Molecular modelling, docking and interaction studies of human-plasmogen and salmonella enolase with enolase inhibitors

Salmonella enteric serovar Typhi Ty2 is a human specific pathogen and an etiological agent for typhoid fever. Most of Salmonella serotypes produce glycogen which has a comparatively minor role in virulence and colonization, but has a more significant role in survival. Enzymes present in glycolytic pathway of bacteria help bacteria to survive by activating other factors inside host. Numerous pathogenic bacteria species intervene with the plasminogen system, and this plasminogen-enolase association may play a critical role in the virulence of S. Typhi by causing direct damage to the host cell extracellular matrix, possibly by enzymic degradation of extracellular matrix proteins or other protein constituents. In this study, molecular modelling of enolase of Salmonella has been accomplished in silico by comparative modelling; we have then analyzed Human alpha enolase which is a homodimer and serves on epithelial cells with our model. Both Structures were docked by D-tartronate semialdehyde phosphate (TSP) and 3-aminoenolpyruvate phosphate (AEP) enolase inhibitors. Our study shows that salmonella enolase and human enolase have different active sites in their structure. This will help in development of new ligands, more suitable for inhibiting bacterial survival inside host as vaccines for typhoid fever are not fully protective. The study also confirmed that enolase Salmonella and Human Plasminogen suggested direct physical interaction between both of them as the activation loop of plasminogen residues showed conformational changes similar to the tissue type plasminogen activator. Various computational biology tools were used for our present study such as Modeller, Molegro Virtual Docker, Grommacs.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane

Key message

An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant.

Abstract

Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA^® and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.     

Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.     

A Monte Carlo study of the self-assembly of bacteriorhodopsin

Bacteriorhodopsin (BR) and specific lipid molecules self-assemble into a quasi two-dimensional lattice structure known as the purple membrane (PM). In the PM, BR molecules exist in a trimeric form with lipid molecules present in the space enclosed by each trimeric unit and in the inter-trimer space. These trimeric units, which have a roughly circular cross-section, are arranged in hexagonal patterns with long-ranged crystalline order. In this work, we investigate the self-assembly of BR in the PM via Monte Carlo simulations of a two-dimensional model of the membrane and proteins. The protein molecules are modeled as 120 degrees sectors of a circle and the lipid molecules enter into the model through effective protein-protein interactions. The sectors cannot overlap with each other, and in addition to this excluded volume interaction there are site-site attractive interactions between specific points of the proteins to mimic interactions between helices on the proteins and lipid-induced interactions. At low values of the attractive well depth, the proteins are found in the monomeric form at all concentrations. At moderate and high values of the attractive well depth, trimers are formed as the concentration increases, and with a further increase in concentration the trimers organize into a hexagonal lattice. The interactions between the proteins and those induced by the intra-trimer lipids play an equally important role in the formation of trimers and the lattice. The lipids in the inter-trimer space cause the trimers to orient in a specific direction in the hexagonal crystal lattice.     

Coemergence of the Amphipathic Helix on Ameloblastin With Mammalian Prismatic Enamel

To investigate correlation between the ameloblastin (Ambn) amino acid sequence and the emergence of prismatic enamel, a notable event in the evolution of ectodermal hard tissues, we analyzed Ambn sequences of 53 species for which enamel microstructures have been previously reported. We found that a potential amphipathic helix (AH) within the sequence encoded by Exon 5 of Ambn appeared in species with prismatic enamel, with a few exceptions. We studied this correlation by investigating synthetic peptides from different species. A blue shift in fluorescence spectroscopy suggested that the peptides derived from mammalian Ambn interacted with liposomes. A downward shift at 222 nm in circular dichroism spectroscopy of the peptides in the presence of liposomes suggested that the peptides of mammals with prismatic enamel underwent a transition from disordered to helical structure. The peptides of species without prismatic enamel did not show similar secondary structural changes in the presence of liposomes. Peptides of mammals with prismatic enamel caused liposome leakage and inhibited LS8 and ALC cell spreading regulated by full-length Ambn. RT-PCR showed that AH is involved in Ambn’s regulation of cell polarization genes: Vangl2, Vangl1, Prickle1, ROCK1, ROCK2, and Par3. Our comprehensive sequence analysis clearly demonstrates that AH motif is closely related to the emergence of enamel prismatic structure, providing insight into the evolution of complex enamel microstructure. We speculate that the AH motif evolved in mammals to interact with cell membrane, triggering signaling pathways required for specific changes in cell morphology associated with the formation of enamel prismatic structure.     

Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two B^og (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening.     

Genetic regulation of life cycle transitions in the brown alga Ectocarpus

The life cycle of an organism is one of its most elemental features, underpinning a broad range of phenomena including developmental processes, reproductive fitness, mode of dispersal and adaptation to the local environment. Life cycle modification may have played an important role during the evolution of several eukaryotic groups, including the terrestrial plants. Brown algae are potentially interesting models to study life cycle evolution because this group exhibits a broad range of different life cycles. Currently, life cycle studies are focused on the emerging brown algal model Ectocarpus. Two life cycle mutants have been described in this species, both of which cause the sporophyte generation to exhibit gametophyte characteristics. The ouroboros mutation is particularly interesting because it induces complete conversion of the sporophyte generation into a functional, gamete-producing gametophyte, a class of mutation that has not been described so far in other systems. Analysis of Ectocarpus life cycle mutants is providing insights into several life-cycle-related processes including parthenogenesis, symmetric/asymmetric initial cell divisions and sex determination.     

Targeted expression of L-myo- inositol 1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka confers multiple stress tolerance in transgenic crop plants

Improving crop tolerance to osmotic stresses is a longstanding goal of agricultural biotechnology. In the present work the PcINO1 gene coding for a salt-tolerant L-myo-inositol-1-phosphate synthase (MIPS) from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice was introgressed into cultivated mustard, Brassica juncea var B85. The transgenic plants demonstrate increased tolerance to salinity and oxidative stress with elevated level of inositol in both roots and shoots. The yield and crop quality of transgenic Brassica plants remain uncompromised and the plants were able to stably grow, set seeds and germinate in saline environments. When targeted to seeds of Nicotiana, PcINO1 was able to improve the seed survival rate under salinity and dehydration. Inositol and its derivatives regulate stress responses in various ways, serving as compatible solutes or signaling molecules. It is implicated that engineering inositol metabolism may affect the plant metabolic network leading to a stress tolerant phenotype as enumerated here in transgenic crop plants. How inositol itself or its derivatives affect the overall metabolic pathways leading to a stress-tolerant phenotype remains an intriguing question for future investigations.