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At least 739 records · Page 41Linked to original sources

PubMed2000

Identification of novel multi-transmembrane proteins from genomic databases using quasi-periodic structural properties.

MOTIVATION: Identification of novel G protein-coupled receptors and other multi-transmembrane proteins from genomic databases using structural features. RESULTS: Here we describe a new algorithm for identifying multi-transmembrane proteins from genomic databases with a specific application to identifying G protein-coupled receptors (GPCRs) that we call quasi-periodic feature classifier (QFC). The QFC algorithm uses concise statistical variables as the 'feature space' to characterize the quasi-periodic physico-chemical properties of multi-transmembrane proteins. For the case of identifying GPCRs, the variables are then used in a non-parametric linear discriminant function to separate GPCRs from non-GPCRs. The algorithm runs in time linearly proportional to the number of sequences, and performance on a test dataset shows 96% positive identification of known GPCRs. The QFC algorithm also works well with short random segments of proteins and it positively identified GPCRs at a level greater than 90% even with segments as short as 100 amino acids. The primary advantage of the algorithm is that it does not directly use primary sequence patterns which may be subject to sampling bias. The utility of the new algorithm has been demonstrated by the isolation from the Drosophila genome project database of a novel class of seven-transmembrane proteins which were shown to be the elusive olfactory receptor genes of Drosophila.

Algorithms↗

PubMed2025

Cilia.Pro database of ciliary proteins from vertebrates, Chlamydomonas, and Caenorhabditis.

Cilia and flagella are microtubule-based organelles that generate force and sense the extracellular environment. In humans, these structures are essential for development, homeostasis, and reproduction, with defects contributing to a wide array of congenital and degenerative disorders. As cilia were present on the last common ancestor of all eukaryotes, research on cilia across model organisms holds significant relevance for understanding human disease. The green alga Chlamydomonas, which diverged from the human lineage with the animal-plant split, shares striking similarities in ciliary structure and function with humans. Two decades ago, our group published the proteome of the Chlamydomonas cilium, identifying hundreds of new ciliary proteins that were organized in an online database. Since then, advances have brought us a more comprehensive understanding of both Chlamydomonas and mammalian cilia. Our database, www.Cilia.Pro, has been continually updated to integrate proteomic, transcriptomic, and genomic data from Chlamydomonas and Caenorhabditis along with humans, and other vertebrates providing a valuable tool for the ciliary research community.

Cilia↗

PubMed1998

In vitro model system for the identification and characterization of proteins involved in inflammatory processes.

An in vitro model featuring important inflammatory cellular states was established, based on the murine monocyte/macrophage cell line RAW 264.7. Macrophages are key players in chronic inflammation, and major parts of the biochemical reactions taking place in vivo, e.g., the production of proinflammatory cytokines, can be triggered in vitro by stimulation of the cells with bacterial lipopolysaccharide (LPS). A mastergel, representing a synthetic image of the expressed basic set of cellular proteins, was designed by a computer-assisted overlay of a statistically significant number of two-dimensional electrophoresis (2-DE) gels of unstimulated RAW 264.7 cells. This image served as a reference for qualitative and quantitative changes in the protein pattern induced by stimulation of the macrophages with LPS. The optimal conditions for LPS stimulation were evaluated by monitoring the expression and secretion of the proinflammatory cytokine tumor necrosis factor-alpha(TNF-alpha). The comparison of the mastergel with the 2-DE gels of LPS-stimulated cells revealed several changes in the protein pattern. In order to prove the relevance of the presented model system, we focused on two low molecular weight proteins, which showed significant changes in the apparent concentration in a 2-DE pattern. These proteins were further characterized by microsequencing of internal peptides. A comparison of the obtained sequences with protein databases identified them as cofilin and keratinocyte lipid-binding protein.

Amino Acid Sequence↗

PubMed2003

Proteomics approach to identify wound-response related proteins from rice leaf sheath.

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.

Amino Acid Sequence↗

PubMed1996

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification.

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Amino Acid Sequence↗

PubMed1999

The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein.

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78,000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full-length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING-H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans, F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.

Amino Acid Sequence↗

PubMed2005

Purification and functional properties of Rab11-FIP2.

Rab11-FIP2 is a 512-amino acid protein that was first identified in a screen for Rab11 interacting proteins. Database analysis revealed that it belongs to a family of proteins characterized by the presence of a highly homologous domain located at its carboxy-termini. This family was termed the Rab11 family of interacting proteins (Rab11-FIPs), as all members have been demonstrated to interact with Rab11. The Rab11-FIPs can be further subdivided into two classes. Rab11-FIP2 belongs to the class I Rab11-FIPs due to the presence of a C2 domain near its amino-terminus. RCP and Rip11 are the other class I family members. A number of proteins that interact directly with Rab11-FIP2, in addition to Rab11, have been identified. These include the EH domain-containing protein Reps1, the AP-2 subunit alpha-adaptin, the actin-based motor protein myosin Vb, and the chemokine receptors CXCR2 and CXCR4. It is hypothesized that Rab11-FIP2 functions to transport cargo, such as chemokine receptors and the EGF receptor, from the endocytic recycling compartment (ERC) to the plasma membrane.

Amino Acid Sequence↗

PubMed1993

Identification of an Escherichia coli protein impurity in preparations of a recombinant pharmaceutical.

A host-cell protein impurity found in preparations of recombinant human acidic fibroblast growth factor (aFGF) was identified. Samples of aFGF examined by western blot analysis employing antiserum raised against an Escherichia coli cell lysate contained an immunoreactive protein with a molecular weight of approximately 26,000. The impurity was chromatographically isolated and the N-terminal sequence was determined. Comparing the sequence to a protein database provisionally identified the isolated impurity as the S3 ribosomal protein of E. coli. Monoclonal antibodies recognizing three separate epitopes of S3 confirmed the identity of the impurity in western blots of aFGF samples. The monoclonal antibodies were also used to estimate S3 levels in various preparations of aFGF.

Amino Acid Sequence↗

PubMed1999

Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains.

A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.

Amino Acid Sequence↗

PubMed1993

The G9a gene in the human major histocompatibility complex encodes a novel protein containing ankyrin-like repeats.

The class III region of the human major histocompatibility complex spans approx. 1.1 Mbp on the short arm of chromosome 6 and is known to contain at least 36 genes. The complete nucleotide sequence of a 3.4 kb mRNA from one of these genes, G9a (or BAT8), has been determined from cDNA and genomic DNA clones. The single-copy G9a gene encodes a protein product of 1001 amino acids with a predicted molecular mass of 111,518 Da. The C-terminal region (residues 730-999) of the G9a protein has been expressed in Escherichia coli as a fusion protein with the 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26). The fusion protein has been used to raise antisera which, in Western-blot analysis, cross-react specifically with an intracellular protein of approx. 98 kDa. The function of the G9a protein is unknown. However, comparison of the derived amino acid sequence of G9a with the protein databases has revealed interesting similarities with a number of other proteins. The C-terminal region of G9a is 35% identical with a 149 amino acid segment of the Drosophila trithorax protein. In addition the G9a protein has been shown to contain six contiguous copies of a 33-amino acid repeat. This repeat, originally identified in the Notch protein of Drosophila and known as the cdc10/SW16 or ANK repeat, is also found in a number of other human proteins and may be involved in intracellular protein-protein interactions.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of a porcine protein kinase gene and relationship to a class of heat shock proteins.

We have determined the genomic sequence of a porcine protein kinase (PPK) gene, including 1,844 bp upstream of the transcription initiation site. The gene spans over 19 kb and consists of 18 exons and 17 introns. The 5' regulatory region contains a characteristic heat shock element in the first intron, a weak heat shock element 1,464 bp upstream of the transcription initiation site, an atypical TATA box, and further consensus sequences typical for eukaryotic promoters such as an SP-1 binding site. Southern blot analysis indicates that PPK exists as a single-copy gene in the porcine haploid genome. The PPK gene is transcribed in all investigated tissues as shown by Northern blotting and reverse transcriptase polymerase chain reaction. Comparison of the protein and cDNA sequences of PPK to other sequences in DNA and protein databases indicates significant homology to a class of heat shock proteins, the glucose-regulated proteins (GRP94). In addition, nucleotide sequences at the 5' terminus of the PPK gene show strong homology to the GRP94 family. Domains highly conserved with human tumor rejection antigen (GP96) or glucose-regulated protein (GRP94) genes are identified within the 5' terminus and the first intron of the PPK gene. These findings suggest that these proteins are either identical or represent a family of closely related proteins.

Animals↗

PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗

PubMed2003

Effective interactions cannot replace solvent effects in a lattice model of proteins.

Protein folding and protein design are among the most challenging problems of the past ten years in biophysics and molecular biology. For a given protein, it is possible to extract, from existing protein databases, a set of specific (i.e., belonging to the investigated protein) effective amino-acid (AA) interactions able to stabilize the native state. On the other hand, attempts to find global effective AA interactions, which would be able to stabilize all proteins at once, failed. Using a simple lattice model where the solvent degrees of freedom are (semi)explicitly taken into account, we show that the absence of global effective AA interactions is due to the solvent and that on this lattice model the solvent effects cannot be reproduced by amino-acid effective interactions.

Hydrophobic and Hydrophilic Interactions↗

PubMed2002

Proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation.

BACKGROUND: Proteomic techniques have recently become available for large-scale protein analysis. The utility of these techniques in identification of urinary proteins is poorly defined. We constructed a proteome map of normal human urine as a reference protein database by using two differential fractionated techniques to isolate the proteins. METHODS: Proteins were isolated from urine obtained from normal human volunteers by acetone precipitation or ultracentrifugation, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry followed by peptide mass fingerprinting. RESULTS: A total of 67 protein forms of 47 unique proteins were identified, including transporters, adhesion molecules, complement, chaperones, receptors, enzymes, serpins, cell signaling proteins and matrix proteins. Acetone precipitated more acidic and hydrophilic proteins, whereas ultracentrifugation fractionated more basic, hydrophobic, and membrane proteins. Bioinformatic analysis predicted glycosylation to be the most common explanation for multiple forms of the same protein. CONCLUSIONS: Combining two differential isolation techniques magnified protein identification from human urine. Proteomic analysis of urinary proteins is a promising tool to study renal physiology and pathophysiology and to determine biomarkers of renal disease.

Acetone↗

PubMed2000

Identification of Protein Spots in Silver-stained Two-dimensional Gels by MALDI-TOF Mass Peptide Map Analysis.

Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including i) In-gel reduction, alkylation and enzymatic digestion ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)) iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrate that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this the procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S.Huwena were analyzed and identified.

Journal Article↗

PubMed2004

[Proteome study of colorectal cancer genesis and hepatic metastasis].

OBJECTIVE: To study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques. METHODS: Using isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins. RESULTS: Significant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP_040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis. CONCLUSION: Loss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.

Calmodulin↗

PubMed2005

[Expression of IgVH and B7-1 in proteome of the human colorectal carcinoma cell lines].

OBJECTIVE: To conduct a proteomic analysis of human colorectal carcinoma cell lines LS174T and SW480 by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). METHODS: The total proteins of human colorectal carcinoma cell lines LS174T and SW480 were separated with 2-DE using immobilized pH gradient strips and analyzed by MALDI-TOF-MS to obtain peptide mass fingerprints (PMFs). Proteins were identified by using Mascot software to search protein databases. RESULTS: Good resolution 2-DE maps were obtained. Some proteins including immunoglobulin heavy chain variable region (IgVH) and co-stimulatory molecule B7-1 were identified. IgVH and B7-1 were confirmed by electrospray ionization tandem spectrometry (ESI-MS/MS) and immunocytochemistry. CONCLUSION: There are IgVH and B7-1 expressions in human colorectal carcinoma cell lines LS174T and SW480. Results obtained will help to elucidate the mechanisms of tumor immune escape.

B7-1 Antigen↗

PubMed2006

[Comparative proteomics research on THP-1 cells infected with Brucella].

Brucella is a facultative intracellular pathogen that survives and multiplies inside host macrophages to cause brucellosis. The response of macrophage plays an essential role in the initiation of immune process following Brucella challenge. Nowadays, proteome approaches have been widely used in many different systems to investigate host-microbe interactions. The effect of pathogen-specific virulence mechanism can now be dissected using bacterial mutants and comparing different species. Attenuated vaccine strain 104M is defective in multification in host macrophage and is cleared relatively rapidly from tissues of the host, whereas virulent strains Brucella abortus 544A can produce chronic infection and cause brucellosis. In order to understand the underlying mechanisms of virulent Brucella intracellular survival, and detect the different-expressed proteins of THP-1 cells after infection with attenuated and virulent strains of Brucella abortus, a comparative proteomics research was conducted. Whole cellular protein profiling of THP-1 cells was presented by two dimensional (2D) electrophoresis and Coomassie Blue staining. After in-gel protein digestion, the different-expressed spots were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). All the peptide mass fingerprints (PMFs) were searched by the program Mascot developed by Matrix Science Ltd. For identifying proteins, database of hemo sapiens was used. A total of 38 proteins with changed expression level were found. These proteins can be grouped into two familes: (1) the expression level increased after infection with 544A; (2) the expression level increased after infection with 104M. Out of the 38 proteins, 10 were mainly in the field of signal transduction, 6 were cytoskeletal proteins, 8 were substance metabolism related proteins and 3 were cell stress and defense associated proteins. Functions of the remaining proteins were unknown. These results provide insight into the changed global protein patterns of THP-1 cells after infection as well as a comprehensive foundation to further study of host-bacterial interaction.

Brucella↗