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Biomedical subjects

M C Peitsch

Publications and source records attributed to M C Peitsch.

At least 37 records · Page 2Linked to original sources
PubMed1996

alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are efficiently phosphorylated in a novel protein kinase C motif.

Protein kinase C (PKC) phosphorylates the catalytic alpha 1 subunit of Na,K-ATPase in purified enzyme preparations and in intact cells. Little is known, however, whether all three known alpha isoforms are substrates for PKC and whether direct phosphorylation is implicated in the modulation of the transport activity of the different Na,K-ATPase isozymes. In this study, we investigated the structural requirements for PKC phosphorylation of alpha 1, alpha 2, and alpha 3 isoforms of different species after expression in Xenopus oocytes. By using a combination of site-directed mutagenesis and computer-assisted protein modeling, we characterized a novel Ser-X-His motif which in concert with more distantly located basic residues acts as an efficient substrate for PKC-mediated phosphorylation in the N-terminus of most Na,K-ATPase alpha 1 isoforms. As indicated by controlled proteolysis, alpha 2 isoforms are also phosphorylated in the N-terminus but to a much lower extent than alpha 1 isoforms containing the Ser-X-His motif. Phosphorylation and phosphoamino acid analysis of fusion proteins containing the wild-type or mutant N-terminus of alpha 2 reveal that Thr-Thr-Ser-X-Asn or Thr-Thr-Ala-X-Asn motifs represent weak targets for PKC phosphorylation. Finally, our data suggest that, with the exception of rat alpha 3, all alpha 3 isoforms from other species are not substrates for PKC. On the basis of the phosphorylation efficiency, we may speculate that only alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are likely candidates for regulatory PKC phosphorylation.

Amino Acid Sequence↗
PubMed1996

Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper).

Several studies have shown that Asp-49 is the residue that controls calcium binding in, and so plays a critical role in the calcium-mediated activation of, low-M(r) group I-III phospholipases A2 (PLA2s). The present paper provides experimental evidence that Asp-49 is not an absolute prerequisite for the enzymic activity of PLA2s, and that proteins with amino acid(s) other than Asp at position 49 can exhibit significant phospholipase activity. The purification, complete amino acid sequence and characterization of ecarpholin S, a PLA2 from Echis carinatus sochureki (saw-scaled viper) venom, is described. This single-chain, 122-amino-acid, basic (pI 7.9) protein is a group II PLA2. Although Asp-49 is replaced by Ser and Tyr-28 by Phe (both of these positions being involved in the Ca(2+)-binding site of PLA2s), the lipolysis of soybean phosphatidylcholine and egg yolk in the presence of 10 mM CaCl2 was 1.5 times and 2.9 times greater respectively with ecarpholin S than with recombinant human group II PLA2. The Ca(2+)-dependencies of the enzymic activities of ecarpholin S and rPLA2 were found to be similar. Ecarpholin S added to washed platelets induced aggregation; the presence of Ca2+ was a prerequisite for this platelet-aggregating effect. Computer modelling of the Ca(2+)-binding site of Ser-49 PLA2 compared with the Asp-49 and Lys-49 forms, for which crystallographic data exist, shows that the Ca(2+)-binding site is sterically blocked by Lys-49 but not by Ser-49; in the latter, the Ser hydroxy group may replace the Asp carboxylate in stabilization of Ca2+ binding. Sequence comparisons of ecarpholin S and other low-M(r) PLA2s predicts the presence of a Ser-49 group in the protein family of low-M(r) PLA2s that is distinct from the Asp-49 and Lys-49 groups.

Amino Acid Sequence↗
PubMed1996

Selectivity and antagonism of chemokine receptors.

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

Amino Acid Sequence↗
PubMed1996

The carboxy-terminal region of human interleukin-5 is essential for maintenance of tertiary structure but not for dimerization.

The C-terminal region of interleukin-5 has previously been suggested to be important for biological activity [Mackenzie et al., (1991), Mol. Immunol. 28, 155-158; Kodama et al. (1991), Biochem. Biophys. Res. Commun. 178, 514-519]. We have investigated this region by making a series of truncation mutants. The proteins were expressed in Escherichia coli, purified from inclusion bodies, and were able to refold with the disulfide homodimeric topology typical of interleukin-5. Analysis of the truncated carboxy-terminal proteins in an interleukin-5-dependent proliferation assay on TF-1 cells showed a rapid loss of activity as the C-terminal was shortened by more than two amino acids. This loss of biological activity correlated with a drop in binding affinity to both the alpha chain of the receptor and the high-affinity complex consisting of the alpha and beta subunits. Analysis of the proteins by 1H-NMR showed that the truncated mutants have higher exchange rates with solvent, indicating a less rigid structure. The carboxy-terminal region is therefore necessary to maintain the stability of the four-helix bundle and to orient correctly the important residues of the fourth helix. Inspection of the structure determined by X-ray crystallography shows that Trp-110 acts as the major residue in anchoring the fourth helix.

Amino Acid Sequence↗
PubMed1996

Increased p34cdc2-dependent kinase activity during apoptosis: a possible activation mechanism of DNase I leading to DNA breakdown.

Cells undergoing apoptosis typically exhibit distinctive morphological characteristics. Early events include the rounding up of the cell, chromatin condensation, nuclear membrane breakdown and blebbing of the cellular membrane. Strikingly similar changes take place in the cell cycle progression, at the entry into mitosis, suggesting a link between mitosis and apoptosis. Here we show that expression of active p34cdc2 at inappropriate phases during the cell cycle leads to morphological changes reminiscent of apoptosis, including DNA degradation. Cells cotransfected with the active mutant of p34cdc2 and DNase I displayed degraded DNA, which was absent in p34cdc2 wild-type and DNase I-transfected cells, in spite of similar DNase activities. Upon induction of apoptosis in thymocytes, transient p34cdc2 activation was detected prior to lamina breakdown and nuclease activation. P34cdc2 activation was also observed during APO-1 (Fas/CD95)-induced apoptosis in a B lymphoblastoma cell line. Our results suggest that unscheduled activation of p34cdc2 may participate in the initiation of the typical apoptotic phenotype.

Animals↗
PubMed1996

Automated modelling of the transmembrane region of G-protein coupled receptor by Swiss-model.

Molecular modelling of the transmembrane helices of G-protein coupled receptors is an increasingly used method to identify the possible three-dimensional environment of key residues. Thereby site-directed mutagenesis experiments, aimed at the understanding of the receptor-ligand interactions, can be designed in a rational way. The modelling methods are however not generally available to experimentalists, and often require expensive software and hardware. To overcome these limitations, we have constructed a World Wide Web server for the automated protein modelling of user-defined transmembrane helices. The service is freely available at this address: http:/(/)expasy.hcuge.ch/swissmod/SWISS-MODEL.++ +html.

Animals↗
PubMed1995

Role of a hydrophobic pocket of the human Y1 neuropeptide Y receptor in ligand binding.

We are investigating the nature of the chemical interactions between the neuropeptide Y (NPY) and its cell surface receptor (Y1). A previous study involving site-directed mutagenesis and computer-aided modelling (Walker et al., 1994) suggested that the C-terminal Tyr36 of NPY, known to be a key residue for receptor binding, might dock at a pocket formed by hydrophobic amino acids of transmembrane domains (TM) 1, 2, 6 and 7 of the Y1 receptor. To investigate which residues were required for ligand binding, we mutated the sequences encoding F41, L43, F96, Y100, F286 and H298 of the human Y1 receptor. The mutant cDNAs were transiently expressed in Hela cells and the ability of the encoded proteins to bind NPY was evaluated. Replacing F41, L43 or F96 with alanines had no effect on NPY binding. On the contrary, Y100, F286 and H298 appeared to be residues critical for ligand binding. In particular, the removal of the hydroxyl group of Y100 (Tyr100-->Phe100 mutation) yielded a protein devoid of affinity for the ligand. The level of expression and the presence on the cell surface of mutants lacking NPY binding activity was assessed by immunological techniques. In addition, we tested the ability of synthetic analogues of neuropeptide Y with substitutions at position 36 to bind to the Y1 receptor. To get spatial insight into the relative positions of the above mentioned residues we constructed a molecular model of the interaction between NPY:Y36 and the elements of the hydrophobic pocket surrounding this residue.

Base Sequence↗
PubMed1995

Identification of key charged residues of human interleukin-5 in receptor binding and cellular activation.

Interleukin-5 (IL-5) is a cytokine that plays a major role in the differentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its receptor and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purified. They were assayed for ability to cause proliferation of the erythroleukaemic cell line TF-1 and the up-regulation of eosinophil adhesion to ICAM-1. In addition, we studied receptor binding using either immobilized recombinant IL-5 receptor alpha-chain or the alpha/beta-receptor complex expressed on TF-1 cells. The key charged residue involved in binding to the beta-chain of the receptor is Glu-12. This residue is in an identical position to those previously identified in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) involved in binding to the receptor beta-chain. The alpha-chain binding site is shown to involve the side chains Arg-90 and Glu-109, located in the second beta sheet and after the end of the fourth helix, respectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF. Understanding the topology of the interaction of IL-5 with its receptor chains will help in the search for rationally designed antagonists of IL-5 function.

Amino Acid Sequence↗
PubMed1995

Genomic organisation and expression of mouse deoxyribonuclease I.

Deoxyribonuclease I (DNase I) has recently been implicated in cell death by apoptosis, a process which is frequently accompanied by chromatin DNA degradation. Despite extensive studies on DNase I, its genomic organisation remained unknown. Here we report for the first time on the intron-exon structure of the DNase I gene. The coding region of mouse DNase I is composed of eight introns and eight exons, spanning 2315 base pairs. The deduced protein sequence is 91.5% identical to its rat counterpart, but does not carry the two mutations (Glu13 to D and V67 to I) responsible for the decrease in actin-binding of rat DNase I. The enzymatic activity of mouse DNase I is found in striated muscle, kidney, intestine, liver, lymphnodes, but not in the heart, spleen or pancreas.

Amino Acid Sequence↗
PubMed1995

Comparative molecular modelling of the Fas-ligand and other members of the TNF family.

A number of proteins with significant similarity to the tumour necrosis factor (TNF) have been identified over the last years. Upon interaction with their cognate receptor (members of the TNF-receptor family), all members of this protein family induce either cell death or proliferation/differentiation of the receptor-bearing cells. One of the last identified members of the TNF family is the apoptosis-inducing ligand of the Fas-receptor, termed Fas-ligand (FasL). Here we report the cloning and sequencing of the mouse cDNA for the FasL. Using knowledge-based protein modelling, we demonstrate that all members of the TNF family form trimeric complexes, and define the residues located at the subunit interfaces. The resulting structurally corrected multiple sequence alignment allows the identification of residues potentially involved in receptor recognition, and should help design mutagenesis experiments for structure-function relationship studies.

Amino Acid Sequence↗
PubMed1995

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Amino Acid Sequence↗