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PubMed2002

In Ssarch of new anti-bacterial target genes: a comparative/structural genomics approach.

We outline a joint academic/industrial (CNRS/AVENTIS) functional genomics project aiming at the discovery of new anti-bacterial gene targets. Starting from all publicly available bacterial genomes, a subset of the most evolutionary conserved protein-coding genes has been identified. We retained genes with clear homolog in E. coli and at least one gram-positive bacterium among B.subtilis, M. tuberculosis, L. lactis or S. pyogenes. This subset was further reduced to genes encoding non-membrane proteins of unknown or hypothetical functions. The 221 E. coli Open Reading Frames (ORFs) identified through this comprehensive bioinformatic analysis are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallisation and X-ray diffraction. Our strategy was designed to focus on promising wide-spectrum targets as well as original biochemical pathways. Bioinformatics is used throughout all phases of project, including the initial large-scale comparative genomics analyses, the purification/expression and crystallisation stages for the detection of helpful sequence-specific features (e.g. cofactor binding motifs, non-structured N- or C- term extremities, etc ), and finally for the interpretation of the structures in conjunction with multiple sequence alignments for the identification of key residues, interaction areas on molecular surfaces, and overall function predictions.

Anti-Infective Agents↗

PubMed2003

Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators.

Resistin is a cysteine-rich protein postulated to be a molecular link between obesity and type 2 diabetes. The aim of this study was to investigate the role of PPAR gamma in the regulation of resistin expression in human primary macrophages. Fluorescent real-time PCR (Taqman) analysis of resistin expression across a range of human tissues showed that resistin is highly expressed in bone marrow compared to other tissues. Taqman analysis and Western blotting showed that rosiglitazone decreased resistin expression at both the mRNA and protein levels in human primary monocyte-derived macrophages in vitro. Resistin expression was reduced by up to 80% after exposure to 100 nM rosiglitazone for 96 h. Bioinformatics analysis of the genomic sequence upstream of the resistin coding sequence identified several putative PPAR response elements of which one was shown to bind PPAR gamma using electrophoretic mobility shift assays. Our data support a direct role for PPAR gamma in the regulation of resistin expression.

Base Sequence↗

PubMed2005

Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica.

The length of the needle ending the Yersinia Ysc injectisome is determined by YscP, a protein acting as a molecular ruler. In addition, YscP is required for Yop secretion. In the present paper, by a systematic deletion analysis, we localized accurately the region required for Yop secretion between residues 405 and 500. As this C-terminal region of YscP has also been shown to control needle length it probably represents the substrate specificity switch of the machinery. By a bioinformatics analysis, we show that this region has a globular structure, an original alpha/beta fold, a P-x-L-G signature and presumably no catalytic activity. In spite of very limited sequence similarities, this structure is conserved among the proteins that are presumed to control the needle length in many different injectisomes and also among members of the FliK family, which control the flagellar hook length. This region thus represents a new protein domain that we called T3S4 for Type III secretion substrate specificity switch. The T3S4 domain of YscP can be replaced by the T3S4 domain of AscP (Aeromonas salmonicida) or PscP (Pseudomonas aeruginosa) but not by the one from FliK, indicating that in spite of a common global structure, these domains need to fit their partner proteins in the secretion apparatus.

Amino Acid Sequence↗

PubMed2004

Molecular and physiological analysis of the role of osmolyte transporters BetL, Gbu, and OpuC in growth of Listeria monocytogenes at low temperatures.

Listeria monocytogenes is a ubiquitous food-borne pathogen found widely distributed in nature as well as an undesirable contaminant in a variety of fresh and processed foods. This ubiquity can be at least partly explained by the ability of the organism to grow at high osmolarity and reduced temperatures, a consequence of its ability to accumulate osmo- and cryoprotective compounds termed osmolytes. Single and multiple deletions of the known osmolyte transporters BetL, Gbu, and OpuC significantly reduce growth at low temperatures. During growth in brain heart infusion broth at 7 degrees C, Gbu and OpuC had a more pronounced role in cryoprotection than did BetL. However, upon the addition of betaine to defined medium, the hierarchy of transporter importance shifted to Gbu > BetL > OpuC. Upon the addition of carnitine, only OpuC appeared to play a role in cryoprotection. Measurements of the accumulated osmolytes showed that betaine is preferred over carnitine, while in the absence of a functional Gbu, carnitine was accumulated to higher levels than betaine was at 7 degrees C. Transcriptional analysis of the genes encoding BetL, Gbu, and OpuC revealed that each transporter is induced to different degrees upon cold shock of L. monocytogenes LO28. Additionally, despite being transcriptionally up-regulated upon cold shock, a putative fourth osmolyte transporter, OpuB (identified by bioinformatic analysis and encoded by lmo1421 and lmo1422), showed no significant contribution to listerial chill tolerance. Growth of the quadruple mutant LO28deltaBCGB (deltabetL deltaopuC deltagbu deltaopuB) was comparable to the that of the triple mutant LO28deltaBCGsoe (deltabetL deltaopuC deltagbu) at low temperatures. Here, we conclude that betaine and carnitine transport upon low-temperature exposure is mediated via three osmolyte transporters, BetL, Gbu, and OpuC.

ATP-Binding Cassette Transporters↗

PubMed2003

Analysis of two large functionally uncharacterized regions in the Methanopyrus kandleri AV19 genome.

BACKGROUND: For most sequenced prokaryotic genomes, about a third of the protein coding genes annotated are "orphan proteins", that is, they lack homology to known proteins. These hypothetical genes are typically short and randomly scattered throughout the genome. This trend is seen for most of the bacterial and archaeal genomes published to date. RESULTS: In contrast we have found that a large fraction of the genes coding for such orphan proteins in the Methanopyrus kandleri AV19 genome occur within two large regions. These genes have no known homologs except from other M. kandleri genes. However, analysis of their lengths, codon usage, and Ribosomal Binding Site (RBS) sequences shows that they are most likely true protein coding genes and not random open reading frames. CONCLUSIONS: Although these regions can be considered as candidates for massive lateral gene transfer, our bioinformatics analysis suggests that this is not the case. We predict many of the organism specific proteins to be transmembrane and belong to protein families that are non-randomly distributed between the regions. Consistent with this, we suggest that the two regions are most likely unrelated, and that they may be integrated plasmids.

Amino Acids↗

PubMed2004

Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

Amino Acid Motifs↗

PubMed2006

G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein.

G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is the first selenoprotein identified in the Golgi apparatus.

Amino Acid Sequence↗

PubMed2026

Increased IL4I1 expression predicts poor survival and modulates the immune microenvironment in acute myeloid leukemia.

BACKGROUND: The immunometabolic enzyme Interleukin-4-induced-1 (IL4I1) is implicated in cancer pathogenesis, yet its specific function and clinical relevance in acute myeloid leukemia (AML) remain unclear. METHODS: Comparative analysis of IL4I1 mRNA levels between AML patients and normal controls was performed using the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. The Kaplan–Meier survival analysis was conducted to evaluate the prognostic value of IL4I1. Functional insights were derived from analyses of differentially expressed genes (DEGs), Gene Set Enrichment Analysis (GSEA), and Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Immune infiltration was evaluated using the ssGSEA, ESTIMATE, quanTIseq and single-cell RNA sequencing (scRNA-seq) analysis. Finally, in vitro and in vivo functional experiments were perfromed to explore the impact of IL4I1 on AML progression and immunoregulation. RESULTS: IL4I1 expression was significantly elevated in AML compared to normal controls (p = 0.0004) and associated with poorer overall survival (p = 0.003). Bioinformatic analysis revealed that IL4I1 was linked to immune-related pathways—including humoral immune response, leukocyte interactions, and chemokine signaling—and to cellular amino acid metabolism. Its expression correlated with immune cell infiltration and checkpoint molecule expression. Experimentally, IL4I1 promoted leukemia cell proliferation in vitro and in vivo (p < 0.05). Furthermore, silencing IL4I1 suppressed M2 macrophage polarization and reduced secretion of inflammatory factors (p < 0.05). CONCLUSIONS: IL4I1 may serve as a potential biomarker for poor prognosis and an attractive target for immune-based therapeutic interventions in AML.

Humans↗

PubMedDate not supplied

Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry.

This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.

Amino Acid Sequence↗

PubMed2004

Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling.

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

Amino Acid Sequence↗

PubMed2006

Evolutionary analysis of fructose 2,6-bisphosphate metabolism.

Fructose 2,6-bisphosphate is a potent metabolic regulator in eukaryotic organisms; it affects the activity of key enzymes of the glycolytic and gluconeogenic pathways. The enzymes responsible for its synthesis and hydrolysis, 6-phosphofructo-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) are present in representatives of all major eukaryotic taxa. Results from a bioinformatics analysis of genome databases suggest that very early in evolution, in a common ancestor of all extant eukaryotes, distinct genes encoding PFK-2 and FBPase-2, or related enzymes with broader substrate specificity, fused resulting in a bifunctional enzyme both domains of which had, or later acquired, specificity for fructose 2,6-bisphosphate. Subsequently, in different phylogenetic lineages duplications of the gene of the bifunctional enzyme occurred, allowing the development of distinct isoenzymes for expression in different tissues, at specific developmental stages or under different nutritional conditions. Independently in different lineages of many unicellular eukaryotes one of the domains of the different PFK-2/FBPase-2 isoforms has undergone substitutions of critical catalytic residues, or deletions rendering some enzymes monofunctional. In a considerable number of other unicellular eukaryotes, mainly parasitic organisms, the enzyme seems to have been lost altogether. Besides the catalytic core, the PFK-2/FBPase-2 has often N- and C-terminal extensions which show little sequence conservation. The N-terminal extension in particular can vary considerably in length, and seems to have acquired motifs which, in a lineage-specific manner, may be responsible for regulation of catalytic activities, by phosphorylation or ligand binding, or for mediating protein-protein interactions.

Animals↗

PubMed2006

Selection of target sites for mobile DNA integration in the human genome.

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn--thus, answering the question "What genomic features affect integration?" requires carefully specifying the length scale of interest.

Binding Sites↗

PubMed2001

Secondary DNA structure analysis of the coding strand switch regions of five Leishmania major Friedlin chromosomes.

As part of the EULEISH international genome project, a region of 74,674 nucleotides from chromosome 21 of Leishmania major Friedlin was subcloned and sequenced; and 31 new coding sequences were predicted. Of particular interest was a unique coding strand switching region covering 1.6 kb of DNA; and this was subjected to further investigation. Bioinformatic analysis of this region revealed an unusually high AT composition, a lack of putative hairpins and a strong curvature of the DNA in agreement with the structural characteristics of similar regions of other Leishmania chromosomes. These observations and a comparison with the secondary DNA structure of four other Leishmania chromosomes and chromosomes of different organisms could suggest a functional role of this region in transcription and mitotic division.

Animals↗

PubMed2005

[Preliminary analysis of retinal gene expression profile of diabetic rat].

OBJECTIVE: Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat and comparing the profiles in order to analyze the possible genes related with diabetic retinopathy. METHODS: The whole retinal transcriptional fragments of non-diabetic rat and 8-week diabetic rat were obtained by restriction fragments differential display-PCR (RFDD-PCR). Bioinformatic analysis of retinal gene expression was performed using soft wares, including Fragment Analysis. After comparison of the expression profiles, the related gene fragments of diabetic retinopathy were initially selected as the target gene of further approach. RESULTS: A total of 3639 significant fragments were obtained. By means of more than 3-fold contrast of fluorescent intensity as the differential expression standard, the authors got 840 differential fragments, accounting for 23.08% of the expressed numbers and including 5 visual related genes, 13 excitatory neruotransmitter genes and 3 inhibitory neurotransmitter genes. At the 8th week, the expression of Rhodopsin kinase, beta-arrestin, Phosducinìrod photoreceptor cGMP-gated channel and Rpe65 as well as iGlu R1-4 were down-regulated. mGluRs and GABA-Rs were all up-regulated, whereas the expression of GlyR was unchanged. CONCLUSION: These results prompt again that the changes in retinal nervous layer of rat have occurred at an early stage of diabetes. The genes expression pattern of visual related genes and excitatory and inhibitory neurotransmitters in rat diabetic retina have been involved in neuro-dysfunctions of diabetic retina.

Animals↗

PubMed2006

Identification and characterization of two mature isoforms of retinoschisin in murine retina.

Retinoschisin (RS) is a 24 kDa secreted protein expressed in retina and is required for the structural and functional integrity of the retina. RS has been predicted to serve as an adhesive protein but the precise molecular mechanism by which it functions in retina is not yet known. During investigations on structural and functional aspects of RS in murine retina using proteomic tools, we identified two isoforms of RS that differed in mass by 200 Da with no apparent change in charge. Mass spectra and amino acid sequence analysis of the tryptic peptides revealed that these isoforms differed by two amino acids at the N-terminus which suggested processing of RS signal sequence at two cleavage sites by signal peptidase as the basic mechanism underlying the occurrence of two mature RS isoforms in retina. Bioinformatic analysis identified two potential cleavage sites (between amino acids 21-22 and 23-24) in RS signal sequence. The flexibility of the signal peptidase to cleave at two sites is correlated to the amino acid composition of the RS signal sequence. This finding represents a rare example of a naturally occurring signal sequence cleavage at more than one site in vivo.

Amino Acid Sequence↗

PubMed2005

Isolation and characterization of a cDNA encoding two novel heat-shock factor OsHSF6 and OsHSF12 in Oryza sativa L.

As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heatshock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes.

Amino Acid Sequence↗

PubMed2004

Analysis of transcriptional regulation of the small leucine rich proteoglycans.

PURPOSE: Small leucine rich proteoglycans (SLRPs) constitute a family of secreted proteoglycans that are important for collagen fibrillogenesis, cellular growth, differentiation, and migration. Ten of the 13 known members of the SLRP gene family are arranged in tandem clusters on human chromosomes 1, 9, and 12. Their syntenic equivalents are on mouse chromosomes 1, 13, and 10, and rat chromosomes 13, 17, and 7. The purpose of this study was to determine whether there is evidence for control elements, which could regulate the expression of these clusters coordinately. METHODS: Promoters were identified using a comparative genomics approach and Genomatix software tools. For each gene a set of human, mouse, and rat orthologous promoters was extracted from genomic sequences. Transcription factor (TF) binding site analysis combined with a literature search was performed using MatInspector and Genomatix' BiblioSphere. Inspection for the presence of interspecies conserved scaffold/matrix attachment regions (S/MARs) was performed using ElDorado annotation lists. DNAseI hypersensitivity assay, chromatin immunoprecipitation (ChIP), and transient transfection experiments were used to validate the results from bioinformatics analysis. RESULTS: Transcription factor binding site analysis combined with a literature search revealed co-citations between several SLRPs and TFs Runx2 and IRF1, indicating that these TFs have potential roles in transcriptional regulation of the SLRP family members. We therefore inspected all of the SLRP promoter sets for matches to IRF factors and Runx factors. Positionally conserved binding sites for the Runt domain TFs were detected in the proximal promoters of chondroadherin (CHAD) and osteomodulin (OMD) genes. Two significant models (two or more transcription factor binding sites arranged in a defined order and orientation within a defined distance range) were derived from these initial promoter sets, the HOX-Runx (homeodomain-Runt domain), and the ETS-FKHD-STAT (erythroblast transformation specific-forkhead-signal transducers and activators of transcription) models. These models were used to scan the genomic sequences of all 13 SLRP genes. The HOX-Runx model was found within the proximal promoter, exon 1, or intron 1 sequences of 11 of the 13 SLRP genes. The ETS-FKHD-STAT model was found in only 5 of these genes. Transient transfections of MG-63 cells and bovine corneal keratocytes with Runx2 isoforms confirmed the relevance of these TFs to expression of several SLRP genes. Distribution of the HOX-Runx and ETS-FKHD-STAT models within 200 kb of genomic sequence on human chromosome 9 and 500 kb sequence on chromosome 12 also were analyzed. Two regions with 3 HOX-Runx matches within a 1,000 bp window were identified on human chromosome 9; one located between OMD and osteoglycin (OGN)/mimecan genes, and the second located upstream of the putative extracellular matrix protein 2 (ECM2) promoter. The intergenic region between OMD and mimecan was shown to coincide with different patterns of DNAse I hypersensitivity sites in MG-63 and U937 cells. ChiP analysis revealed that this region binds Runx2 in U937 cells (mimecan transcript note detectable), but binds Pitx3 in MG-63 cells (expressing high level of mimecan), thereby demonstrating its functional association with mimecan expression. Upon comparing the predictions of S/MARs on the relevant chromosomal context of human chromosomes 9 and 12 and their rodent equivalents, no convincing evidence was found that the tandemly arranged genes build a chromosomal loop. CONCLUSIONS: Twelve of 13 known SLRP genes have at least one HOX-Runx module match in their promoter, exon 1, intron 1, or intergenic region. Although these genes are located in different clusters on different chromosomes, the common HOX-Runx module could be the basis for co-regulated expression.

Animals↗

PubMed2003

Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists.

TGF-beta family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.

Animals↗