Hydrophobicity Plots Research Articles

UPF1 promotes rapid degradation of m6A-containing RNAs

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

Hyper-Variable Spike Protein of Omicron Corona Virus and Its Differences with Alpha and Delta Variants: Prospects of RT-PCR and New Vaccine

NCBI SARS-CoV-2 Database was analyzed between November-December, 2021 to decipher the spread of Delta corona virus variants in the USA and compared with highly transmissible new omicron variant recently originated in South Africa. Presently, B.1.617.2 and AY.103 lineages Delta variants with spike protein L452R, T478K, P681R mutations and F157/R158 two amino acids deletions were predominant in the USA and superseded the deadly outbreaks of B.1.1.7 Alpha variant with deletions of H69, V70 and Y145 amino acids as well as N501Y, and D614G highly transmissible mutations. Interestingly, omicron variant has six H69, V70, V143, Y144, Y145, L212 immune-escape deletions as well as 29 mutations in the spike protein including most deadly N501Y (Y498 in omicron) and D614G (G611 in omicron). This indicated that omicron variant was originated by combination among B.1.1.7, AY.X and B.1.617.2 lineages. A unique three amino acids (EPE) insertion at 215 position of spike protein was detected to compensate six deletions suggesting further recombination events. Three Serine residues were mutated at amino acids 371 (S=L, L368 in omicron), 373 (S=P, P370 in Omicron), 375 (S=F, F372 in omicron) but compensated at 446 (G=S, S443 in omicron) and 496 (G=S, S493 in omicron) at the RBD domain of omicron virus. The three amino acids (ERS) deletion at position 30 in the N-protein acts as another signature of omicron virus. Omicron variant has less mutation in the 2/3 5’-end of the genome that codes for ORF1ab poly-protein but dominant P4715L mutation in the RNA-dependent RNA polymerase. However, overall amino acid composition, alipathic index, and instability index were found fairly constant although hydrophobic plot gave some difference between spike protein of Wuhan and omicron corona viruses. BLAST search detected 20nt and 19nt perfect match of hyper-variable 22957-22977nt region comprising 488-493 amino acids (NH2-PLRSYS-CO2H) of the spike protein of omicron virus with the ch-2 of Seladonia tumulorum or ch-16 of Steromphala cineraria respectively. A primer set designed from the RBD domain of spike gene did not detected the omicron genome by BLAST search but primers from the constant regions of the genome worked well. Such hyper-variation in the spike protein suggested that DNA vaccine or mRNA vaccine using spike gene of corona virus may not efficiently protect omicron virus infection and attenuated whole corona virus vaccine will be safer vaccine.

An integrative bioinformatics analysis of caprine GNAI3 gene

Goat is the most preferred domesticated animal in Indian subcontinent. However, the climatic change-induced heat stresscauses a formidable challenge for maintaining optimum productivity. G protein subunit alpha i3 (GNAI3) is one of the genes that may have significant role in heat tolerance mechanism in goats. The caprine GNAI3 gene was searched for homology analysis and its three dimensional protein structure was predicted followed by its validation through in silico approach. Nucleotide sequence-based phylogenetic tree analysis showed that the caprine GNAI3 gene has close evolutionary relationship with that of Ovis aries. Homology modelling of caprine GNAI3 protein was done in MODELLER 9.18 (P1), PHYRE2 (P2), GENO3D (P3) and SWISS MODEL (P4). The modelled structures were further validated after observing the Ramachandran and hydrophobicity plots. In the best of three dimensional protein structure (P4 as produced by SWISS MODEL), 330 (98.8%), three (0.9%) and one (0.3%) amino acid residues were found in favoured region, allowed region and outlier region, respectively. Degree of hydrophobicity of the generated protein structures revealed the presence of alternate hydrophobic and hydrophilic regions. The ligand receptor interaction site of the predicted 3D model was traced out using Discovery Studio 3.5. STRING database revealed protein interactions with Plcb1, Plcb2, Plcb3 and other proteins of G family such as Gnb1, Gnb2, Gnb3,Gnb4, Gng2, Gng4 and Gpsm1. KEGG pathway maps revealed interaction with eNOS, iNOS, VEGF and MAPK, which are reported to be transcribed in response to heat stress. Thus, caprine GNAI3 can be used as a possible biomarker for studying heattolerance mechanism in goats.

Peptide Combination Generator: a Tool for Generating Peptide Combinations.

Peptides are used as reagents both for basic research and diagnostic purposes. Therefore, there is a need for novel methods for the design of peptide molecules with a particular specific physiochemical profile. The properties of the peptides are governed by the nature of amino acids constituting the peptide. There is a lack of a web server or tools which could predict all the possible combinations of the peptides generated because of the combinations of amino acids based on the physiochemical properties. We have developed a peptide combination generator (PepCoGen), a web server for generating all the possible combinations of peptides by varying the amino acids having similar physiochemical properties at a particular position. It also predicts other properties of the peptides including molecular weight, charge, solubility, hydrophobic plot, and isoelectric point, and random three-dimensional models for each generated combination.

Biochemistry and computer generated graph comparison of the structural and nonstructural proteins of spanish-1918 Influenza, pandemic-2009, and bird flu viruses.

The potential emergence of deadly pandemic influenza viruses is unpredictable and most have emerged with no forewarning. The distinct epidemiological and pathological patterns of the Spanish (H1N1), pandemic-2009 (H1N1), and avian influenza (H5N1), known as bird flu, viruses may allow us to develop a 'template' for possible emergence of devastating pandemic strains. Here, we provide a detailed molecular dissection of the structural and nonstructural proteins of this triad of viruses. GenBank data for three representative strains were analyzed to determine the polymorphic amino acids, genetic distances, and isoelectric points, hydrophobicity plot, and protein modeling of various proteins. We propose that the most devastating pandemic strains may have full-length PB1-F2 protein with unique residues, highly cleavable HA, and a basic NS1. Any newly emerging strain should be compared with these three strains, so that resources can be directed appropriately.

Repeats in S1 Proteins: Flexibility and Tendency for Intrinsic Disorder.

An important feature of ribosomal S1 proteins is multiple copies of structural domains in bacteria, the number of which changes in a strictly limited range from one to six. For S1 proteins, little is known about the contribution of flexible regions to protein domain function. We exhaustively studied a tendency for intrinsic disorder and flexibility within and between structural domains for all available UniProt S1 sequences. Using charge–hydrophobicity plot cumulative distribution function (CH-CDF) analysis we classified 53% of S1 proteins as ordered proteins; the remaining proteins were related to molten globule state. S1 proteins are characterized by an equal ratio of regions connecting the secondary structure within and between structural domains, which indicates a similar organization of separate S1 domains and multi-domain S1 proteins. According to the FoldUnfold and IsUnstruct programs, in the multi-domain proteins, relatively short flexible or disordered regions are predominant. The lowest percentage of flexibility is in the central parts of multi-domain proteins. Our results suggest that the ratio of flexibility in the separate domains is related to their roles in the activity and functionality of S1: a more stable and compact central part in the multi-domain proteins is vital for RNA interaction, terminals domains are important for other functions.

In silico analysis of caprine superoxide dismutase 1 (SOD1) gene

Climatic change induced heat stress causes a formidable challenge for maintaining optimum productivity in goats. The SOD1 gene (superoxide dismutase 1) is one of the genes that regulates the heat tolerance capacity. This study aimed to in silico characterize this gene. Sequence based phylogenetic tree analysis showed the caprine SOD1 gene has close evolutional relationship with that of sheep. STRING database reveals its interaction with SOD2 (Superoxide dismutase [Mn], mitochondrial), ATOX1 (Copper transport protein), RAC1 (Ras-related C3 botulinum toxin substrate 1), GPX2 (Glutathione peroxidase 2), GPX4 (Glutathione peroxidase 4), GPX5, GPX6, CAT (Catalase). The KEGG pathway maps gave interaction with eNOS, iNOS, VEGF and MAPK in which gene transcription modulates response to heat stress. The three dimensional protein structure was predicted using Modeller, Phyre 2, and Swiss Model. Structure validation was done observing the Ramachandran Plot and Hydrophobicity Plot in Discovery Studio considering amino acid residues in favoured region.

Structural proteomics: Topology and relative accessibility of plant lipid droplet associated proteins

Lipid droplets are the major stock of lipids in oleaginous plant seeds. Despite their economic importance for oil production and biotechnological issues (biofuels, lubricants and plasticizers), numerous questions about their formation, structure and regulation are still unresolved. To determine water accessible domains of protein coating at lipid droplets surface, a structural proteomic approach has been performed. This technique relies on the millisecond timescale production of hydroxyl radicals by the radiolysis of water using Synchrotron X-ray white beam. Thanks to the evolution of mass spectrometry analysis techniques this approach allows the creation of a map of the solvent accessibility for proteins difficult to study by other means. Using these results, a S3 oleosin water accessibility map is proposed. This is the first time that such a map on an oleosin co-purified with plant lipid droplets and other associated protein is presented. Biological significanceLipid droplet associated proteins function is linked to stability, structure and probably formation and lipid mobilization of droplets. Structure of these proteins in their native environment, at the interface between bulk water and the lipidic core of these organelles is only based on hydrophobicity plot. Using hydroxyl radical footprinting and proteomics approaches we studied water accessibility of one major protein in these droplets: S3 oleosin of Arabidopsis thaliana seeds.

Insights into eukaryotic evolution from transmembrane domain lengths

Biological membranes, comprised of proteins anchored by their trans-membrane domains (TMDs) creating a semi-permeable phase with lipid constituents, serve as ‘checkposts’ for not only intracellular trafficking in eukaryotic cells but also for material transactions of all living cells with external environments. Hydropathy (or hydrophobicity) plots of ‘bitopic’ proteins (i.e. having single alpha-helical TMDs) are routinely utilized in biochemistry texts for predicting their TMDs. The number of amino acids (i.e. TMD length) embedded as alpha-helices may serve as indicators of thickness of biological membranes in which they reside under assumptions that are universally applied for fixing window sizes for identifying TMDs using hydropathy plots. In this work we explore variations in thickness of different eukaryotic biological membranes (reflected by TMD lengths of their resident proteins) over evolutionary time scales. Rigorous in silico analyses of over 23,000 non-redundant membrane proteins residing in different subcellular locations from over 200 genomes of fungi, plants, non-mammalian vertebrates and mammals, reveal that differences in plasma membrane and organellar TMD lengths have decreased over time (scales) of eukaryotic cellular evolution. While earlier work has indicated decreasing differences in TMD lengths with increasing ‘perceived’ organismal complexity, this work is the first report on TMD length variations as a function of evolutionary time of eukaryotic cellular systems. We report that differences in TMD lengths of bitopic proteins residing in plasma membranes and other intra-cellular locations have decreased with evolutionary time, suggesting better/more avenues of intracellular trafficking in the emergence of eukaryotic organisms.

Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga

Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, ε, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6–Asa6, Asa6–Asa8, Asa6–Asa9, Asa8–Asa8 and Asa8–Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.

ZIKA VIRUS SERENE PROTEASE COMPLEX (NS2B-NS3) INHIBITION BY 2-AMINO-5-{[(1Z)-AMINO({[(Z)-BENZOYL]IMINO})METHYL]AMINO}-N-(5-AMINO-7-{[CARBAMOYL(PHENYL)METHYL]AMINO}-6-OXOHEPTYL)PENTANAMIDE, IN SILICO STUDIES

Objective: The present in silico study is taken to report 2-amino-5-{[(1Z) -amino ({[(Z) -benzoyl] imino}) methyl] amino} -N-(5-amino-7-{[carbamoyl (phenyl) methyl] amino} -6-oxoheptyl) pentanamide as Zika virus (ZIKV) NS2B-NS3 protease inhibitor.Methods: In silico studies performed on online docking servers. NS2B-NS3 serine protease from ZIKV with PDB ID: 5GJ4 a hydrolase with total structure weight of 102878.54 is selected as the target. Docking server is used for carrying out docking calculations. Lamarckian genetic algorithm and the Solis and Wets local search methods are used for performing docking simulations. Free energy calculations, hydrogen bond (HB) formation, polar and hydrophobic interactions and HB plot are studied in this study.Results: Binding pocket is found on a serine protease NS2B chain A. Binding site predictions propose NKK as the suitable ligand for binding, which has structure closely related to the proposed ligand2-amino-5-{[(1Z) -amino ({[(Z) -benzoyl] imino}) methyl] amino} -N-(5-amino-7-{[carbamoyl (phenyl) methyl] amino} -6-oxoheptyl) pentanamide. Free energy of binding is - 4.08 kcal/Mol and inhibition constant (Ki) is very less 1.02 mm. The ligand binds to chain A of NS2B and chain B of NS3 serine protease. The legend is bound to serine protease complex through strong HB, formed between THR 60 (A) and N6 of ligand, GLU62 (A) and N8 of ligand, ARG 55 (A) and N3 of ligand and ASN108 (B) and N7 of ligand apart from polar and hydrophobic interactions.Conclusion: Docking studies performed establishes the proposed ligand2-amino-5-{[(1Z)-amino ({[(Z)-benzoyl] imino}) methyl] amino} -N-(5- amino-7-{[carbamoyl (phenyl) methyl] amino}-6-oxoheptyl) pentanamide as a molecule which can be used for the inhibition of ZIKV NS2B-NS3 serine protease.Keywords: Zika virus, NS2B-NS3 protease, Inhibition, In silico.

Diversity in VP3, NSP3, and NSP4 of rotavirus B detected from Japanese cattle

Bovine rotavirus B (RVB) is an etiological agent of diarrhea mostly in adult cattle. Currently, a few sequences of viral protein (VP)1, 2, 4, 6, and 7 and nonstructural protein (NSP)1, 2, and 5 of bovine RVB are available in the DDBJ/EMBL/GenBank databases, and none have been reported for VP3, NSP3, and NSP4. In order to fill this gap in the genetic characterization of bovine RVB strains, we used a metagenomics approach and sequenced and analyzed the complete coding sequences (CDS) of VP3, NSP3, and NSP4 genes, as well as the partial or complete CDS of other genes of RVBs detected from Japanese cattle. VP3, NSP3, and NSP4 of bovine RVBs shared low nucleotide sequence identities (63.3–64.9% for VP3, 65.9–68.2% for NSP3, and 52.6–56.2% for NSP4) with those of murine, human, and porcine RVBs, suggesting that bovine RVBs belong to a novel genotype. Furthermore, significantly low amino acid sequence identities were observed for NSP4 (36.1–39.3%) between bovine RVBs and the RVBs of other species. In contrast, hydrophobic plot analysis of NSP4 revealed profiles similar to those of RVBs of other species and rotavirus A (RVA) strains. Phylogenetic analyses of all gene segments revealed that bovine RVB strains formed a cluster that branched distantly from other RVBs. These results suggest that bovine RVBs have evolved independently from other RVBs but in a similar manner to other rotaviruses. These findings provide insights into the evolution and diversity of RVB strains.

A Python Based Hydrophilicity Plot to Assess the Exposed and Buried Regions of a Protein

Predicting the antigenic regions of a protein is of prime importance in assessing the states of a polypeptide chain as exposed or buried regions. This can be achieved by calculating and plotting the hydrophilicity of a protein using values of Hoop-Woods scale. Hence, in this paper, we report a hydrophilicity plot of Hoop-Woods scale amino acid sequence of a protein on its x-axis, and degree of hydrophobicity or hydrophilicity on its y-axis using python language as architecture by utilizing various functional attributes such as scipy, matplot and numpy modules. A case study has been reported using siallidase-4 enzyme. The program code can be available on request.

Polymorphic membrane protein I of Chlamydia trachomatis: prokaryotic expression, purification, antibody preparation and identification

Objective To clone and express the polymorphic membrane protein I (PmpI) gene of Chlamydia trachomatis (Ct) , and to assess the immunogenicity and biological characteristics of PmpI. Methods A bioinformatic software was used to analyze the sequence of the PmpI gene of Ct, and to predict B cell epitopes in PmpI. With Ct serovar D DNA as the template, PCR was performed to amplify the N-terminal region (from position 90 to 1464) of the PmpI gene, which was cloned into a prokaryotic expression vector pET28a to express the recombinant protein PmpI. A Ni-ion affinity chromatography column was used to purify the recombinant protein, which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies. Western blot analysis was conducted to evaluate the immunogenicity of this protein. Results A comprehensive analysis was carried out on the secondary structure, flexible regions, hydrophilicity plot, antigenic index and surface probability plot of the protein, which suggested that PmpI had 8 dominant B-cell epitopes. The product of PCR targeting the PmpI gene of Ct serovar D showed a total length of 1 375 bp. The recombinant prokaryotic expression vector pET28a-PmpI was successfully constructed. A recombinant protein with a relative molecular mass of approximately 50 000 was successfully expressed after isopropyl β-d-1-thiogalactopyranoside (IPTG) induction, and purified by affinity chromatography. Polyclonal antibodies against the recombinant protein were successfully prepared. Conclusion The N-PmpI protein of Ct serovar D is cloned and expressed successfully, laying a foundation for further studies on its biological functions. Key words: Chlamydia trachomatis; Bacterial outer membrane proteins; Cloning, molecular; Polymorphic membrane protein

The hydrophilicity vs. ion interaction selectivity plot revisited: The effect of mobile phase pH and buffer concentration on hydrophilic interaction liquid chromatography selectivity behavior

This work systematically investigates the selectivity changes on many HILIC phases from wwpH 3.7–6.8, at 5 and 25mM buffer concentrations. Hydrophilicity (kcytosine/kuracil) vs. ion interaction (kBTMA/kuracil) selectivity plots developed by Ibrahim et al. (J. Chromatogr. A 1260 (2012) 126–131) are used to investigate the effect of mobile phase changes on the selectivity of 18 HILIC columns from various classes. “Selectivity change plots” focus on the change in hydrophilicity and ion interaction that the columns exhibit upon changing mobile phase conditions. In general, the selectivity behavior of most HILIC columns is dominated by silanol activity. Minimal changes in selectivity are observed upon changing pH between wwpH 5 and 6.8. However, a reduction in ionic interaction is observed when the buffer concentration is increased at wwpH≥5.0 due to ionic shielding. Reduction of the wwpH to<5.0 results in decreasing cation exchange activity due to silanol protonation. Under all eluent conditions, the majority of phases show little change in their hydrophilicity.

Genetic characteristics of porcine epidemic diarrhea virus in Chinese mainland, revealing genetic markers of classical and variant virulent parental/attenuated strains

Since October 2010, porcine epidemic diarrhea (PED) caused by variant porcine epidemic diarrhea virus (PEDV) has led great economic losses to the global pig industry, especially in China. To study the genetic characteristics of PEDV strains in Chinese mainland, a total of 603 clinical samples from nine provinces/districts of Chinese mainland from January 2014 to December 2015 were collected for RT-PCR detection and 1-1323bp of S gene of 91 isolates and ORF3 gene of 46 isolates were sequenced. The results showed that the variant PEDV were the dominant pathogens of viral diarrhea diseases in these areas. Six novel variant PEDV strains (FJAX1, FJAX2, HeNPDS1, HeNPDS2, HeNPY3, and HeNPY4) with two amino acids (aa) deletion at the 56–57 aa of S protein were identified. A total of 405 Chinese PEDV strains were subjected to phylogenetic and phylogeographic analysis. The results revealed that the subgroup Va in variant PEDV group were the dominant subgroup and the spread trend of variant PEDV strains seemed to be from the southeast coastal districts to other coastal districts and interior districts. The N-terminal of S gene (1–750bp), to some extent, could represent S1 or full length S gene for phylogenetic, similarity, antigen index, hydrophilicity plot, and differentiation analyses. The 404–472bp of S gene contained the three genetic markers, i.e., “TAA” insertion at 404–405bp, “ACAGGT” deletion at 430–435bp, and “ATA” deletion at 455–457bp can be used to differentiate the classical and variant virulent parental/attenuated PEDV strains and help us to learn the infectious and genetic characteristics of PEDV strains more convenient and cheaper. This study has important implication for understanding the infectious, genetic, and evolutionary aspects of PEDV strains in Chinese mainland.

Immunoinformatics and Similarity Analysis of House Dust Mite Tropomyosin

Background: Dermatophagoides farinae and Dermatophagoides pteronyssinus are house dust mites (HDM) that they cause severe asthma and allergic symptoms. Tropomyosin protein plays an important role in mentioned immune and allergic reactions to HDMs. Here, tropomyosin protein from Dermatophagoides spp. was comprehensively screened in silico for its allergenicity, antigenicity and similarity/conservation. M aterials and Methods: The amino acid sequences of D . farinae tropomyosin, D. pteronyssinus and other mites were retrieved. We included alignments and evaluated conserved/ variable regions along sequences, constructed their phylogenetic tree and estimated overall mean distances. Then, followed by with prediction of linear B-cell epitope based on different approaches, and besides in-silico evaluation of IgE epitopes allergenicity (by SVMc, IgE epitope, ARPs BLAST, MAST and hybrid method). Finally, comparative analysis of results by different approaches was made. R es ults: Alignment results revealed near complete identity between D. farina and D. pteronyssinus members, and also there was close similarity among Dermatophagoides spp. Most of the variations among mites' tropomyosin were approximately located at amino acids 23 to 80, 108 to 120, 142 to 153 and 220 to 230. Topology of tree showed close relationships among mites in tropomyosin protein sequence, although their sequences in D. farina , D. pteronyssinus and Psoroptes ovis are more similar to each other and clustered. Dermanyssus gallinae (AC: Q2WBI0) has less relationship to other mites, being located in a separate branch. Hydrophilicity and flexibility plots revealed that many parts of this protein have potential to be hydrophilic and flexible. Surface accessibility represented 7 different epitopes. Beta-turns in this protein are with high probability in the middle part and its two terminals. Kolaskar and Tongaonkar method analysis represented 11 immunogenic epitopes between amino acids 7-16. From comparative analysis of predicted probable consensus epitope regions by machine learning approaches these epitopes were gained: AA 23-48 , AA 59-80 , AA 91-110 , AA 114-143 , AA 154-168 , AA 182-200 , AA 208-225 , and AA 254-272 . Prediction of allergenic proteins by AlgPred server showed 10 matches for IgE epitope, and prediction by hybrid approach showed that IgE epitope is undoubtedly the major allergen. Conclusion: Immunoinformatic approaches in allergenic protein analysis are now reliable tools for explanation/interpretation of clinically observed complexities. Results of present study, would help in HDM immunotherapy against several species of parasites as a wide range epitopic desensitization or prevention (vaccine) regime.

Identifying a C-terminal ATP binding sites-based novel Hsp90-Inhibitor in silico: A plausible therapeutic approach in Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is a progressive brain disorder, which gradually and irreversibly destroys the intellectual and cognitive abilities of the brain. Heat shock protein 90 (Hsp90α) is a molecular chaperone which was found to regulate the function of number of client proteins including tau that is involved in the cause of the AD. Inhibition of Hsp90α by C-Terminal domain (CTD) ATP binding-site blockage might be used as an effective treatment strategy against the disease via degradation of tau proteins that are involved in the progression of the disease. Till date, a variety of drugs have been identified as Hsp90α inhibitors, which include Novobiocin, Clorobiocin, Epigallocatechingallate (EGCG) and Derrubone. However, which drug among the four binds to the CTD ATP binding site strongly and what are the specific residue responsible for such binding, have not been reported so far. HypothesisWe hypothesize that binding site for ATP of Hsp90α CTD contains multiple ATP binding sites. We also hypothesize that a drug which can bind to the ATP binding site of CTD strongly can inhibit Hsp90α function which is in turn redirects towards the proteasomal degradation of diseased client protein like tau in AD. Such inhibition will find a novel therapeutic approach in the treatment of AD. Experimental designThe identification of ATP binding site of Hsp90α CTD was done using various software tools like Hex 6.3, CastP, protein Hydrophobicity plots, ATPint and LigPlot+ v.1.4.5. Docking experiments were conducted between Hsp90αCTD and its inhibitors at these ATP binding site using the Autodock 4.0. The docking energies were further compared to obtain the most effective Hsp90α inhibitor of CTD. ResultsFrom our experiments, Leucine (Leu) 665, Leu 666 and Leu 694 were predicted to be located in CTD ATP binding site. Furthermore, docking studies were performed of various Hsp90α inhibitors like Novobiocin, Clorobiocin, Epigallocatechingallate (EGCG) and Derrubone with the previously recognized ATP binding residues of CTD i.e. Leu 665, Leu 666 and Leu 694. The docking results of Derrubone showed the highest binding energy at all the three sites of ATP interaction. Additionally, Derrubone showed the best binding energy at Leu 666 (−7.53kcal/mol) compared to Leu 665 (−7.20kcal/mol) and Leu 694 (−6.67kcal/mol). ConclusionBased on our findings, we propose that the recognized sites i.e. Leu665, Leu 666 and Leu694 could possibly be the binding sites of Hsp90α CTD for ATP and the Hsp90 inhibitors. It was predicted that Derrubone could bind with CTD of Hsp90α strongly and resulted tau protein degradation which might be considered to be a therapeutic approach in AD.

Intestinal expression of peptide transporter 1 (PEPT1) at different life stages of Japanese eel, Anguilla japonica

The expression of peptide transporter 1 (PEPT1) was investigated at the different life stages of Japanese eel, Anguilla japonica. The cDNA encoding Japanese eel PEPT1 was cloned and sequenced. The hydrophilicity plot analysis of its deduced amino acid sequence showed high similarities with topological features of known PEPT1 molecules in other species. Tissue distribution analysis confirmed that PEPT1 mRNA was detected specifically in the anterior and posterior intestines of adult eel. In eel larvae at 13days post hatching (dph), PEPT1 mRNA expression was mainly detected in the intestinal tract regions. The trypsinogen mRNA was only detected in the gastric region including the pancreas. Intense immunoreaction for PEPT1 was observed in the apical membrane of the intestinal epithelial cells of both larval and adult eel. These results indicated that PEPT1 was an intestine-specific transporter, which was localized at the luminal side of the epithelial cells, suggesting that di/tri-peptide absorption via PEPT1 takes place in the eel intestine. According to the ontogenetic analyses by quantitative PCR, PEPT1 and trypsinogen mRNA expressions were simultaneously increased at 5-7 dph. It is thus assumed that nutrient absorption systems in the intestinal tracts of larvae become functional at this age.

Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.