Swapping N-terminal regions among tick evasins reveals cooperative interactions influencing chemokine binding and selectivity

Class A tick evasins are natural chemokine-binding proteins that block the signaling of multiple chemokines from the CC subfamily through their cognate receptors, thus suppressing leukocyte recruitment and inflammation. Development of tick evasins as chemokine-targeted anti-inflammatory therapeutics requires an understanding of the factors controlling their chemokine recognition and selectivity. To investigate the role of the evasin N-terminal region for chemokine recognition, we prepared chimeric evasins by interchanging the N-terminal regions of four class A evasins, including a newly identified evasin, EVA-RPU02. We show through chemokine binding analysis of the parental and chimeric evasins that the N-terminal region is critical for chemokine binding affinity and selectivity. Notably, we found some chimeras were unable to bind certain cognate chemokine ligands of both parental evasins. Moreover, unlike any natural evasins characterized to date, some chimeras exhibited specific binding to a single chemokine. These results indicate that the evasin N terminus interacts cooperatively with the “body” of the evasin to enable optimum chemokine recognition. Furthermore, the altered chemokine selectivity of the chimeras validates the approach of engineering the N termini of evasins to yield unique chemokine recognition profiles.     

p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility

Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8–amino acid motif (533–541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41–amino acid region containing 14-3-3– and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER–mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER–mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER–mitochondria association and mitochondria motility.     

Identification and Characterization of a Novel Phosphodiesterase from the Metagenome of an Indian Coalbed

Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn²⁺ was required for the activity of PdeM, but addition of metals (Mn²⁺ or Fe³⁺) did not induce oligomerization. Further increase in concentration of Mn²⁺ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.     

Effect of Moringa oleifera leaves on hematological profile of fluorosis affected rats

Fluorosis is a metabolic disease that is endemic in nearly 25 countries with India being one of the most affected. It primarily affects the bone and the teeth. Moringa oleifera (MO) leaves are known to reduce the effect of fluorosis on various tissues. Therefore, it is of interest to document the effect of Moringa oleifera leaves on the hematological profile of fluorosis affected rats. Twenty four Sprague Dawley rats were housed two per cage in a room with 12 hours light and 12 hours dark cycle. The rats were allowed to adjust to the laboratory environment for about one to two weeks before the beginning of the study. This study reveals that MO leaves is effective in reducing the plasma fluoride content. It also helps in improving the Hb % and RBC count in fluorosis affected rats. Data shows that Moringa olifera leaves powder is effective in reducing the plasma fluoride content. It also helps in improving the Hemoglobin percentage & Red Blood Cell count in fluorosis affected rats.     

Computational analysis of the ESX-1 region of Mycobacterium tuberculosis: insights into the mechanism of type VII secretion system

Type VII secretion system (T7SS) is a recent discovery in bacterial secretion systems. First identified in Mycobacterium tuberculosis, this secretion system has later been reported in organisms belonging to the Actinomycetales order and even to distant phyla like Firmicutes. The genome of M. tuberculosis H37Rv contains five gene clusters that have evolved through gene duplication events and include components of the T7SS secretion machinery. These clusters are called ESAT-6 secretion system (ESX) 1 through 5. Out of these, ESX-1 has been the most widely studied region because of its pathological importance. In spite of this, the overall mechanism of protein translocation through ESX-1 secretion machinery is not clearly understood. Specifically, the structural components contributing to the translocation through the mycomembrane have not been characterized yet. In this study, we have carried out a comprehensive in silico analysis of the genes known to be involved in ESX-1 secretion pathway and identified putative proteins having high probability to be associated with this particular pathway. Our study includes analysis of phylogenetic profiles, identification of domains, transmembrane helices, 3D folds, signal peptides and prediction of protein-protein associations. Based on our analysis, we could assign probable novel functions to a few of the ESX-1 components. Additionally, we have identified a few proteins with probable role in the initial activation and formation of mycomembrane translocon of ESX-1 secretion machinery. We also propose a probable working model of T7SS involving ESX-1 secretion pathway.     

Constitutive expression and localization of cytochrome P-450 1A1 in rat and human brain: presence of a splice variant form in human brain

Cytochrome P-450 function as mono-oxygenases and metabolize xenobiotics. CYP1A1, a cytochrome P-450 enzyme, bioactivates polycyclic aromatic hydrocarbons to reactive metabolite(s) that bind to DNA and initiate carcinogenesis. Northern and immunoblot analyses revealed constitutive expression of Cyp1a1 and CYP1A1 in rat and human brain, respectively. CYP1A1 mRNA and protein were localized predominantly in neurons of cerebral cortex, Purkinje and granule cell layers of cerebellum and pyramidal neurons of CA1, CA2, and CA3 subfields of the hippocampus. RT-PCR analyses using RNA obtained from autopsy human brain samples demonstrated the presence of a splice variant having a deletion of 87 bp of exon 6. This splice variant was present in human brain, but not in the liver from the same individual, and was absent in rat brain and liver. Structural modeling indicated broadening of the substrate access channel in the brain variant. The study demonstrates the presence of a unique cytochrome P-450 enzyme in human brain that is generated by alternate splicing. The presence of distinct cytochrome P-450 enzymes in human brain that are different from well-characterized hepatic forms indicates that metabolism of xenobiotics including drugs could occur in brain by pathways different from those known to occur in liver.     

Characterization of neutralizing profiles in HIV-1 infected patients from whom the HJ16, HGN194 and HK20 mAbs were obtained

Several new human monoclonal antibodies (mAbs) with a neutralizing potential across different subtypes have recently been described. Three mAbs, HJ16, HGN194 and HK20, were obtained from patients within the HIV-1 cohort of the Institute of Tropical Medicine (ITM). Our aim was to generate immunization antibodies equivalent to those seen in plasma. Here, we describe the selection and characterization of patient plasma and their mAbs, using a range of neutralization assays, including several peripheral blood mononuclear cell (PBMC) based assays and replicating primary viruses as well as cell line based assays and pseudoviruses (PV). The principal criterion for selection of patient plasma was the activity in an 'extended incubation phase' PBMC assay. Neutralizing Abs, derived from their memory B cells, were then selected by ELISA with envelope proteins as solid phase. MAbs were subsequently tested in a high-throughput HOS-PV assay to assess functional neutralization. The present study indicates that the strong profiles in the patients' plasma were not solely due to antibodies represented by the newly isolated mAbs. Although results from the various assays were divergent, they by and large indicate that neutralizing Abs to other epitopes of the HIV-1 envelope are present in the plasma and synergy between Abs may be important. Thus, the spectrum of the obtained mAbs does not cover the range of cross-reactivity seen in plasma in these carefully selected patients irrespective of which neutralization assay is used. Nevertheless, these mAbs are relevant for immunogen discovery because they bind to the recombinant glycoproteins to which the immune response needs to be targeted in vivo. Our observations illustrate the remaining challenges required for successful immunogen design and development.     

Use of a zinc fluorophore to measure labile pools of zinc in body fluids and cell-conditioned media

Here we describe a rapid and sensitive zinquin-based fluorometric assay that enables one to monitor levels of labile Zn(II) in body fluids, buffers, and cell-conditioned culture media as well as changes in these pools in disease. Labile pools of Zn(II) are free or loosely bound pools and more tightly bound but zinquin-accessible pools in contrast to the fixed pools of Zn(II) within metalloproteins. In human plasma, mean labile Zn(II) was 8.1 microM (SEM 0.53; n = 81) and constituted about 70% of the total plasma Zn(II) and >90% of human plasma albumin Zn(II). Plasma labile Zn(II) was significantly depleted after 7 days of Zn(II) deprivation in mice, despite only small changes in body weight. Labile Zn(II) concentrations were also measured in the induced sputum plugs, saliva, and urine of normal adults and were 1.30 microM (SEM 0.27; n = 73), 0.11 microM (SEM 0.11; n = 6), and 0.23 microM (SEM 0.08; n = 8), respectively. Urinary labile Zn(II) concentration was significantly increased in some patients with type II diabetes mellitus (overall mean was 0.90 microM, SEM 0.30; n = 12). The technique may be particularly useful in assessing extracellular Zn(II) levels in diseases associated with altered Zn(II) homeostasis, identifying those subjects most in need of Zn(II) supplementation, and defining the optimum concentrations of available Zn(II) in buffers and culture media.     

Pairwise alignment of protein interaction networks.

With an ever-increasing amount of available data on protein-protein interaction (PPI) networks and research revealing that these networks evolve at a modular level, discovery of conserved patterns in these networks becomes an important problem. Although available data on protein-protein interactions is currently limited, recently developed algorithms have been shown to convey novel biological insights through employment of elegant mathematical models. The main challenge in aligning PPI networks is to define a graph theoretical measure of similarity between graph structures that captures underlying biological phenomena accurately. In this respect, modeling of conservation and divergence of interactions, as well as the interpretation of resulting alignments, are important design parameters. In this paper, we develop a framework for comprehensive alignment of PPI networks, which is inspired by duplication/divergence models that focus on understanding the evolution of protein interactions. We propose a mathematical model that extends the concepts of match, mismatch, and gap in sequence alignment to that of match, mismatch, and duplication in network alignment and evaluates similarity between graph structures through a scoring function that accounts for evolutionary events. By relying on evolutionary models, the proposed framework facilitates interpretation of resulting alignments in terms of not only conservation but also divergence of modularity in PPI networks. Furthermore, as in the case of sequence alignment, our model allows flexibility in adjusting parameters to quantify underlying evolutionary relationships. Based on the proposed model, we formulate PPI network alignment as an optimization problem and present fast algorithms to solve this problem. Detailed experimental results from an implementation of the proposed framework show that our algorithm is able to discover conserved interaction patterns very effectively, in terms of both accuracies and computational cost.