogenic assays using BaF3 cells transfected with specific FGFR isoforms allow for the quantification of FGF1 and FGF2 induced responses independent of conflicting influences. As such, this system enables a systematic investigation into the role of cell surface HSGAGs on FGF signaling. We demonstrate this approach using cell surface-derived HSGAGs and find that distinct HSGAGs elicit differential FGF response patterns through FGFR1c and FGFR3c. We conclude that this assay system can be used to probe the ability of distinct HSGAG species to regulate the activity of specific FGF-FGFR pairs.     

Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.     

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.     

Effects of diesel exhaust, heavy metals and pesticides on various organ systems: possible mechanisms and strategies for prevention and treatment

Environmental pollutants have a significant impact on the ecosystem and disrupt balance between environment, human and non-human components that result in deleterious effects to all forms of life. Identifying environmental factors for potential imbalance are extremely crucial for devising strategies for combating such toxic dysregulation. Automobile exhaust (in air), heavy metals (in food and water) and pesticides (in air, food, soil and water) are the most common environmental pollutants and their short and long-term exposures can cause hazardous effects in humans leading to systemic disorders involving lungs, kidney and immune systems. Mechanisms involved in genesis of such toxic effects have revealed complex, interactive pathways. Strategies for the protection of homeostasis and health, viz., general preventive measures, nutritional supplements and herbal agents have been described, to counter these pollutants induced damaging effects on various body systems.