PROCHECK Programs Research Articles

Three-Dimensional Structure of Human Epididymis Protein 4 (HE4): A Protein Modelling of an Ovarian Cancer Biomarker Through In Silico Approach

The Human Epididymis Protein 4 (HE4) biomarker has been extensively investigated for its potential in diagnosing ovarian cancer (OC). For the application of diagnostic techniques and drug delivery, it is crucial to understand the protein tertiary structure. However, the Protein Data Bank (PDB) does not currently contain the three-dimensional (3D) structure of HE4. Therefore, an in silico analysis was conducted to model the HE4 protein using AlphaFold, I-TASSER, and Robetta servers, with the sequence retrieved from UniProt (ID: Q14508). These three servers employed deep learning algorithms, threading templates, and de novo methods, respectively. Subsequently, Molecular Dynamics (MD) simulation using the GROMACS software package improved each 3D structure model, resulting in optimized and refined structures: RF1, RF2, and RF3. PROCHECK and ERRAT programs were employed to assess the structure quality. The Ramachandran plots from PROCHECK indicated that 100% of residues were within the allowed regions for all servers except for I-TASSER. For the refined structures, RF1 and RF3, all residues were concentrated within the allowed regions. According to the ERRAT program, the RF1 model exhibited the highest overall quality factor of 97.701, followed by RF3 and AlphaFold models with scores of 94.643 and 93.750, respectively. After these validations, RF1 emerged as the most accurately predicted 3D structure of HE4 and has one tunnel identified by CAVER 3.0 tool that facilitates the transportation of small particles to the active site, supported by FTsite and PrankWeb binding site predictions. This model holds potential for various computational studies, including the development of OC diagnostic kits. It will enhance our comprehension of the interactions between the protein and other biomolecules.

Molecular interactions of paraben family of pollutants with embryonic neuronal proteins of Danio rerio: A step ahead in computational toxicity towards adverse outcome pathway

The paraben family of endocrine disruptors exhibit persistent behaviours in aquatic matrices, having bio-accumulative effects and necessitating toxicity analysis and safe use, as well as prevention of food web penetration. In this study, the toxicity effects of 9 different parabens (Methyl, Ethyl, Propyl, Butyl, Heptyl, Isopropyl, Isobutyl, benzyl parabens and p-hydroxybenzoic acid) were studied against 17 neuronal proteins (Neurog1, Ascl1a, DLA, Syn2a, Ntn1a, Pitx2, and SoxB1, Her/Hes, Zic family) expressed during the early embryonic developmental stage of Danio rerio. The neuronal genes were selected as a biomarker to study the inhibitory effects on the cascade of genes expressed in the early developmental stage. The study uses trRossetta software to predict protein structures of neuronal genes, followed by structural refinement, energy minimisation, and active site prediction, evaluated using energy value, RC plot and ERRAT scores of PROCHECK and ERRAT programs. Compared to raw structures, highly confident predicted structures and quality scores were observed for refined protein with few exceptions. Based on the polarity and charge of the aminoacids, the probable pockets were identified using active site prediction, which were then used for molecular docking analysis. Further, the ADMET analysis, ligand likeliness and toxicological test revealed the paraben family of compounds as one of the most susceptible toxic and mutagenic compounds. The molecular docking results showed an interesting pattern of increasing binding affinity with increase in the carbon chains of paraben molecules. Benzyl Paraben showed higher binding affinities across all 17 neuronal proteins. Finally, gene co-occurrence/co-expression and protein-protein interaction studies using the STRING database depict that all proteins are functionally related and play essential roles in standard biological processes or pathways, conserved and expressed in diverse organisms. The interaction between paraben compounds and neuronal genes indicates high risks of inhibiting reactions in embryonic stages, emphasising the need for effective treatment measures and strict regulations.

Characterisation of the Putative Antigenic Genes of the Outer Membrane Proteins of Pasteurella multocida B:2 Strain PMTB2.1 through in silico Analysis

Outer membrane proteins (OMPs), usually found in Gram-negative bacteria, have long been shown to elicit immune responses in infected hosts. This tendency of OMPs to generate immune reactions makes them ideal candidates for vaccine development against pathogenic bacteria. Pasteurella multocida is a Gram-negative pathogen responsible for the economically significant veterinary disease, hemorrhagic septicemia (HS). HS is an endemic and highly fatal disease affecting buffaloes and cattle. In Malaysia, outbreaks of this disease cost about half a million USD each year. Thus, despite current treatment and prevention measures, HS is a prevalent issue that needs to be overcome. Pasteurella multocida subsp. multocida PMTB2.1, a Malaysian strain of the pathogen, has recently had its entire genome sequenced after being isolated from HS outbreaks in the region. Antigenic OMPs from this strain have since been identified and published for further characterisation. LptD, Wza, and TbpA are integral membrane proteins, while Pal is a peripheral membrane protein that has not been characterised in-depth. This study, therefore, aims to analyse these OMPs through in silico methods. First, protein homology modelling was performed using SWISS-MODEL, whereafter, the structures generated were validated using the SWISS-MODEL structure assessment page, PROCHECK, ERRAT, and PROSA programs. The Pal, Wza, and TbpA structures were good models, while the LptD structure was found to be a near-good model based on the validation performed. Analyses using BCPREDS, NetMHCpan4.1, and NetBoLAIIpan1.0 revealed that these four OMPs could potentially elicit humoral and cellular immune responses.

In silico detection and characterization of novel virulence proteins of the emerging poultry pathogen Gallibacterium anatis.

The pathogen Gallibacterium anatis has caused heavy economic losses for commercial poultry farms around the world. However, despite its importance, the functions of its hypothetical proteins (HPs) have been poorly characterized. The present study analyzed the functions and structures of HPs obtained from Gallibacterium anatis (NCTC11413) using various bioinformatics tools. Initially, all the functions of HPs were predicted using the VICMpred tool, and the physicochemical properties of the identified virulence proteins were then analyzed using Expasy's ProtParam server. A virulence protein (WP_013745346.1) that can act as a potential drug target was further analyzed for its secondary structure, followed by homology modeling and three-dimensional (3D) structure determination using the Swiss-Model and Phyre2 servers. The quality assessment and validation of the 3D model were conducted using ERRAT, Verify3D, and PROCHECK programs. The functional and phylogenetic analysis was conducted using ProFunc, STRING, KEGG servers, and MEGA software. The bioinformatics analysis revealed 201 HPs related to cellular processes (n = 119), metabolism (n = 61), virulence (n = 11), and information/storage molecules (n = 10). Among the virulence proteins, three were detected as drug targets and six as vaccine targets. The characterized virulence protein WP_013745346.1 is proven to be stable, a drug target, and an enzyme related to the citrate cycle in the present pathogen. This enzyme was also found to facilitate other metabolic pathways, the biosynthesis of secondary metabolites, and the biosynthesis of amino acids.

Three Dimension Structure Modeling of The Superoxide Dismutase (SOD) of Rice (Oryza sativa) Using Fold Recognition Method Using Phyre2 Web Server

Determining the 3D structure of proteins using laboratory instrumentation is time-consuming and expensive. The in silico method can be used as an alternative to predict the 3D structure of proteins, such as the fold recognition method. This study aims to create a 3D structural model of rice's (Oryza sativa) protein superoxide dismutase (SOD). The 3D structure modeling of the protein was carried out with the Phyre2 web server. The protein sequence was obtained from the UniProt KB database with the code A0A6F8FUX1. The results showed that the suitable template used to build the model was the template with the code c1unfX. The c1unfX template has a coverage value of 80%, 100% confidence, and i.d. of 51%. Validation of the model with the PROCHECK program showed that the most favored area on the Ramachandran Plot was 87.8%, and the disallowed area was 1.1%. The disallowed area, which is still below 15%, indicates that the three-dimensional structure model of the SOD protein built from the c1unfX template has good a value .

Pemodelan Calponin Ikan Gabus (Channa striata) dengan Phyre2 dan Interaksi dengan Protein Lain

Knowledge of the three-dimensional structure of proteins is very important to understand their character and function at the molecular level. Determination of protein structure in the laboratory is expensive and relatively difficult because it requires sophisticated instruments and takes a long time. As an alternative, an in silico approach can be used to predict the three-dimensional structure of proteins, namely the fold recognition method. The purpose of this study was to create a three-dimensional structure model of snakehead fish (Channa striata) calponin protein using the Phyre2 web server. The target protein sequence was obtained from UniProt KB with code K9LH64. The results of the study showed that the template for building the model was the c1wynA code. The results of the model (MP) evaluation obtained a coverage value was 45%, a confidence value was 100%, and an identity value was 69%. Validation of the model (MP) using the PROCHECK program showed the model was in the most favoured residue plot of 82.4%. This model deserves to be used as a model for calponin protein of snakehead fish, because the disallowed area was below 15%. The results of the analysis of interactions with other proteins using STRING-DB obtained the interaction model (MP) with Actin assembly-inducing protein (ACTA2) was 0.791; and Tropomyosin (TPM1) was 0.786. The results of molecular docking using PatchDock obtained the value of Atomic Contact Energy (ACE) for the calponin protein model (MP)-ACTA2 was 372.76 kJ/mol; Calponin model protein (MP)-TPM1 was 406.93 kJ/mol. The interactions that occured in the calponin model (MP)-ACTA2 were hydrogen bonds at residues Gly-41, Glu-60, Arg-64, Arg-66, Pro-67, Gly-68, Lys-71 and hydrophobic bonds at Arg-64, Lys- 71, Lys-72, Ile-73, Va-138. The interactions model of calponin model (MP)-TPM1 were hydrogen bonding at residues Ser-13, Lys-71, Ala-95, Tyr-96, Ser-136, Arg-137, Arg-150 and hydrophobic bonds at residues Leu-12, Ala-14, Lys-71, Ala-95, Val-138, Arg-150, Phe-152. Keywords: fold recognition, Phyre2 , 3D structure protein, calponin, protein-protein interaction

In Silico Designing and Interaction of Coumarin-Amino Acid(s) Conjugates with Integrin Like Protein of Cryptococcus neoformans: Insights on Antifungal Drug Design

In the present work, a library of 117 coumarin-amino acid(s) conjugates was designed, and molecular docking study was performed to investigate their possible role as fungal integrin like receptor antagonists. The objective of this study is in-silico designing and docking of coumarin-amino acid conjugates against integrin like protein of Cryptococcus neoformans. In the absence of a crystallographic structure of integrin of the fungal pathogen, a homology model of protein OXH63806.1 of Cryptococcus neoformans was developed using the currently available X-ray structure of human integrin as a template. The quality of the 3D model obtained by homology modeling was evaluated by the PROCHECK program. A docking study using coumarin-amino acid(s) conjugates as ligands on the binding site of the modeled receptor was carried out to understand the protein-ligand binding interactions. Some of the compounds have shown very good binding energies ranging from -10.32 to -10.94 Kcal/mol towards the target receptor. These results may be helpful in the designing and development of new antifungal agents as integrin like protein antagonists.

Protein-protein Interaction and Molecular Dynamics of Iturin A Gene on Effector Proteins of Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary, the fungal pathogen causes late blight, which results in devastating economic loss among the Solanaceae. The bacillus lipopeptides show the antagonistic activity against the many plant pathogens, among bacillus lipopeptides reported as the antifungal gene. Hence, to understand the in silico antifungal activity, we have selected gene iturin A (AXN89987) produced by Bacillus spp to check the molecular dynamics study with the effector proteins of the P. infestanse. In this concern, known effector proteins of P. infestans were subjected to the protein-protein interaction followed by simulation. Iturin A gene was amplified using the soil bacterium Bacillus subtilis with gene-specific primers, cloned into pTZ 57R/T vector and confirmed by sequencing. To get better insights, the protein model was developed for Iturin A using Modeller 9.17, using PDB structure of ID 4MRT (Phosphopantetheine transferase Sfp) and 1QR0 (4'-phosphopantetheinyl moiety of coenzyme A) as a template, it shared the identity 72% and expected P-value: 3e-121, respectively. The model quality was assessed using ProSA and PROCHECK programs. The potency of modelled protein against effector proteins of P. infestans were evaluated in silico using the HADDOCK server and the results showed the high affinity of towards the effector protein Host ATG8 (PDB-5L83). Finally, the simulation was performed to the docked conformation of with Host ATG8 to further understand the stability of the complex using the Desmond program. Altogether, the protein-protein interaction and simulation study propose a new methodology and to uncover possible antifungal activity of iturin A against effector proteins of P. infestans.

In silico structural modeling and analysis of physicochemical properties of curcumin synthase (CURS1, CURS2, and CURS3) proteins of Curcuma longa

BackgroundPharmaceutically important curcuminoid synthesis in C. longa is controlled by CURS1, CURS2, and CURS3 genes. The present study detected the physicochemical properties and structural characteristics including the secondary and 3D structure of CURS proteins. The primary, secondary, and tertiary structure of the CURS proteins were modeled and characterized using multiple bioinformatics tools such as ExPasy ProtParam tools, self-optimized prediction method with alignment (SOPMA), PSIPRED, and SWISS-MODEL. The predicted secondary structure of curcumin synthase provided an α-helix and random coil as the major components. The reliability of the modeled structure was confirmed using PROCHECK and QMEAN programs. ResultsThe molecular weight of CURS1 is 21093.19 Da, theoretical pI as 4.93, and an aliphatic index of 99.19. Molecular weight of CURS2 and CURS3 proteins are 20266.13 Da and 20629.52 Da, theoretical pI as 5.28 and 4.96, and an aliphatic index of 89.30 and 86.37, respectively. In the predicted secondary structure of CURS proteins, alpha helices and random coils of CURS1, CUR2, and CURS3 were 42.72, 41.38, and 44.74% and 24.87, 31.03, and 17.89, respectively. The extended strands were 16.24, 19.40, and 17.89. QMEAN Z-score is − 0.83, − 0.89, and − 1.09 for CURS1, CURS2, and CURS3, respectively. ConclusionPrediction of the 3D model of a protein by in silico analysis is a highly challenging aspect to confirm the NMR or X-ray crystallographic data. This report can contribute to the understanding of the structure, physicochemical properties, structural motifs, and protein-protein interaction of CURS1, CUR2, and CURS3.

Isoform-Specific Role of Akt Kinase in Cancer and its Selective Targeting by Potential Anticancer Natural Agents

Background: Akt kinase is a serine/threonine kinase that plays an important role in different cellular processes such as cell proliferation, apoptosis, glucose metabolism, transcription, and cell migration. It has three isoforms (Akt1, 2, and 3) that have distinct and sometimes contrasting functions in different cancers. However, to date, most of the inhibitors are directed against Akt kinase generally which would not serve the purpose due to the lack of isoform selectivity and offtarget toxicity. Therefore, the present study is an elementary step towards the demarcation of the natural inhibitors available from food sources and dietary supplements using in silico methods. Objective: To demarcate the natural agents and general Akt kinase inhibitors into Akt isoformspecific inhibitors. Methods: The genetic alterations data for Akt isoforms were obtained from The Cancer Genome Atlas datasets. The protein sequence alignment was achieved using PRALINE program. The modeling of Akt3 protein and its evaluation was performed by ModWeb Server and PROCHECK program, respectively. The docking was performed by using Schrödinger Glide software. Results: Differential pattern of genetic alterations of Akt isoforms was observed in different cancers. The protein sequence alignment has shown both the conserved as well as the non- conserved region of Akt isoforms. The structure of Akt3 was successfully modeled and evaluated. Finally, with the help of molecular docking, the natural agents and general Akt inhibitors have been segregated into Akt isoform-specific inhibitors based on the derived Glide Score (GScore). Conclusion: Isoform-specific inhibition of Akt would have huge clinical significance and research should be commenced in preclinical and clinical settings.

In Silico Studies on Anti-Stress Compounds of Ethanolic Root Extract of Hemidesmus indicus L.

Alternative medicine is available for those diseases which cannot be treated by conventional medicine. Ayurveda and herbal medicines are important alternative methods in which the treatment is done with extracts of different medicinal plants. This work is concerned with the evaluation of anti-stress bioactive compounds from the ethanolic root extract of Hemidesmus indicus. Gas chromatography and Mass Spectrum studies are used to identify the compounds present in the ethanolic extract based on the retention time, area. In order to perform docking studies, Vasopressin model is generated using modeling by Modeller 9v7. Vasopressin structure is developed based on the crystal structure of neurophysin-oxytocin from Bos taurus (PDB ID: 1NPO_A) collected from the PDB data bank. Using molecular dynamics simulation methods, the final predicted structure is obtained and further analyzed by verifying 3D and PROCHECK programs, confirmed that the final model is reliable. The identified compounds are docked to vasopressin for the prediction of anti-stress activity using GOLD 3.0.1 software. The predicted model of Vasopressin structure is stabilized and confirmed that it is a reliable structure for docking studies. The results indicated ARG4, THR7, ASP9, ASP26, ALA32, ALA 80 in Vasopressin are important determinant residues in binding as they have strong hydrogen bonding with phytocompounds. Among the 21 phytocompounds identified and docked, molecule Deoxiinositol, pentakis- O-(trimethylsilyl) showed the best docking results with Vasopressin. The identified compounds were used for anti-stress activity by insilico method with Vasopressin which plays an important role in causing stress and hence selected for inhibitory studies with phytocompounds. The phytocompounds are inhibiting vasopressin through hydrogen bodings and are important in protein-ligand interactions. Docking results showed that out of twenty-one compounds, Deoxiinositol, pentakis-O-(trimethylsilyl) showed best docking energy to the Vasopressin.

Comparative modeling and mutual docking of structurally uncharacterized heat shock protein 70 and heat shock factor-1 proteins in water buffalo

Aim:In this study, a wide range of in silico investigation of Bubalus bubalis (BB) heat shock protein 70 (HSP70) and heat shock factor-1 (HSF1) has been performed, ranging from sequence evaluation among species to homology modeling along with their docking studies to decipher the interacting residues of both molecules.Materials and Methods:Protein sequences of BB HSP70 and HSF1 were retrieved from NCBI database in FASTA format. Primary and secondary structure prediction were computed using Expasy ProtParam server and Phyre2 server, respectively. TMHMM server was used to identify the transmembrane regions in HSP70. Multiple sequence alignment and comparative analysis of the protein was carried out using MAFFT and visualization was created using ESPript 3.0. Phylogenetic analysis was accomplished by COBALT. Interactions of HSP70 with other proteins were studied using STRING database. Modeller 9.18, RaptorX, Swiss-Modeller, Phyre2, and I-TASSER were utilized to design the three-dimensional structure of these proteins followed by refinement; energy minimization was accomplished using ModRefiner and SPDBV program. Stereochemical quality along with the accuracy of the predicted models and their visualization was observed by PROCHECK program of PDBsum and UCSF Chimera, respectively. ClusPro 2.0 server was accessed for the docking of the receptor protein with the ligand.Results:The lower value of Grand Average of Hydropathy indicates the more hydrophilic nature of HSP70 protein. Value of the instability index (II) classified the protein as stable. No transmembrane region was reported for HSP70 by TMHMM server. Phylogenetic analysis based on multiple sequence alignments (MSAs) by COBALT indicated more evolutionarily closeness of Bos indicus (BI) with Bos taurus as compared to BI and BB. STRING database clearly indicates the HSF1 as one of the interacting molecules among 10 interacting partners with HSP 70. The best hit of 3D model of HSP70 protein and HSF1 was retrieved from I-TASSER and Phyre2, respectively. Interacting residues and type of bonding between both the molecules which were docked by ClusPro 2.0 were decoded by PIC server. Hydrophobic interactions, protein-protein main-chain-side-chain hydrogen bonds, and protein-protein side-chain-side-chain hydrogen bonds were delineated in this study.Conclusion:This is the first-ever study on in silico interaction of HSP70 and HSF1 proteins in BB. Several bioinformatics web tools were utilized to study secondary structure along with comparative modeling, physicochemical properties, and protein-protein interaction. The various interacting amino acid residues of both proteins have been indicated in this study.

Insilico Characterization, Phylogenetic Analysis and Homology based 3D Structural Modeling of HSPA4 Heat Shock Protein Family a (Hsp70) Member 4 from Homo Sapiens (Human)

HSP70 are the specialized Heat Shock Proteins that show their novelty presence inside the deepest part of Cellular Component Networks of Chaperons and involve with various heat Shock repairing up mechanisms as main Catalytic Components. They actively participate in proper alignment and resistance of all types pressures by their proper dealing with substrates and proper exposure of their hydrophobic peptide portions inside of their substrate molecules. The exchange of information cum process of information occurs between the cell components through HSP70’s low level of affinity ATP bind sites and High Affinity level of ADP binding sites. Therefore, ATP hydrolysis process plays a key role in fundamental mechanisms, in vitro and as well as in in vivo repairing mechanisms for the functioning of Chaperone like functionalities of HSP 70 proteins. In the current study analysis, Homology Modeling methods permits us for modeling the 3D structure of the protein by taking backbone of experimentally determined 3D-Structures of homologous proteins extracted in various structural formats like PDBs. We worked with HSPA4 heat shock protein family A (HSP 70) part 4 from Homo sapiens ( Human ) and conducted 3-D structure prediction using an Automated Swiss Model Generation by employing the crystal structures of Template molecule. PDB Blast and Template search for required protein were conducted with help of all the parameters with respect to Imapact and Psi Impact angles and that leads to for most noteworthy sequence identity, structural alignments and functionalities. Finally, the homology modeling were performed by using SWISS modeler and modeled proteins were fine tuned by using the Ramachandran Plot, by using PROCHECK and 3D-Check Software Programs.

Epitope-Based Vaccine Designing of Nocardia asteroides Targeting the Virulence Factor Mce-Family Protein by Immunoinformatics Approach.

Nocardia asteroides is the main causative agent responsible for nocardiosis disease in immunocompromised patient viz. Acquired Immunodeficiency Syndrome (AIDS), malignancy, diabetic, organ recipient and genetic disorders. The virulence factor and outer membrane protein pertains immense contribution towards the designing of epitopic vaccine and limiting the robust outbreak of diseases. While epitopic based vaccine element carrying B and T cell epitope along with adjuvant is highly immunoprophylactic in nature. Present research equips immunoinformatics to figure out the suitable epitopes for effective vaccine designing. The selected epitopes VLGSSVQTA, VNIELKPEF and VVPSNLFAV amino acids sequence are identified by HLA-DRB alleles of both MHC class (MHC-I and II) molecules. Simultaneously, these also accessible to B-cell, confirmed through the ABCPred server. Antigenic property expression is validated by the Vaxijen antigenic prediction web portal. Molecular docking between the epitopes and T cell receptor delta chain authenticate the accurate interaction between epitope and receptor with significantly low binding energy. Easy access of epitopes to immune system also be concluded as transmembrane nature of the protein verified by using of TMHMM server. Appropriate structural identity of the virulence factor Mce-family protein generated through Phyre2 server and subsequently validated by ProSA and PROCHECK program suite. The structural configuration of theses epitopes also shaped using DISTILL web server. Both the structure of epitopes and protein will contribute a significant step in designing of epitopic vaccine against N. asteroides. Therefore, such immunoinformatics based computational drive definitely provides a conspicuous impel towards the development of epitopic vaccine as a promising remedy of nocardiosis

Modeling of the Phytophthora capsici cellulose synthase 3 and its inhibitors activity assay.

Phytophthora capsici is a devastating pathogen for crop. Cellulose synthase 3 (CesA3) is a target for many potential fungicides such as valinamide derivatives. However, the 3-dimensional structure (3-DS) of CesA3 in Phytophthora capsici was still unknown. Here CesA3 protein sequence was retrieved from the NCBI protein sequence database We did the 3-DS structural modeling for CesA3 and used molecular dynamics to optimize the model. The model was further validated by the Ramachandran plot in PROCHECK program. Two series of new valinamide compound were synthesized and tested for its biological activity. The docking data obtained by the model perfectly matched with the biometric data, indicating that the model is valid. Moreover, docking study data revealed the mechanism of action of inhibitors on target enzymes. The 3-DS structural model was analyzed from the perspective of the biocide receptor, the structure of the target protein and the mechanism of action of the compound. It provides a new perspective for the design of new fungicides. © 2019 Society of Chemical Industry.

Computational characterization of epitopic region within the outer membrane protein candidate in Flavobacterium columnare for vaccine development

Gram-negative bacteria is the main causative agents for columnaris disease outbreak to finfishes. The outer membrane proteins (OMPs) candidate of Flavobacterium columnare bacterial cell served a critical component for cellular invasion targeted to the eukaryotic cell and survival inside the macrophages. Therefore, OMPs considered as the supreme element for the development of promising vaccine against F. columnare. Implies advanced in silico approaches, the predicted 3-D model of targeted OMPs were characterized by the Swiss model server and validated through Procheck programs and Protein Structure Analysis (ProSA) web server. The protein sequences having B-cell binding sites were preferred from sequence alignment; afterwards the B cell epitopes prediction was prepared using the BCPred and amino acid pairs (AAP) prediction algorithms modules of BCPreds. Consequently, the selected antigenic amino acids sequences (B-cell epitopic regions) were analyzed for T-cell epitopes determination (MHC I and MHC II alleles binding sequence) performing the ProPred 1 and ProPred server respectively. The epitopes (9 mer: IKKYEPAPV, YGPNYKWKF and YRGLNVGTS) within the OMPs binds to both of the MHC classes (MHC I and MHC II) and covered highest number of MHC alleles are characterized. OMPs of F. columnare being conserved across serotypes and highly immunogenic for their exposed epitopes on the cell surface as a potent candidate focus to vaccine development for combating the disease problems in commercial aquaculture. The portrayed epitopes might be beneficial for practical designing of abundant peptide-based vaccine development against the columnaris through boosting up the advantageous immune responses.Communicated by Ramaswamy H. Sarma

Alkaloids and Leishmania donovani UDP-Galactopyarnosemutase: A Novel Approach in Drug Designing Against Visceral Leishmaniasis.

The unsatisfactory treatment options for Visceral Leishmaniasis (VL), need identification of new drug targets. Among natural products, Alkaloids have been proved to be highly effective against number of diseases. In Leishmania, UDP-galactopyranosemutase (UGM) is a critical enzyme required for cell wall synthesis and thus a drug target for structure based drug designing against L. donovani. The aim was to build the homology model of UDP galactopyransemutase and investigate the interaction of selected alkaloids with this modeled UDP galactopyranosemutase by molecular docking. Since no crystal structure record has been found with this protein, a homology modeling was performed and a three dimensional structure of L. donovani UGM was created using MODELLER v9.9, structure quality was validated using PROCHECK and QMEAN programs which confirms that the structure is reliable. Further Molecular docking was performed with previously reported15 alkaloids. It was found that Protopine with a binding energy of -12.39Kcal/mole, binds at Flavin adenine dinucleotide (FAD) biding site. It was concluded that Protopine, an alkaloid could interrupt the functional aspect of L. donovani UGM and thus may be useful for drug designing studies. These finding would contribute to the understanding of the effect of drug on the parasite.

Pharmacophore-Based Virtual Screening of Novel Inhibitors and Docking Analysis for CYP51A from Penicillium italicum.

Sterol 14α-demethylases from Cytochrome P450 family (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. The 3D structure of CYP51A from Penicillium italicum (PiCYP51A) was constructed through homology modeling based on the crystal structure of human CYP51A (PDB: 3LD6). Molecular dynamics (MD) simulation was operated to relax the initial model and followed by quality assessment using PROCHECK program. On the basis of the docking information on the currently available CYP51s with the patent demethylase inhibitors (DMIs), pharmacophore-based virtual screening combined with docking analysis was performed to pick out twelve new compounds from ZINC database. Six hits revealed in the ligand database suggested potential ability to inhibit PiCYP51A. Compared to patent fungicide triazolone, the top three lead compounds had similar or higher affinity with the target enzyme, and accordingly, exhibited comparable or lower EC50 values to P. italicum isolates. The results could provide references for de novo antifungal drug design.

An in silico approach for characterization of encoded protein from Rdr1, a black spot resistance gene in Rosa multiflora

Black spot disease is the most common diseases of landscape roses and is caused by Diplocarpon rosae Wolf. In Rosa multiflora, the screening of black spot-resistant tetraploids and diploids demonstrated the presence of dominant genes Rdr1 and Rdr2. A previous study has identified muRdr1H as the only TIR-NBS-LRR gene, which could restore black spot resistance in the susceptible genotypes. In this study, we selected the protein encoded by muRdr1H gene, AEE43932.1, for computational analysis. Its primary, secondary, and tertiary structures were obtained for further analysis. The results showed that the query protein was acidic, stable, and hydrophilic. The secondary structure prediction showed that the query protein consisted of 37 beta strands, 26 alpha-helices, and 28 coils and is probably localized in the cell membrane and cytoplasm. Five three-dimensional (3D) models were predicted by I-TASSER and validated. We found that model A showed the best results with the highest confidence score (−0.90) as well as enzyme commission-related confidence score and ligation binding sites (LI). The models developed were validated using PROCHECK program. These analyses validated that the predicted model A was the best and reliable enough to be used for future studies related to black spot resistance in Rosaceae plants.

Cloning, Characterization and Bioinformatics Analysis of Novel Cytosine Deaminase from Escherichia coli AGH09

Cytosine deaminase (CDase) catalyzes the deamination of cytosine and 5-fluorocytosine (5-FC) to uracil and 5-fluorouracil (5-FU), respectively. The enzyme is used for cancer therapy, as cancerous cells are not able to synthesize cytosine via a de-novo pathway. In order to characterize the Escherichia coli AGH09 CDase, a strong CDase producing strain, was isolated from the urinary infection patients. The DNA fragment encoding the CDase was cloned into pET22b vector and sequenced. The presented paper uses bioinformatics and experimental approaches to describe the physiochemical, functional and structural properties of CDase. The results revealed that the enzyme was active in pH ranging from 6.0 to 9.0 along with an optimal activity at 47 °C. Moreover, in the presence of Cu++, Zn++ and Fe++ ions, the enzyme activity was increased; however, the Mn++ and Na+ ions had negatively influence on CDase activity. Bioinformatics analysis showed the enzyme had no signal sequences and disulfide bond along with no allergenicity. The aliphatic index (94.59) and grand average of hydropathicity index (−0.21) calculated by ProtParam server indicated that CDase may be stable for a wide range of temperature and has good solubility in water. Secondary structure analysis revealed that most of the recombinant CDase has predominant α-helical structures (37.24 %) and Random coils (39.58 %). The three dimensional structure of CDase is predicted by homology modeling using the known CDase crystal structures of E. coli (3O7U) as template using Geno3D2 Web Server. The model was evaluated with PROCHECK and WHAT IF programs. These findings suggested our enzyme might be a novel CDase for deployment in cancer treatment. Moreover, our data provides such beneficial information for a better drug formulation with proper storage shelf life storage.