Biomedical subjects

O Burlet-Schiltz

Publications and source records attributed to O Burlet-Schiltz.

6 recordsLinked to original sources

PubMed2001

Structural and functional identification of major histocompatibility complex class I-restricted self-peptides as naturally occurring molecular mimics of viral antigens. Possible role in CD8+ T cell-mediated, virus-induced autoimmune disease.

Structural similarity (molecular mimicry) between viral epitopes and self-peptides can lead to the induction of autoaggressive CD4(+) as well as CD8(+) T cell responses. Based on the flexibility of T cell receptor/antigen/major histocompatibility complex recognition, it has been proposed that a self-peptide could replace a viral epitope for T cell recognition and therefore participate in pathophysiological processes in which T cells are involved. To address this issue, we used, as a molecular model of viral antigen, the H-2D(b)-restricted immunodominant epitope nucleoprotein (NP)-(396-404) (FQPQNGQFI) of lymphocytic choriomeningitis virus (LCMV). We identified peptide sequences from murine self-proteins that share structural and functional homology with LCMV NP-(396-404) and that bound to H-2D(b) with high affinity. One of these self-peptides, derived from tumor necrosis factor receptor I (FGPSNWHFM, amino acids 302-310), maintained LCMV-specific CD8(+) T cells in an active state as observed both in vitro in cytotoxic assays and in vivo in a model of virus-induced autoimmune diabetes, the rat insulin promoter-LCMV NP transgenic mouse. The natural occurrence and molecular concentration at the surface of H-2(b) spleen cells of tumor necrosis factor receptor I-(302-310) were determined by on-line micro-high pressure liquid chromatography/mass spectrometry and supported its biological relevance.

Adoptive Transfer↗

PubMed2001

Identification of neuropeptide FF-related peptides in rodent spinal cord.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

Amino Acid Sequence↗

PubMed2000

Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.

By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

Antigen Presentation↗

PubMed1996

Structural basis of extended spectrum TEM beta-lactamases. Crystallographic, kinetic, and mass spectrometric investigations of enzyme mutants.

The E166Y and the E166Y/R164S TEM-1 beta-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-A resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalosporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the omega-loop. These structural observations strongly suggest that the link between the position of the omega-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased omega-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparisons of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis.

Amino Acid Sequence↗

PubMed1995

Mass spectral kinetic study of acylation and deacylation during the hydrolysis of penicillins and cefotaxime by beta-lactamase TEM-1 and the G238S mutant.

The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.

Acylation↗

PubMed1995

The asparagine to aspartic acid substitution at position 276 of TEM-35 and TEM-36 is involved in the beta-lactamase resistance to clavulanic acid.

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.

Base Sequence↗