Allostery and conformational free energy changes in human tryptophanyl‐tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of allostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side‐chains and then to compute the ligand‐induced population shift. Finally, we obtain the free‐energy landscape of the protein in equilibrium, characterizing the free‐energy minima accessed by the protein complexes. We have chosen human tryptophanyl‐tRNA synthetase (hTrpRS), a protein responsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis of novel 3-amino and 29-hydroxamic acid derivatives of glycyrrhetinic acid as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. So far, no selective 11β-HSD2 inhibitor has been developed and neither animal studies nor clinical trials have been reported based on 11β-HSD2 inhibition. Starting from the lead compound glycyrrhetinic acid, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Several hydroxamic acid derivatives showed high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1.     

Biodegradation of feather keratin with a PEGylated protease of Chryseobacterium gleum

Present study deals with the covalent modification of keratinolytic protease of Chryseobacterium gleum with higher enzyme activity, improved stability, non-immunogenicity and reusability. Protease of C. gleum showing feather degradation ability was modified by covalent attachment to polyethylene glycol. This modification culminated the change in electrophoretic mobility of protease in acrylamide gel. The modified enzyme showed 1.4 times more catalytic activity with better stability than native in aqueous system containing whole feathers as keratin. It showed improved pH, thermal, storage and solvent stability with a broadened range of pH (7–9) and temperature (25–50 °C) than native. The differentiation between modified and native enzyme was authenticated through UV–vis spectroscopy, SEM, XRD, FTIR and DSC. This modification of protease proved to be non-immunogenic in rats. The enzyme extracted after first run could be used for several cycles which clearly demonstrated its reusability in catalytic bioprocess of keratin degradation.     

Adaptation of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> to increased NaCl concentration due to dominant beneficial mutations

New beneficial mutations, combined with selection, were responsible for quick adaptation of Drosophila melanogaster to a novel environment. Using a highly inbred homozygous stock of D. melanogaster, we observed that in thirty generations the original stock had evolved to resist a previously toxic level of dietary salt (NaCl) and to produce a significantly higher number of progeny when reared in elevated salt concentrations. Survival in higher salt-stressed environments was due to new dominant genetic changes on the second and third chromosomes.     

Expression, purification, and characterisation of nesiritide using an E. coli expression system

Nesiritide, the recombinant human brain natriuretic peptide, is involved in the regulation of cardiovascular homeostasis and has been approved for treatment of patients with congestive heart failure. We prepared a synthetic cDNA construct of Nesiritide to generate a fusion protein with an affinity handle and 41 amino acid peptide of β-galactosidase. The fusion protein was expressed mainly in the inclusion bodies and accounted for approximately 20% of total cellular protein. After purification by Ni-IDA affinity chromatography and renaturation, the fusion protein was cleaved with purified recombinant enterokinase. Nesiritide was purified by pH precipitation/ion exchange chromatography followed by source phenyl chromatography to obtain protein with > 99% purity (determined by RPHPLC) and a mass of 3,464 Daltons. The potency (ED₅₀) of the purified protein was equivalent to that of Natrecor (Innovator formulation). Analytical methods were developed to identify oxidised, reduced and other related impurities. The expression strategy described in this work allows the convenient generation of high yield Nesiritide and enabled ease of purification.     

The bladder tumor suppressor protein TERE1 (UBIAD1) modulates cell cholesterol: implications for tumor progression

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression.     

Screening of Enterobacterial Contamination During Gelatin Production and its Effect on Pharmaceutical Grade Gelatin

Summary Gelatin, an animal protein derived from collagen has many industrial applications; mainly in food and pharmaceutical products. In this study, enterobacterial contamination of gelatin during different stages of its manufacturing was examined. Since gelatin is extracted in the form of liquor by hot water treatment of ossein and undergoes a complex series of processing stages before being finally blended and packaged off as dry gelatin product, contamination at any stage affects the quality of the final product. In total, 142 samples of gelatin were analysed for the presence of enteric bacteria, which were obtained from different stages of gelatin processing. The Enterobacteriaceae-positive samples were processed and these enterobacterial species were isolated and identified as Proteus mirabilis, Serratia marcescens, E. coli, Salmonella typhi, Klebsiella oxytoca, Klebsiella pneumoniae, Shigella flexneri and Serratia liquefaciens. These were tested for gelatinase activity. The eight species producing maximum gelatinolysis were selected and protease zymography was performed using 1% (w/v) gelatin as a substrate in 7.5% (w/v) acrylamide gel and their molecular weights were determined. The effect of these bacteria on the quality of gelatin was assessed chromatographically in terms of change in amino acid content of gelatin broth inoculated by these species, which showed a marked change with length of incubation. Since these enteric bacteria possess pathogenic properties, some of them are gelatinolytic and also have a significant effect on the quality and amino acid content of gelatin, they are of great concern both for the manufacturers as well as for the consumers.     

Microhyla laterite sp. nov., A New Species of Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from a Laterite Rock Formation in South West India

In recent times, several new species of amphibians have been described from India. Many of these discoveries are from biodiversity hotspots or from within protected areas. We undertook amphibian surveys in human dominated landscapes outside of protected areas in south western region of India between years 2013–2015. We encountered a new species of Microhyla which is described here as Microhyla laterite sp. nov. It was delimited using molecular, morphometric and bioacoustics comparisons. Microhyla laterite sp. nov. appears to be restricted to areas of the West coast of India dominated by laterite rock formations. The laterite rock formations date as far back as the Cretaceous-Tertiary boundary and are considered to be wastelands in-spite of their intriguing geological history. We identify knowledge gaps in our understanding of the genus Microhyla from the Indian subcontinent and suggest ways to bridge them.     

Effective suppression of HIV-1 by artificial bispecific miRNA targeting conserved sequences with tolerance for wobble base-pairing

The high genetic diversity and mutability of HIV pose a major problem for RNAi-mediated antiviral therapy. Simultaneous targeting of multiple highly conserved viral sequences has been suggested for durable cross-clade inhibition. Here we validate the approach of co-targeting two conserved sequences in the Tat and Vif genes. When coexpressed as artificial microRNA from a PolII driven miR-155-based vector, the sequences together mediated effective and sustained inhibition of HIV replication without virus breakout. To understand the nature of this efficient control, we analyzed genome sequences of 625 HIV-1 isolates in the Los Alamos Sequence database. Interestingly most natural variants were capable of wobble binding with the Tat/Vif siRNAs. Efficient silencing of reporter luciferase constructs bearing these variants residues verified that the Tat/Vif sequences together tolerated wobble binding and mediated functional RNAi. We propose the rationale of targeting highly conserved HIV sequences where wobble substitutions permit functional RNAi for global HIV repression.     

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.