A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Structural bioinformatics analysis of the family of human ubiquitin-specific proteases

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Easy Bioinformatics Analysis (EBiAn): a package for manipulating and analysis of short biological sequences

The work of biochemists and molecular biologists often is dependent or extremely favored by a preliminary computer analysis. Thus, the development of an efficient and friendly computational tool is very important. In this work, we developed a package of programs in Javascript language which can be used online or locally. The programs depend exclusively of Web browsers and are compatible with Internet Explorer, Opera, Mozilla Firefox and Google Chrome. With the EBiAn package it is can perform the main analysis and manipulation of DNA, RNA, proteins and peptides sequences. The programs can be freely accessed and adapted or modified to generate new programs.

Bioinformatics analysis of the Pedobacter sp. NL19 genome

The last decades of the 20th century defined the genetic engineering advent, climaxing in the development of techniques, such as PCR and Sanger sequencing. This, permitted the appearance of new techniques to sequencing whole genomes, identified as next-generation sequencing. One of the many applications of these techniques is the in silico search for new secondary metabolites, synthesized by microorganisms exhibiting antimicrobial properties. The peptide antibiotics compounds can be classified in two classes, according to their biosynthesis, in ribosomal or nonribosomal peptides. Lanthipeptides are the most studied ribosomal peptides and are characterized by the presence of lanthionine and methylanthionine that result from posttranslational modifications. Lanthipeptides are divided in four classes, depending on their biosynthetic machinery. In class I, a LanB enzyme dehydrate serine and threonine residues in the C-terminus precursor peptide. Then, these residues undergo a cyclization step performed by a LanC enzyme, forming the lanthionine rings. The cleavage and the transport of the peptide is achieved by the LanP and LanT enzymes, respectively. Although, in class II only one enzyme, LanM, is responsible for the dehydration and cyclization steps and also only one enzyme performs the cleavage and transport, LanT. Pedobacter sp. NL19 is a Gram-negative bacterium, isolated from sludge of an abandon uranium mine, in Viseu (Portugal). Antibacterial activity in vitro was detected against several Gram-positive and Gram-negative bacteria. Sequencing and in silico analysis of NL19 genome revealed the presence of 21 biosynthetic clusters for secondary metabolites, including nonribosomal and ribosomal peptides biosynthetic clusters. Four lanthipeptides clusters were predicted, comprising the precursor peptides, the modifying enzymes (LanB and LanC), and also a bifunctional LanT. This result revealed the hybrid nature of the clusters, comprising characteristics from two distinct classes, which are poorly described in literature. The phylogenetic analysis of their enzymes showed that they clustered within the bacteroidetes clade. Furthermore, hybrid gene clusters were also found in other species of this phylum, revealing that it is a common characteristic in this group. Finally, the analysis of NL19 colonies by MALDI-TOF MS allowed the identification of a 3180 Da mass that corresponds to the predicted mass of a lanthipeptide encoded in one of the clusters. However, this result is not fully conclusive and further experiments are needed to understand the full potential of the compounds encoded in this type of clusters. In conclusion, it was determined that NL19 strain has the potential to produce diverse secondary metabolites, including lanthipeptides that were not functionally characterized so far.

Association between prostinogen (KLK15) genetic variants and prostate cancer risk and aggressiveness in Australia and a meta-analysis of GWAS data

Background: Kallikrein 15 (KLK15)/Prostinogen is a plausible candidate for prostate cancer susceptibility. Elevated KLK15 expression has been reported in prostate cancer and it has been described as an unfavorable prognostic marker for the disease. Objectives: We performed a comprehensive analysis of association of variants in the KLK15 gene with prostate cancer risk and aggressiveness by genotyping tagSNPs, as well as putative functional SNPs identified by extensive bioinformatics analysis. Methods and Data Sources: Twelve out of 22 SNPs, selected on the basis of linkage disequilibrium pattern, were analyzed in an Australian sample of 1,011 histologically verified prostate cancer cases and 1,405 ethnically matched controls. Replication was sought from two existing genome wide association studies (GWAS): the Cancer Genetic Markers of Susceptibility (CGEMS) project and a UK GWAS study. Results: Two KLK15 SNPs, rs2659053 and rs3745522, showed evidence of association (p, 0.05) but were not present on the GWAS platforms. KLK15 SNP rs2659056 was found to be associated with prostate cancer aggressiveness and showed evidence of association in a replication cohort of 5,051 patients from the UK, Australia, and the CGEMS dataset of US samples. A highly significant association with Gleason score was observed when the data was combined from these three studies with an Odds Ratio (OR) of 0.85 (95% CI = 0.77-0.93; p = 2.7610 24). The rs2659056 SNP is predicted to alter binding of the RORalpha transcription factor, which has a role in the control of cell growth and differentiation and has been suggested to control the metastatic behavior of prostate cancer cells. Conclusions: Our findings suggest a role for KLK15 genetic variation in the etiology of prostate cancer among men of European ancestry, although further studies in very large sample sets are necessary to confirm effect sizes.

Analysis of kinase gene expression in the frontal cortex of suicide victims: Implications of fear and stress

Suicide is a serious public health issue that results from an interaction between multiple risk factors including individual vulnerabilities to complex feelings of hopelessness, fear, and stress. Although kinase genes have been implicated in fear and stress, including the consolidation and extinction of fearful memories, expression profiles of those genes in the brain of suicide victims are less clear. Using gene expression microarray data from the Online Stanley Genomics Database 1 and a quantitative PCR, we investigated the expression profiles of multiple kinase genes including the calcium calmodulin-dependent kinase (CAMK), the cyclin-dependent kinase, the mitogen-activated protein kinase (MAPK), and the protein kinase C (PKC) in the prefrontal cortex (PFC) of mood disorder patients died with suicide (N = 45) and without suicide (N = 38). We also investigated the expression pattern of the same genes in the PFC of developing humans ranging in age from birth to 49 year (N = 46). The expression levels of CAMK2B, CDK5, MAPK9, and PRKCI were increased in the PFC of suicide victims as compared to non-suicide controls (false discovery rate, FDR-adjusted p < 0.05, fold change >1.1). Those genes also showed changes in expression pattern during the postnatal development (FDR-adjusted p < 0.05). These results suggest that multiple kinase genes undergo age-dependent changes in normal brains as well as pathological changes in suicide brains. These findings may provide an important link to protein kinases known to be important for the development of fear memory, stress associated neural plasticity, and up-regulation in the PFC of suicide victims. More research is needed to better understand the functional role of these kinase genes that may be associated with the pathophysiology of suicide

Analysis of differential gene expression based on Bayesian estimation of variance

Gene expression is arguably the most important indicator of biological function. Thus identifying differentially expressed genes is one of the main aims of high throughout studies that use microarray and RNAseq platforms to study deregulated cellular pathways. There are many tools for analysing differentia gene expression from transciptomic datasets. The major challenge of this topic is to estimate gene expression variance due to the high amount of ‘background noise’ that is generated from biological equipment and the lack of biological replicates. Bayesian inference has been widely used in the bioinformatics field. In this work, we reveal that the prior knowledge employed in the Bayesian framework also helps to improve the accuracy of differential gene expression analysis when using a small number of replicates. We have developed a differential analysis tool that uses Bayesian estimation of the variance of gene expression for use with small numbers of biological replicates. Our method is more consistent when compared to the widely used cyber-t tool that successfully introduced the Bayesian framework to differential analysis. We also provide a user-friendly web based Graphic User Interface for biologists to use with microarray and RNAseq data. Bayesian inference can compensate for the instability of variance caused when using a small number of biological replicates by using pseudo replicates as prior knowledge. We also show that our new strategy to select pseudo replicates will improve the performance of the analysis. - See more at: http://www.eurekaselect.com/node/138761/article#sthash.VeK9xl5k.dpuf

AnaBench: a Web/CORBA-based workbench for biomolecular sequence analysis

Affiliation: Département de biochimie, Faculté de médecine, Université de Montréal

Proteornics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructurall protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Population and Computational Analysis of the MGEA6 P521A Variation as a Risk Factor for Familial Idiopathic Basal Ganglia Calcification (Fahr`s Disease)

Familial idiopathic basal ganglia calcification, also known as ""Fahr`s disease"" (FD), is a neuropsychiatric disorder with autosomal dominant pattern of inheritance and characterized by symmetric basal ganglia calcifications and, occasionally, other brain regions. Currently, there are three loci linked to this devastating disease. The first one (IBGC1) is located in 14q11.2-21.3 and the other two have been identified in 2q37 (IBGC2) and 8p21.1-q11.13 (IBGC3). Further studies identified a heterozygous variation (rs36060072) which consists in the change of the cytosine to guanine located at MGEA6/CTAGE5 gene, present in all of the affected large American family linked to IBGC1. This missense substitution, which induces changes of a proline to alanine at the 521 position (P521A), in a proline-rich and highly conserved protein domain was considered a rare variation, with a minor allele frequency (MAF) of 0.0058 at the US population. Considering that the population frequency of a given variation is an indirect indicative of potential pathogenicity, we screened 200 chromosomes in a random control set of Brazilian samples and in two nuclear families, comparing with our previous analysis in a US population. In addition, we accomplished analyses through bioinformatics programs to predict the pathogenicity of such variation. Our genetic screen found no P521A carriers. Polling these data together with the previous study in the USA, we have now a MAF of 0.0036, showing that this mutation is very rare. On the other hand, the bioinformatics analysis provided conflicting findings. There are currently various candidate genes and loci that could be involved with the underlying molecular basis of FD etiology, and other groups suggested the possible role played by genes in 2q37, related to calcium metabolism, and at chromosome 8 (NRG1 and SNTG1). Additional mutagenesis and in vivo studies are necessary to confirm the pathogenicity for variation in the P521A MGEA6.

Expression, purification and spectroscopic analysis of an HdrC: An iron-sulfur cluster-containing protein from Acidithiobacillus ferrooxidans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bioinformatics tools and databases for the study of human growth hormone

Advances in novel molecular biological diagnostic methods are changing the way of diagnosis and study of metabolic disorders like growth hormone deficiency. Faster sequencing and genotyping methods require strong bioinformatics tools to make sense of the vast amount of data generated by modern laboratories. Advances in genome sequencing and computational power to analyze the whole genome sequences will guide the diagnostics of future. In this chapter, an overview of some basic bioinformatics resources that are needed to study metabolic disorders are reviewed and some examples of bioinformatics analysis of human growth hormone gene, protein and structure are provided.

Proof of concept : A bioinformatic and serological screening method for identifying new peptide antigens for Chlamydia trachomatis related sequelae in women

This study aimed to identify new peptide antigens from Chlamydia (C.) trachomatis in a proof of concept approach which could be used to develop an epitope-based serological diagnostic for C. trachomatis related infertility in women. A bioinformatics analysis was conducted examining several immunodominant proteins from C. trachomatis to identify predicted immunoglobulin epitopes unique to C. trachomatis. A peptide array of these epitopes was screened against participant sera. The participants (all female) were categorized into the following cohorts based on their infection and gynecological history; acute (single treated infection with C. trachomatis), multiple (more than one C. trachomatis infection, all treated), sequelae (PID or tubal infertility with a history of C. trachomatis infection), and infertile (no history of C. trachomatis infection and no detected tubal damage). The bioinformatics strategy identified several promising epitopes. Participants who reacted positively in the peptide 11 ELISA were found to have an increased likelihood of being in the sequelae cohort compared to the infertile cohort with an odds ratio of 16.3 (95% c.i. 1.65 – 160), with 95% specificity and 46% sensitivity (0.19-0.74). The peptide 11 ELISA has the potential to be further developed as a screening tool for use during the early IVF work up and provides proof of concept that there may be further peptide antigens which could be identified using bioinformatics and screening approaches.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.