Presence Of Signal Peptides Research Articles

In silico prediction of CD8+ and CD4+ T cell epitopes in Leishmania major proteome: Using immunoinformatics

The leishmaniases are NDTs (neglected tropical diseases) that affect people all over the world. They are brought on by protozoans from the genus Leishmania and disseminated by phlebotomine flies that are afflicted with the disease. The best option to manage and lower the incidence of these diseases has been thought by the creation of a safe and effective vaccination. This research used an in silico based mining approach to look for high potential epitopes that might bind to MHC Class I and MHC Class II molecules (mainly; HLA-A*02:01 & HLA-DRB1*03:01) from human population in order to promote vaccine development. Based on the presence of signal peptides, GPI anchors, antigenicity predictions, and a subtractive proteomic technique, we have screened 17 putative antigenic proteins from the 8083 total proteins of L. major. After that thorough immunogenic epitope prediction were done using IEDB-AR tools. We isolated five immunogenic epitopes (three 9-mer & two 15-mer) from five antigenic proteins through docking and MD simulation analysis. Finally, these five anticipated epitopes, viz., TLPEIPVNV, ELMAPVFGL, TLAAAVALL, NSINIRLDGVTSAGF and NVPLVVDASSLFRVA have considerably stronger binding potential with their respective alleles and may trigger immunological responses. The goal of this work was to identify MHC restricted epitopes for CD8+ and CD4+ T cells activation using immunoinformatics in order to identify potential vaccine candidates against L. major parasites.

Characterization of β-glucosidases from Meridianimaribacter sp. CL38

The production of second-generation biofuel requires a huge amount of freshwater. It is estimated that at least three gal of freshwater is used to produce one gal of biofuel. The replacement of freshwater with seawater serves as a potential alternative in biofuel generation. Therefore, salt-tolerant enzymes play an important role in saccharification and fermentation process. Halophilic β-glucosidase is one of the key enzymes for the process. In this study, the β-glucosidase of halophile Meridianimaribacter sp. CL38 isolated from mangrove soil was characterized. Strain CL38 achieved maximum production of β-glucosidase at 12th hour of growth. The β-glucosidase showed highest activity at 2% (w/v) NaCl while highly stable at salt concentration ranging from 1-2% (w/v) (more than 96% of relative activity). Its β-glucosidase activity remained active in the presence of 5mM Mn2+, Mg2+, Ca2+ ions, and 1% (v/v) Tween-20 and Tween-80. The draft genome sequence of strain CL38 was retrieved from GenBank database and submitted to dbCAN meta server for CAZymes annotation. Strain CL38 harbors 44 GHs and GH3 are annotated as β-glucosidases. The β-glucosidases of Meridianimaribacter flavus (99.61%) and Mesoflavibacter sabulilitoris (97.44%) showed the closest identity with Bgl3a and Bgl3b protein sequences from strain CL38, respectively. Glycoside hydrolase family 3 domain was identified in both enzymes via InterPro scan server. The presence of signal peptides indicated that both enzymes were secreted extracellularly. Five motifs were identified in Bgl3a and Bgl3b, with the active site (nucleophile) found at Asp296 and Asp297, respectively. Collectively, these β-glucosidases could be potentially used in the biofuel production, in particular the lignocellulosic biomass pretreatment process. This is the first attempt to characterize the β-glucosidase in genus Meridianimaribacter as so far none of the lignocellulolytic enzymes from this genus were characterized.

Peritrophin-like Genes Are Associated with Delousing Drug Response and Sensitivity in the Sea Louse Caligus rogercresseyi.

Caligus rogercresseyi is the main ectoparasite that affects the salmon industry in Chile. The mechanisms used by the parasite to support its life strategy are of great interest for developing control strategies. Due to the critical role of insect peritrophins in host-parasite interactions and response to pest control drugs, this study aimed to identify and characterize the peritrophin-like genes present in C. rogercresseyi. Moreover, the expression of peritrophin-like genes was evaluated on parasites exposed to delousing drugs such as pyrethroids and azamethiphos. Peritrophin genes were identified by homology analysis among the sea louse transcriptome database and arthropods peritrophin-protein database obtained from GenBank and UniProt. Moreover, the gene loci in the parasite genome were located. Furthermore, peritrophin gene expression levels were evaluated by RNA-Seq analysis in sea louse developmental stages and sea lice exposed to delousing drugs deltamethrin, cypermethrin, and azamethiphos. Seven putative peritrophin-like genes were identified in C. rogercresseyi with high homology with other crustacean peritrophins. Differences in the presence of signal peptides, the number of chitin-binding domains, and the position of conserved cysteines were found. In addition, seven peritrophin-like gene sequences were identified in the C. rogercresseyi genome. Gene expression analysis revealed a stage-dependent expression profile. Notably, differential regulation of peritrophin genes in resistant and susceptible populations to delousing drugs was found. These data are the first report and characterization of peritrophin genes in the sea louse C. rogercresseyi, representing valuable knowledge to understand sea louse biology. Moreover, this study provides evidence for a deeper understanding of the molecular basis of C. rogercresseyi response to delousing drugs.

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity.IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics.

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.

Genome-Wide Prediction of Vaccine Candidates for Leishmania major: An Integrated Approach.

Despite the wealth of information regarding genetics of the causative parasite and experimental immunology of the cutaneous leishmaniasis, there is currently no licensed vaccine against it. In the current study, a two-level data mining strategy was employed, to screen the Leishmania major genome for promising vaccine candidates. First, we screened a set of 25 potential antigens from 8312 protein coding sequences, based on presence of signal peptides, GPI anchors, and consensus antigenicity predictions. Second, we conducted a comprehensive immunogenic analysis of the 25 antigens based on epitopes predicted by NetCTL tool. Interestingly, results revealed that candidate antigen number 1 (LmjF.03.0550) had greater number of potential T cell epitopes, as compared to five well-characterized control antigens (CSP-Plasmodium falciparum, M1 and NP-Influenza A virus, core protein-Hepatitis B virus, and PSTA1-Mycobacterium tuberculosis). In order to determine an optimal set of epitopes among the highest scoring predicted epitopes, the OptiTope tool was employed for populations susceptible to cutaneous leishmaniasis. The epitope (127SLWSLLAGV) from antigen number 1, found to bind with the most prevalent allele HLA-A⁎0201 (25% frequency in Southwest Asia), was predicted as most immunogenic for all the target populations. Thus, our study reasserts the potential of genome-wide screening of pathogen antigens and epitopes, for identification of promising vaccine candidates.

Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis.

Plant diseases caused by fungi and oomycetes pose an increasing threat to food security and ecosystem health worldwide. These filamentous pathogens, while taxonomically distinct, modulate host defense responses by secreting effectors, which are typically identified based on the presence of signal peptides. Here we show that Phytophthora sojae and Verticillium dahliae secrete isochorismatases (PsIsc1 and VdIsc1, respectively) that are required for full pathogenesis. PsIsc1 and VdIsc1 can suppress salicylate-mediated innate immunity in planta and hydrolyse isochorismate in vitro. A conserved triad of catalytic residues is essential for both functions. Thus, the two proteins are isochorismatase effectors that disrupt the plant salicylate metabolism pathway by suppressing its precursor. Furthermore, these proteins lack signal peptides, but exhibit characteristics that lead to unconventional secretion. Therefore, this secretion pathway is a novel mechanism for delivering effectors and might play an important role in host–pathogen interactions.

A Novel Predicted Calcium-Regulated Kinase Family Implicated in Neurological Disorders

The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca2+-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca2+-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins.

Predicting membrane protein types by incorporating protein topology, domains, signal peptides, and physicochemical properties into the general form of Chou’s pseudo amino acid composition

The type information of un-annotated membrane proteins provides an important hint for their biological functions. The experimental determination of membrane protein types, despite being more accurate and reliable, is not always feasible due to the costly laboratory procedures, thereby creating a need for the development of bioinformatics methods. This article describes a novel computational classifier for the prediction of membrane protein types using proteins’ sequences. The classifier, comprising a collection of one-versus-one support vector machines, makes use of the following sequence attributes: (1) the cationic patch sizes, the orientation, and the topology of transmembrane segments; (2) the amino acid physicochemical properties; (3) the presence of signal peptides or anchors; and (4) the specific protein motifs. A new voting scheme was implemented to cope with the multi-class prediction. Both the training and the testing sequences were collected from SwissProt. Homologous proteins were removed such that there is no pair of sequences left in the datasets with a sequence identity higher than 40%. The performance of the classifier was evaluated by a Jackknife cross-validation and an independent testing experiments. Results show that the proposed classifier outperforms earlier predictors in prediction accuracy in seven of the eight membrane protein types. The overall accuracy was increased from 78.3% to 88.2%. Unlike earlier approaches which largely depend on position-specific substitution matrices and amino acid compositions, most of the sequence attributes implemented in the proposed classifier have supported literature evidences. The classifier has been deployed as a web server and can be accessed at http://bsaltools.ym.edu.tw/predmpt.

The Zebrafish Thrombospondin 3 and 4 Genes ( thbs3 and thbs4 ): cDNA and Protein Structure

The cDNA sequences of the zebrafish thrombospondin 3 gene (thbs3) and thrombospondin 4 gene (thbs4) have been determined, and the encoded amino acid sequences (Thbs3 and Thbs4) have been analyzed. Thbs3 cDNA specifies a protein of 962 amino acid residues (MW 105,787), while Thbs4 cDNA specifies a protein of 949 residues (MW 103,989). Both proteins are highly acidic with Asp + Glu contents of 15.8% (Thbs3) and 16.9% (Thbs4). The acidic amino acids are clustered in the region of the Type III repeats and part of the C-terminal domain. The presence of signal peptides indicates that Thbs3 and Thbs4 function as secreted, extracellular proteins. The two polypeptides contain homologous sequences with two cysteines that are likely to form interchain disulfide bonds. Potential glycosylation sites were identified, five for Thbs3 and three for Thbs4. The polypeptides share the domain structure characteristic of the thrombospondin 3/thrombospondin 4/COMP subgroup of thrombospondin proteins. This domain structure consists of an amino-terminal thrombospondin N-terminal-like domain (TSPN domain), four Type II (EGF-like) repeats, seven Type III (calcium-binding) repeats, and a carboxyl terminal region. In aligning the Thbs3 and Thbs4 domain sequences with those of other thrombospondins, a gradient of sequence homology is observed that increases from N-terminus to C-terminus.

Molecular cloning, by a novel approach, of a cDNA encoding a putative olfactory protein in the labial palps of the moth Cactoblastis cactorum

We have used the CapFinder technology, without the library construction step, to amplify and clone full-length cDNAs expressed in the labial palps (CO 2-sensing organs) of the moth Cactoblastis cactorum. The validity of our approach is exemplified by the sequence analysis of a 597-bp cDNA clone, designated CLP-1, that contains a 390-bp open reading frame (ORF) flanked by motifs characteristic to a full-length cDNA. The ORF in CLP-1 encodes a predicted polypeptide that is 47% identical to a novel protein, OS-D, found exclusively in the olfactory antennal segment of Drosophila melanogaster. Both CLP-1 and OS-D have primary structures that do not bear sequence similarity to any previously characterised proteins including odorant-binding proteins (OBPs) in vertebrates and pheromone-binding proteins (PBPs) in moths. Although they share features common to OBPs and PBPs, such as the presence of signal peptides and cysteine motifs, they clearly belong to a distinct class of olfactory proteins that appear to be unique to insects. The relative abundance of the CLP-1 message in the labial palps of females leads to the suggestion that this protein is involved in the CO 2-sensing cascade. Our results suggest that the experimental procedure can be used as an alternative, rapid method to identify genes expressed in a particular organ, or tissue, especially in situations when the amount of available tissue is a limiting factor.