Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

The complete sequence of a 9000 bp fragment of the right arm of Saccharomyces cerevisiae chromosome VII contains four previously unknown open reading frames

We report the sequence of a 9000 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete previously unknown open reading frames, which were named G7587, G7589, G7591 and G7594 following standard rules for provisional nomenclature. Outstanding features of some of these proteins were the homology of the putative protein coded by G7589 with proteins involved in transcription regulation and the transmembrane domains predicted in the putative protein coded by G7591.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

trEST, trGEN and Hits: access to databases of predicted protein sequences.

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits. isb-sib.ch).

MyHits: a new interactive resource for protein annotation and domain identification.

The MyHits web server (http://myhits.isb-sib.ch) is a new integrated service dedicated to the annotation of protein sequences and to the analysis of their domains and signatures. Guest users can use the system anonymously, with full access to (i) standard bioinformatics programs (e.g. PSI-BLAST, ClustalW, T-Coffee, Jalview); (ii) a large number of protein sequence databases, including standard (Swiss-Prot, TrEMBL) and locally developed databases (splice variants); (iii) databases of protein motifs (Prosite, Interpro); (iv) a precomputed list of matches ('hits') between the sequence and motif databases. All databases are updated on a weekly basis and the hit list is kept up to date incrementally. The MyHits server also includes a new collection of tools to generate graphical representations of pairwise and multiple sequence alignments including their annotated features. Free registration enables users to upload their own sequences and motifs to private databases. These are then made available through the same web interface and the same set of analytical tools. Registered users can manage their own sequences and annotations using only web tools and freeze their data in their private database for publication purposes.

Schistosoma mansoni heat shock protein 70 elicits an early humoral immune response in S. mansoni infected baboons

A study was undertaken to search for DNA recombinant Schistosoma mansoni proteins responsible for eliciting an antibody response from the host at a very early phase after infection. A S. mansoni adult worm cDNA expression library was screened using pooled sera from baboons with four weeks of infection. Based on their specific reactivity with the S. mansoni infected sera and no reactivity when tested against the pre-infection sera from the same baboons, four clones were selected for further studies. Sequence analysis revealed that they were homologous to the S. mansoni heat shock protein 70 (hsp70). The insert sizes of the four selected clones varied from 1150 to 2006 bp. The preliminary characterization for antibody reactivity against a panel of baboon sera showed that the longest clone was the most reactive, eight out of eight acute and three out of four chronic sera reacting positively to this clone. The shortest clone was the least reactive. Our results suggest that the S. mansoni hsp70 elicits an early and strong antibody response in baboons and that antibodies to this protein can be detected in chronically infected animals. Therefore S. mansoni hsp70 may be a valid target for immunodiagnosis. However further studies are needed to identify the portion of the hsp70 that best fits the requirements for a valuable diagnostic antigen.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Mutation analysis of genes that control the G1/S cell cycle in melanoma: TP53, CDKN1A, CDKN2A, and CDKN2B.

BACKGROUND The role of genes involved in the control of progression from the G1 to the S phase of the cell cycle in melanoma tumors in not fully known. The aim of our study was to analyse mutations in TP53, CDKN1A, CDKN2A, and CDKN2B genes in melanoma tumors and melanoma cell lines METHODS We analysed 39 primary and metastatic melanomas and 9 melanoma cell lines by single-stranded conformational polymorphism (SSCP). RESULTS The single-stranded technique showed heterozygous defects in the TP53 gene in 8 of 39 (20.5%) melanoma tumors: three new single point mutations in intronic sequences (introns 1 and 2) and exon 10, and three new single nucleotide polymorphisms located in introns 1 and 2 (C to T transition at position 11701 in intron 1; C insertion at position 11818 in intron 2; and C insertion at position 11875 in intron 2). One melanoma tumor exhibited two heterozygous alterations in the CDKN2A exon 1 one of which was novel (stop codon, and missense mutation). No defects were found in the remaining genes. CONCLUSION These results suggest that these genes are involved in melanoma tumorigenesis, although they may be not the major targets. Other suppressor genes that may be informative of the mechanism of tumorigenesis in skin melanomas should be studied.

Molecular analysis of non-melanoma skin cancer

SUMMARY:Cylindroma, trichoepithelioma and spiradenoma are benign tumors of hair follicle. They are caused by mutations and loss of heterozygosity in the CYLD gene. CYLD is a ubiquitously expressed, but the tumors are restricted to skin, suggesting that the tumorigenesis is influenced by skin-specific regulators and probably by mutations in other genes. The objectives of the thesis were to analyze the molecular mechanisms leading to the aforementioned tumors. In the first project, we have identified five new mutations in CYLD gene in tive families affected with different combinations of these skin appendage tumors. F our of these mutations caused the introduction of a premature stop codon in CYLD protein sequence, but one was a missense mutation changing aspartic acid 681 into glycine (D68lG), in patients exhibiting multiple trichoepitheliomas. CYLD is a deubiquitinase which can downregulate NF-κB and INK pathways through the deubiquitination of TRAF2, for example. We showed that the CYLD-D681G mutant was unable to remove polyubiquitin chains from TRAF2. We also proved that CYLD-D68lG could not inhibit TRAP 2- or TNFα- mediated NF-κB or INK activations in 293T cells. These results underlined the importance of the D68l residue for the enzymatic activity of CYLD. TRAP-interacting protein (TRIP), which is a E3-Ubiquitin ligase, is a partner of CYLD. In the second project of the thesis, we studied the function of TRIP in the epidermis. We found that TRIP was a nucleolar protein in cultured human primary keratinocytes (HEK) and HeLa cells, and was detected in the midbody of HeLa cells. Moreover, TRIP expression was shown to be downregulated through a PKC-dependent mechanism before induction of keratinocyte differentiation. We also proved that TRIP was upregulated in basal cell carcinomas. Furthermore, TRIP was found to be important for keratinocyte survival and proliferation through the regulation of the Gl/S transition. Our results suggest that TRIP may be involved in keratinocyte tumorigenesis.RÉSUMÉ :Les cylindromes, trichoépithéliomes et spiradénomes sont des tumeurs bénignes du follicule pileux causées par des mutations et une perte d'hétérozygotie du gène CYLD. CYLD est ubiquitaire mais les tumeurs sont limitées à la peau, suggérant que la tumorigénèse est influencée par des protéines spécifiques de la peau et par des mutations dans d'autres gènes. Les objectifs de la thèse étaient d'2malyser les mécanismes moléculaires aboutissant à la formation de ces tumeurs. Dans le premier projet, cinq nouvelles mutations du gène CYLD ont été identifiées chez cinq familles présentant différentes combinaisons des tumeurs citées ci- dessus. Quatre de ces mutations causaient I' introduction d'un codon stop prématuré dans la séquence protéique, mais une était une mutation «misser1se» changeant l'aspartate 681 en résidu glycine (D68lG) chez des patients présentant des trichoépithéliomes multiples. CYLD est une déubiquitinase qui inhibe les voies de signalisation de NF-κB et JNK, en déubiquitinant notamment TRAF2. Nous avons montré que la protéine mutante CYLD- D68lG ne pouvait pas cliver la chaîne de poly-ubiquitines liée à TRAF2. CYLD-D68lG était aussi incapable d'inhiber l'activation de NF-κB ou de JNK induite par TRAF2 ou TNF-o dans les cellules 293T. Ces résultats ont donc souligné l'impo1tance du résidu D68l pour l'activité de CYLD. «TRAF-interacting protein (TRIP)», qui est une «E3-ubiquitin-ligase», est un partenaire de CYLD. Dans le second proj et de la thèse, nous avons étudié la fonction de TRIP dans l'épidenne. Nous avons montrépque TRIP était nucléolaire dans les cellules HeLa et les kératinocytes primaires humains en culture et était détectée dans le «midbody» des cellules HeLa. Nous avons prouvé que l'ARNm de TRIP était diminué avant l'induction de la différentiation des kératinocytes, par un mécanisme dépendent de la protéine kinase C, tandis qu'il était augmenté dans les carcinomes baso-cellulaires. Nous avons aussi montré que TRIP influençait la prolifération et la survie des kératinocytes en régulant la transition G1/S, Nos résultats suggèrent que TRIP est peut-être impliquée dans la tumorigénèse des kératinocytes. 7

Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients.

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.

Ongoing and future developments at the Universal Protein Resource.

The primary mission of Universal Protein Resource (UniProt) is to support biological research by maintaining a stable, comprehensive, fully classified, richly and accurately annotated protein sequence knowledgebase, with extensive cross-references and querying interfaces freely accessible to the scientific community. UniProt is produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is comprised of four major components, each optimized for different uses: the UniProt Archive, the UniProt Knowledgebase, the UniProt Reference Clusters and the UniProt Metagenomic and Environmental Sequence Database. UniProt is updated and distributed every 4 weeks and can be accessed online for searches or download at http://www.uniprot.org.

From SNPs to pathways: integration of functional effect of sequence variations on models of cell signalling pathways

Background: Single nucleotide polymorphisms (SNPs) are the most frequent type of sequence variation between individuals, and represent a promising tool for finding genetic determinants of complex diseases and understanding the differences in drug response. In this regard, it is of particular interest to study the effect of non-synonymous SNPs in the context of biological networks such as cell signalling pathways. UniProt provides curated information about the functional and phenotypic effects of sequence variation, including SNPs, as well as on mutations of protein sequences. However, no strategy has been developed to integrate this information with biological networks, with the ultimate goal of studying the impact of the functional effect of SNPs in the structure and dynamics of biological networks. Results: First, we identified the different challenges posed by the integration of the phenotypic effect of sequence variants and mutations with biological networks. Second, we developed a strategy for the combination of data extracted from public resources, such as UniProt, NCBI dbSNP, Reactome and BioModels. We generated attribute files containing phenotypic and genotypic annotations to the nodes of biological networks, which can be imported into network visualization tools such as Cytoscape. These resources allow the mapping and visualization of mutations and natural variations of human proteins and their phenotypic effect on biological networks (e.g. signalling pathways, protein-protein interaction networks, dynamic models). Finally, an example on the use of the sequence variation data in the dynamics of a network model is presented. Conclusion: In this paper we present a general strategy for the integration of pathway and sequence variation data for visualization, analysis and modelling purposes, including the study of the functional impact of protein sequence variations on the dynamics of signalling pathways. This is of particular interest when the SNP or mutation is known to be associated to disease. We expect that this approach will help in the study of the functional impact of disease-associated SNPs on the behaviour of cell signalling pathways, which ultimately will lead to a better understanding of the mechanisms underlying complex diseases.

Sequence determinants of a microtubule tip localization signal (MtLS).

Microtubule plus-end-tracking proteins (+TIPs) specifically localize to the growing plus-ends of microtubules to regulate microtubule dynamics and functions. A large group of +TIPs contain a short linear motif, SXIP, which is essential for them to bind to end-binding proteins (EBs) and target microtubule ends. The SXIP sequence site thus acts as a widespread microtubule tip localization signal (MtLS). Here we have analyzed the sequence-function relationship of a canonical MtLS. Using synthetic peptide arrays on membrane supports, we identified the residue preferences at each amino acid position of the SXIP motif and its surrounding sequence with respect to EB binding. We further developed an assay based on fluorescence polarization to assess the mechanism of the EB-SXIP interaction and to correlate EB binding and microtubule tip tracking of MtLS sequences from different +TIPs. Finally, we investigated the role of phosphorylation in regulating the EB-SXIP interaction. Together, our results define the sequence determinants of a canonical MtLS and provide the experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms.