Kinetics of Ligand-Receptor Interaction Reveals an Induced-Fit Mode of Binding in a Cyclic Nucleotide-Activated Protein

Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼10⁷ M⁻¹ s⁻¹ are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.     

MiADMSA Protects Arsenic-Induced Oxidative Stress in Human Keratinocyte ‘HaCaT’ Cells

Arsenic toxicity may lead to skin manifestations and arsenic accumulation in keratinised tissue. Thus human keratinocytes has been extensively used to study dermal effects of arsenic exposure. The present study was aimed to investigate time and dose-dependent effects of arsenic using HaCaT cell line. Another major focus of the study was to evaluate if treatment with monoisoamyl dimercaptosuccinic acid (MiADMSA) offers protection against arsenic-induced oxidative stress and apoptotic cell death using HaCaT cells. HaCaT cell lines were incubated to three different concentrations of arsenic (10, 30 and 50 μM) for 24 h to identify the toxic dose by measuring oxidative stress variables. Later, MiADMSA pre-incubation for an hour preceded arsenic exposure (30 μM). We evaluated cell morphology, lactate dehydrogenase, glutathione linked enzyme and antioxidant enzyme activities to measure oxidative stress status, while MTT assay and caspase 9 and 3 levels were determined for cell viability and apoptotic status. The present study suggests arsenic-induced toxicity in a concentration-dependant manner. Arsenic also caused a significant increase in lactate dehydrogenase accompanied by an elevated antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and caspase activity). Interestingly, pre-treatment of cell with MiADMSA elicited significant protection against arsenic-induced oxidative stress and apoptotic cell death. The present findings are of clinical relevance and suggest MiADMSA to be a promising candidate in protecting skin against arsenic-induced toxic effects, which need further exploration using in vivo experimental models.     

Oxime-dipeptides as anticholinesterase,reactivator of phosphonylated-serine of AChE catalytic triad: probing the mechanistic insight by MM-GBSA,dynamics simulations and DFT analysis

Neuropathological cascades leading to reduced cholinergic transmission in Alzheimer’s disease led to development of AChE-inhibitors. Although lethal dose of some inhibitors cause interruption with AChE mediated mechanism but reversible AChE inhibitors can assist in protection from inhibition of AChE and hence in an aim to probe potential molecules as anticholinesterase and as reactivators, computationally structure-based approach has been exploited in this work for designing new 2-amino-3-pyridoixime-dipeptides conjugates. We have combined MD simulations with flexible ligand docking approach to determine binding specificity of 2-amino-3-pyridoixime dipeptides towards AChE (PDB 2WHP). PAS residues are found to be responsible for oxime-dipeptides binding along with π–π interactions with Trp86 and Tyr286, hydrogen bonding with side chains of Asp74 and Tyr341 (Gscore –10.801 and MM-GBSA free energy –34.89?kcal/mol). The docking results depicted complementary multivalent interactions along with good binding affinity as predicted from MM-GBSA analysis. The 2-amino-3-pyridoxime-(Arg-Asn) AChE systems subjected to MD simulations under explicit solvent systems with NPT and NVT ensemble. MD simulations uncovered dynamic behavior of 2-amino-3-pyridoxime-(Arg-Asn) and exposed its mobile nature and competence to form strong long range-order contacts towards active site residues to approach inhibited serine residue and facilitated via large contribution from hydrogen bonding and water bridges along with slow and large movements of adjacent important residues. In an effort to evaluate the complete potential surface profile, 2-amino-3-pyridoxime induced reactivation pathway of sarin–serine adduct has been investigated by the DFT approach at the vacuum MO6/6–311G (d, p) level along with the Poisson-Boltzmann solvation model and found to be of relatively low energy barrier. The pKa evaluation has revealed the major deprotonated 2-amino-3-pyridoixime species having pKa of 6.47 and hence making 2-amino-3-pyridoxime-(Arg-Asn) potential anticholinesterase and reactivator for AChE under the physiological pH.     

Full-length human glutaminase in complex with an allosteric inhibitor

Glutaminase (GLS1/2) catalyzes the conversion of L-glutamine to L-glutamate and ammonia. The level of a splice variant of GLS1 (GAC) is elevated in certain cancers, and GAC is specifically inhibited by bis-2-(5-phenylacetimido-1,2,4,thiadiazol-2-yl)ethyl sulfide (BPTES). We report here the first full-length crystal structure of GAC in the presence and absence of BPTES molecules. Two BPTES molecules bind at an interface region of the GAC tetramer in a manner that appears to lock the GAC tetramer into a nonproductive conformation. The importance of these loops with regard to overall enzymatic activity of the tetramer was revealed by a series of GAC point mutants designed to create a BPTES resistant GAC.     

Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton

Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.     

What Are the Reasons for Poor Uptake of HIV Testing among Patients with TB in an Eastern India District?

Background

National policy in India recommends HIV testing of all patients with TB. In West Bengal state, only 28% of patients with TB were tested for HIV between April-June, 2010. We conducted a cross-sectional survey to understand patient, provider and health system related factors associated with low uptake of HIV testing among patients with TB.

Methods

We reviewed TB and HIV program records to assess the HIV testing status of patients registered for anti-TB treatment from July-September 2010 in South-24-Parganas district, West Bengal, assessed availability of HIV testing kits and interviewed a random sample of patients with TB and providers.

Results

Among 1633 patients with TB with unknown HIV status at the time of diagnosis, 435 (26%) were tested for HIV within the intensive phase of TB treatment. Patients diagnosed with and treated for TB at facilities with co-located HIV testing services were more likely to get tested for HIV than at facilities without [RR = 1.27, (95% CI 1.20–3.35)]. Among 169 patients interviewed, 67 reported they were referred for HIV testing, among whom 47 were tested. During interviews, providers attributed the low proportion of patients with TB being referred and tested for HIV to inadequate knowledge among providers about the national policy, belief that patients will not test for HIV even if they are referred, shortage of HIV testing kits, and inadequate supervision by both programs.

Discussion

In West Bengal, poor uptake of HIV testing among patients with TB was associated with absence of HIV testing services at sites providing TB care services and to poor referral practices among providers. Comprehensive strategies to change providers’ beliefs and practices, decentralization of HIV testing to all TB care centers, and improved HIV test kit supply chain management may increase the proportion of patients with TB who are tested for HIV.     

Non-Alcoholic Fatty Liver Disease Is Closely Associated with Sub-Clinical Inflammation: A Case-Control Study on Asian Indians in North India

Objectives

Association between sub-clinical inflammation and non-alcoholic fatty liver disease (NAFLD) has not been studied in Asian Indians. In this case-control study, we aimed to analyse association of NAFLD with the sub-clinical inflammation and metabolic profile in Asian Indians in north India.

Methods

Ultrasound diagnosed 120 cases of NAFLD were compared to 152 healthy controls without NAFLD. Anthropometric profile [body mass index (BMI), waist circumference (WC), hip circumference (HC)], high-sensitivity C-reactive protein (hs-CRP), metabolic profile [fasting blood glucose (FBG), lipid profile] and hepatic function tests [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were recorded.

Results

Metabolic parameters [FBG, total cholesterol (TC), serum triglycerides (TG),low-density lipoprotein (LDL-c)], hs-CRP and prevalence of the metabolic syndrome were higher in cases as compared to controls (p-value<0.05 for all). The median (range) of hs-CRP (mg/L) for cases [2.6(0.2–13.4)] were significantly higher than in controls [1.4(0.03–11.4), p = 0.01]. Similarly, higher values of hs-CRP were obtained when subgroups of cases with obesity, abdominal obesity and the metabolic syndrome were compared to controls [2.75 (0.03–14.3) vs. 1.52 (0.04–14.3), p = 0.0010; 2.8 (0.03–14.3) vs. 1.5 (0.06–14.3), p = 0.0014 and 2.7 (0.5–14.3) vs. 1.6 (0.06–8.5), p = 0.0013, respectively. On multivariate logistic regression analysis BMI (p = 0.001), WC (p = 0.001), FBG (p = 0.002), TC (p = 0.008), TG (p = 0.002), blood pressure (p = 0.005), metabolic syndrome (p = 0.001) and hs-CRP (p = 0.003) were significantly and independently associated with NAFLD. After adjusting for significant variables, the association between high hs-CRP and NAFLD remained large and statistically significant [adjusted OR = 1.17, 95% confidence interval (CI) = 1.05–1.29]. An increase in 1 mg/dl of hs-CRP level calculated to increase the risk of developing NAFLD by 1.7 times as compared to controls after adjusting for significant variables associated with NAFLD.

Conclusions

In this cohort of Asian Indians in North India, presence of NAFLD showed independent relationships with sub-clinical inflammation.     

Fructose-induced modifications of myoglobin: Change of structure from met (Fe3+) to oxy (Fe2+) form

We studied structural modifications of metmyoglobin (Mb) after short-term (6 days) and long-term (30 days) glycation by fructose (fructation). Fructation caused gradual changes in the structure of the protein with respect to increased absorbance at 280 nm, enhanced fluorescence emission (with excitation at 285 nm), increased surface accessible tryptophan residues and reduced α-helix content and change in tertiary structure. However, long-term fructation changed Mb to oxymyoglobin (MbO2), as demonstrated by different spectroscopic (absorption, fluorescence, circular dichroic and electron paramagnetic resonance) studies and trifluoperazine-induced oxygen release experiment. Fructation appeared to modify Arg139 to arg-pyrimidine, which exhibited antioxidative activity and might be involved in the conversion of met (Fe3+) to oxy (Fe2+) form of myoglobin.