Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT₁R and CysLT₂R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT₁R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD₄ and LTE₄-induced calcium influx through CysLT₁R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT₁R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.     

Identifying potential entry inhibitors for emerging Nipah virus by molecular docking and chemical-protein interaction network

Abstract

Nipah Virus (NiV) is a newly emergent paramyxovirus that has caused various outbreaks in Asian countries. Despite its acute pathogenicity and lack of approved therapeutics for human use, there is an urgent need to determine inhibitors against NiV. Hence, this work includes prospection of potential entry inhibitors by implementing an integrative structure- and network-based drug discovery approach. FDA-approved drugs were screened against attachment glycoprotein (NiV-G, PDB: 2VSM), one of the prime targets to inhibit viral entry, using a molecular docking approach that was benchmarked both on CCDC/ASTEX and known NIV-G inhibitor set. The predicted small molecules were prioritized on the basis of topological analysis of the chemical-protein interaction network, which was inferred by integrating the drug-target network, NiV-human interaction network, and human protein-protein interaction network. A total of 17 drugs were predicted to be NiV-G inhibitors using molecular docking studies that were further prioritized to 3 novel leads???Nilotinib, Deslanoside and Acetyldigitoxin???on the basis of topological analysis of inferred chemical-protein interaction network. While Deslanoside and Acetyldigitoxin belong to an already known class of anti-NiV inhibitors, Nilotinib belongs to Benzenoids chemical class that has not been reported hitherto for developing anti-NiV inhibitors. These identified drugs are expected to be successful in further experimental evaluation and therefore could be used for anti-Nipah drug discovery. Apart, we also obtained various insights into the underlying chemical-protein interaction network, based on which several important network nodes were predicted. The applicability of our proposed approach was also demonstrated by prospecting for anti-NiV phytochemicals on an independent dataset.

Communicated by Ramaswamy H. Sarma     

Common variant in FUT2 gene is associated with levels of vitamin B12 in Indian population

Vitamin B₁₂ is an essential micronutrient synthesized by microorganisms. Mammals including humans have evolved ways for transport and absorption of this vitamin. Deficiency of vitamin B₁₂ (either due to low intake or polymorphism in genes involved in absorption and intracellular transport of this vitamin) has been associated with various complex diseases. Genome-wide association studies have recently identified several common single nucleotide polymorphisms (SNPs) in fucosyl transferase 2 gene (FUT2) to be associated with levels of vitamin B₁₂—the strongest association was with a non-synonymous SNP rs602662 in this gene. In the present study, we attempted to replicate the association of this SNP (rs602662) in an Indian population since a significant proportion has been reported to have low levels of vitamin B₁₂ in this population. A total of 1146 individuals were genotyped for this SNP using a single base extension method and association with levels of vitamin B₁₂ was assessed in these individuals. Regression analysis was performed to analyze the association considering various confounding factors like for age, sex, diet, hypertension, diabetes mellitus and coronary artery disease status. We found that the SNP rs602662 was significantly associated with the levels of vitamin B₁₂ (p value < 0.0001). We also found that individuals adhering to a vegetarian diet with GG (homozygous major genotype) have significantly lower levels of vitamin B₁₂ in these individuals. Thus, our study reveals that vegetarian diet along with polymorphism in the FUT2 gene may contribute significantly to the high prevalence of vitamin B₁₂ deficiency in India.     

Tryptophan to Glycine mutation in the position 116 leads to protein aggregation and decreases the stability of the LITAF protein

Mutations in the gene-encoding vesicle lipopolysaccharide-induced tumor necrosis factor (LITAF) protein cause Charcot–Marie–Tooth type 1C (CMT1C) disease, a neurological disorder. The LITAF gene is mapped to chromosome number 16 and can be found at cytogenetic location 16p13 of the chromosome. CMT1C-linked small integral membrane protein of lysosome/late endosome mutants are loss-of-function mutants that act in a dominant negative manner to impair endosomal trafficking, leading to prolonged extracellular signal-regulated kinases 1/2 signaling downstream of ErbB activation. Mutation W116G in the LITAF decreases the stability of the protein and also interrupts the functioning of gene. We have analyzed the single nucleotide polymorphism (SNP) results of 28 nsSNPs obtained from dbSNP. We also carried out multiple molecular dynamics simulations of 200 ns and obtained results of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent-accessible surface area, H-bond, and principal component analysis to check and prove the stability of both the wild type and the mutant. The protein was then checked for its aggregation and the results showed loss of helix. The loss of helix leads to the instability of the protein.     

An Anionic Phospholipid Enables the Hydrophobic Surfactant Proteins to Alter Spontaneous Curvature

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.     

Agonist Leukadherin-1 Increases CD11b/CD18-Dependent Adhesion Via Membrane Tethers

Integrin CD11b/CD18 is a key adhesion receptor that mediates leukocyte migration and immune functions. Leukadherin-1 (LA1) is a small molecule agonist that enhances CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. Here, we used single-molecule force spectroscopy to investigate the biophysical mechanism by which LA1-activated CD11b/CD18 mediates leukocyte adhesion. Between the two distinct populations of CD11b/CD18:ICAM-1 complex that participate in cell adhesion, the cytoskeleton(CSK)-anchored elastic elements and the membrane tethers, we found that LA1 enhanced binding of CD11b/CD18 on K562 cells to ICAM-1 via the formation of long membrane tethers, whereas Mn²⁺ additionally increased ICAM-1 binding via CSK-anchored bonds. LA1 activated wild-type and LFA1^−/− neutrophils also showed longer detachment distances and time from ICAM-1-coated atomic force microscopy tips, but significantly lower detachment force, as compared to the Mn²⁺-activated cells, confirming that LA1 primarily increased membrane-tether bonds to enhance CD11b/CD18:ICAM-1 binding, whereas Mn²⁺ induced additional CSK-anchored bond formation. The results suggest that the two types of agonists differentially activate integrins and couple them to the cellular machinery, providing what we feel are new insights into signal mechanotransduction by such agents.     

Assessment of yield based selection under managed field stress condition for breeding for rice yield improvement under drought

The progress in development and dissemination of drought tolerant lines has been slow as compared to the increasing drought prevalence in the rice growing regions. Significant amount of work has been done in the past on drought resistance traits in rice crop, still the benefit of improved drought tolerant rice cultivars reaching the farmer’s field is not very high and ways to expedite the development of drought tolerant and productive rice cultivars needs to be addressed. In this article, an assessment of easily practicable approach of managed stress screening and prospect of direct selection for yield under drought stress is discussed. Also the large effect yield QTLs identified for grain yield under drought stress field conditions is being reviewed for successful introgression into elite genetic background for developing drought tolerant cultivars with improved yield for the drought prone target environment.     

Interaction between mosquito-larvicidal Lysinibacillus sphaericus binary toxin components: Analysis of complex formation

The two components (BinA and BinB) of Lysinibacillus sphaericus binary toxin together are highly toxic to Culex and Anopheles mosquito larvae, and have been employed world-wide to control mosquito borne diseases. Upon binding to the membrane receptor an oligomeric form (BinA2.BinB2) of the binary toxin is expected to play role in pore formation. It is not clear if these two proteins interact in solution as well, in the absence of receptor. The interactions between active forms of BinA and BinB polypeptides were probed in solution using size-exclusion chromatography, pull-down assay, surface plasmon resonance, circular dichroism, and by chemically crosslinking BinA and BinB components. We demonstrate that the two proteins interact weakly with first association and dissociation rate constants of 4.5 × 10³ M^?1 s^?1 and 0.8 s^?1, resulting in conformational change, most likely, in toxic BinA protein that could kinetically favor membrane translocation of the active oligomer. The weak interactions between the two toxin components could be stabilized by glutaraldehyde crosslinking. The cross-linked complex, interestingly, showed maximal Culex larvicidal activity (LC₅₀ value of 1.59 ng mL^?1) reported so far for combination of BinA/BinB components, and thus is an attractive option for development of new bio-pesticides for control of mosquito borne vector diseases.     

Identification of Penicillin-binding proteins employing support vector machines and random forest

Penicillin-Binding Proteins are peptidases that play an important role in cell-wall biogenesis in bacteria and thus maintaining bacterial infections. A wide class of β-lactam drugs are known to act on these proteins and inhibit bacterial infections by disrupting the cell-wall biogenesis pathway. Penicillin-Binding proteins have recently gained importance with the increase in the number of multi-drug resistant bacteria. In this work, we have collected a dataset of over 700 Penicillin-Binding and non-Penicillin Binding Proteins and extracted various sequence-related features. We then created models to classify the proteins into Penicillin-Binding and non-binding using supervised machine learning algorithms such as Support Vector Machines and Random Forest. We obtain a good classification performance for both the models using both the methods.