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Biomedical subjects

M C Peitsch

Publications and source records attributed to M C Peitsch.

At least 19 recordsLinked to original sources
PubMed2001

MDB: a database system utilizing automatic construction of modules and STAR-derived universal language.

MOTIVATION: The value of information greatly increases if stored in databases. The objective was to construct a multi-purpose database system primarily designed to store and provide access to three-dimensional structures of biological molecules including theoretical models. RESULTS: A dictionary defining data format and structure for three-dimensional models of biological molecules (MDB dictionary) was developed. The dictionary was written using universal, standardized data description language. This language can be applied to describe data with no restrictions on their origin or type, including metadata. Thus both the data definitions (format) and database descriptions are created using the uniform language and processed with universal software. A database and data design technique that allowed use of dictionaries to automatically construct relational databases was developed. This technique was employed to construct the MDB database system. Data design developed and applied in the MDB project makes it possible to carry out data curation utilizing the database engine to identify errors. It also allows storage and query of data at different levels of consistency with the standard format specifications, i.e. both the correctly formatted data, and data that requires further curation. AVAILABILITY: The MDB dictionary is available at http://www.gwer.ch/proteinstructure/mdb and as part of the PDB resources at http://pdb.rutgers.edu/mmcif/.

Computational Biology↗
PubMed2000

Protein structure computing in the genomic era.

Functional analysis of the proteins discovered in fully sequenced genomes represents the next major challenge of life science research. Computational methods play a crucial role in this activity and, among them, comparative protein modelling is of great assistance during the rational design of mutagenesis experiments. Our aim over the last several years has been to further the use of 3-D model structures in this field. Therefore, we have developed a comparative protein modelling environment composed of the Swiss-PdbViewer (sequence to structure workbench and viewing program), SWISS-MODEL (internet-based server for model generation) and a database of a model generated with 3DCrunch.

Computational Biology↗
PubMed2000

Automated protein modelling--the proteome in 3D.

Functional analysis of the proteins discovered in fully sequenced genomes represent the next major challenge of life science research. Computational methods play an increasingly important role in this activity. Among them, comparative protein modelling will play a major role in this challenge, especially in the light of the Structural Genomics programmes about to be started around the world. In recent years, much progress has been made in automating these methods, enabling the production of models for genome scale problems. In this review we discuss how protein models can be applied to functional analysis, as well as some of the current issues and limitations inherent to these methods.

Animals↗
PubMed1999

Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.

Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.

Animals↗
PubMed1998

New insulin-like proteins with atypical disulfide bond pattern characterized in Caenorhabditis elegans by comparative sequence analysis and homology modeling.

We have identified three new families of insulin homologs in Caenorhabditis elegans. In two of these families, concerted mutations suggest that an additional disulfide bond links B and A domains, and that the A-domain internal disulfide bond is substituted by a hydrophobic interaction. Homology modeling remarkably confirms these predictions and shows that despite this atypical disulfide bond pattern and the absence of C-like peptide, all these proteins may adopt the same fold as the insulin. Interestingly, whereas we identified 10 insulin-like peptides, only one insulin-like-receptor (daf-2) has been found. We propose that these insulin-related peptides may correspond to different activators or inhibitors of the daf-2 insulin-regulating pathway.

Amino Acid Sequence↗
PubMed1997

Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3.

To contribute to the development of the transcription map of human chromosome 21 (HC21), we have used exon trapping from pools of HC21-specific cosmids. Using selected trapped exons, we have identified a novel gene (named TMPRSS2) that encodes a multimeric protein with a serine protease domain. The TMPRSS2 3.8-kb mRNA is expressed strongly in small intestine and weakly in several other tissues. The full-length cDNA encodes a predicted protein of 492 amino acids that contains the following domains: (i) A serine protease domain (aa 255-492) of the S1 family that probably cleaves at Arg or Lys residues. (ii) An SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) of group A (6 conserved Cys). This type of domain is involved in the binding to other cell surface or extracellular molecules. (iii) An LDLRA (LDL receptor class A) domain (aa 113-148). This type of domain forms a binding site for calcium. (iv) A predicted transmembrane domain (aa 84-106). No typical signal peptide was recognized. The gene was mapped to 21q22.3 between markers ERG and D21S56 in the same P1 as MX1. The physiological role of TMPRSS2 and its involvement in trisomy 21 phenotypes or monogenic disorders that map to HC21 are unknown.

Amino Acid Sequence↗
PubMed1997

Characterisation of macrophage inflammatory protein-5/human CC cytokine-2, a member of the macrophage-inflammatory-protein family of chemokines.

A human monocyte-activating CC chemokine has been identified based on sequences in an expressed sequence tag (EST) cDNA database. The protein shows highest sequence identity to the macrophage inflammatory protein (MIP) group of chemokines, particularly MIP-3 (76.7%) and MIP-1alpha (75.4%), and has been named MIP-5. Model building confirms that the protein has a similar three dimensional structure to other chemokines, but has an additional third disulphide bond. Northern blot analysis and reverse-transcriptase PCR show that the mRNA for MIP-5 is expressed at a high levels in liver, intestine and in lung leukocytes. MIP-5 induces chemotaxis of human monocytes, T-lymphocytes and, to a lesser degree, eosinophils at nanomolar concentrations; it has no effect on neutrophil migration. In receptor-binding assays, MIP-5 shows IC50 values of 12 nM for competition with 125I-MIP-1alpha for binding to CC-chemokine receptor (CCR)1, and 2.5 nM for competition with 125I-MCP-3 for binding to CCR3. It shows no ability to compete with ligand for binding to the two interleukin (IL)-8 receptors (CXC-chemokine receptors 1 and 2) or to CCR2, CCR4 or CCR5. Consistent with this binding data, MIP-5 was only able to induce calcium fluxes in CHO cells stably transfected with CCR1 or CCR3.

Amino Acid Sequence↗
PubMed1997

Spatial orientation of the alpha and betac receptor chain binding sites on monomeric human interleukin-5 constructs.

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with Kd values ranging from 20 to 200 microM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the betac contact residue on the first helix, Glu12, to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu12 on the first helix, and alternative mechanisms are discussed.

Binding Sites↗
PubMed1997

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.

Amino Acid Sequence↗
PubMed1997

Amino acid sequence of the alpha subunit and computer modelling of the alpha and beta subunits of echicetin from the venom of Echis carinatus (saw-scaled viper).

Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.

Amino Acid Sequence↗
PubMed1997

SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.

Comparative protein modeling is increasingly gaining interest since it is of great assistance during the rational design of mutagenesis experiments. The availability of this method, and the resulting models, has however been restricted by the availability of expensive computer hardware and software. To overcome these limitations, we have developed an environment for comparative protein modeling that consists of SWISS-MODEL, a server for automated comparative protein modeling and of the SWISS-PdbViewer, a sequence to structure workbench. The Swiss-PdbViewer not only acts as a client for SWISS-MODEL, but also provides a large selection of structure analysis and display tools. In addition, we provide the SWISS-MODEL Repository, a database containing more than 3500 automatically generated protein models. By making such tools freely available to the scientific community, we hope to increase the use of protein structures and models in the process of experiment design.

Amino Acid Sequence↗
PubMed1997

The chemokine information source: identification and characterization of novel chemokines using the WorldWideWeb and expressed sequence tag databases.

The chemokine superfamily is a large group of more than 30 small proteins. Many of these were originally identified because of their role in the selective recruitment and activation of leukocytes during inflammation. More recently, some of the chemokine receptors and ligands have been implicated in the mechanism of viral infection for primate lentiviruses such as HIV-1. From the original identification of interleukin-8 (IL-8; the most studied member of the superfamily), the number of new family members has mushroomed over the last few years. Two events have dramatically altered the speed at which sequence information concerning novel chemokines has become available to the scientific community. First, many groups have been obtaining large amounts of sequence information from cDNA libraries by sequencing the clones at random, generating expressed sequence tags (ESTs). Although these ESTs are relatively short, typically less than 500 bases, this amount of sequence is usually sufficient to obtain the entire open reading frame for chemokines. Second, there has been a rapid growth in the use of the WorldWideWeb by bioinformatics groups. The Web was originally set up by the European Centre for Particle Physics (CERN) in Geneva as a method of transferring data between collaborating groups throughout the world. It has enabled biologists throughout the world to have almost instantaneous access both to the databases containing the EST sequences and to the automated tools that are required for searching such databases. With such methods we have been able to rapidly identify more than 10 new human chemokines from public domain databases. In addition to the known categories of chemokines, which are named C, CC, and CXC based on the spacings of N-terminal cysteine residues, we have been able to identify the first member of a novel chemokine subfamily, with a novel CXXXC cysteine spacing. Furthermore, we can subdivide the CC chemokines into monocyte chemotactic protein and macrophage inflammatory protein families based on their sequence identity levels, but also their clustering on the human genome, as identified on other Web sites. The rapid availability of all this data has reduced the amount of time spent on conventional gene identification, enabling us to move quickly on to trying to understand the biology and physiological relevance of these molecules. The novel chemokine sequences obtained and alignments with existing members of the superfamily are now contained within a Chemokine Information Source on an open access server, allowing further searching of chemokine sequences and increasing the availability of such data to the scientific community.

Amino Acid Sequence↗
PubMed1997

Clinical spectrum of X-linked hyper-IgM syndrome.

We report the clinical and immunologic features and outcome in 56 patients with X-linked hyper-IgM syndrome, a disorder caused by mutations in the CD40 ligand gene. Upper and lower respiratory tract infections (the latter frequently caused by Pneumocystis carinii), chronic diarrhea, and liver involvement (both often associated with Cryptosporidium infection) were common. Many patients had chronic neutropenia associated with oral and rectal ulcers. The marked prevalence of infections caused by intracellular pathogens suggests some degree of impairment of cell-mediated immunity. Although lymphocyte counts and in vitro proliferation to mitogens were normal, a defective in vitro proliferative response to antigens was observed in some patients, and additional defects of cell-mediated immunity may be presumed on the basis of current knowledge of CD40-ligand function. All patients received regular infusions of immunoglobulins. Four patients underwent liver transplantation because of sclerosing cholangitis, which relapsed in there. Three patients underwent bone marrow transplantation. Thirteen patients (23%) died of infection and/or liver disease. X-linked hyper-IgM syndrome, once considered a clinical variant of hypogammaglobulinemia, is a severe immunodeficiency with significant cellular involvement and a high mortality rate.

Bone Marrow Transplantation↗
PubMed1997

Structure and bioactivity of recombinant human CTAP-III and NAP-2.

Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the alpha-granules of platelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli. We have purified and renatured these recombinant proteins. The integrity of the recombinant proteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10(-7) M, whereas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC50 of 2 x 10(-8) M in chemotaxis, an EC50 of 3 x 10(-8) M for shape change, and could displace IL-8 from neutrophils with a Kd of 7.5 x 10(-9) M. CTAP-III had no activity in these assays. The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies. We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix-helix interaction that could stabilize the association of two CTAP-III dimers.

Amino Acid Sequence↗
PubMed1997

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1).

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.

Amino Acid Sequence↗
PubMed1997

Large scale protein modelling and model repository.

Knowledge-based molecular modelling of proteins has proven useful in many instances including the rational design of mutagenesis experiments, but it has been generally limited by the availability of expensive computer hardware and software. To overcome these limitations, we have developed the SWISS-MODEL server for automated knowledge-based protein modelling. The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known 3D-structure. We now use the software framework of the server to generate large collections of protein models. To store these models, we have established the SWISS-MODEL Repository, a new database for protein models generated by theoretical approaches. This repository is directly integrated with SWISS-PROT and other databases through the ExPASy World-Wide Web server (URL is http:(/)/www.expasy.ch).

Amino Acid Sequence↗