Jean-François Chich

Lactic acid bacteria and proteomics: current knowledge and perspectives.

Lactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics.

Intracellular esterase from Lactococcus lactis subsp. lactis NCDO 763: Purification and characterization

The presence of esterolytic activities in the intracellular extract of Lactococcus lactis subsp. lactis NCDO 763 was investigated. One major activity hydrolyzing β-naphthyl butyrate was detected. This activity was purified to homogeneity. The enzyme was a homotrimer; the molecular mass of the monomer was estimated by SDS-PAGE to be 29kDa; by mass spectrometry, it was 29655 ± 30 Da. The pI of the molecule was 4.5. The enzyme was active on para-nitrophenyl esters from C2 to C12 and on ortho-nitrophenyl butyrate, with a maximum activity on p-nitrophenyl butyrate. The optimum activity on this substrate was found at pH 8.0 and at 55 °C; kinetic parameters for this substrate were measured at 55 °C and were KM = 0.11 mM and Vmax = 8.17 μmol min−1. The enzyme was strongly inhibited by Pefabloc, diisopropyl fluoro-phosphate and 3,4-dichloroisocoumarin, demonstrating that it was a serine esterase. The amino-terminus of the enzyme was sequenced. The role of this enzyme in cheese ripening is discussed.

Identification of the active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis

The active site serine of the X‐prolyl dipeptidyl aminopeptidase from Lactococcus lactis (PepX) was identified. The enzyme was labeled by [3H]DFP, treated by CNBr and the resulting peptides were separated by reverse‐phase‐HPLC. The main radiolabeled peptide was sequenced. Ser‐348, in the following sequence, Gly‐Lys‐Ser‐Tyr‐Leu‐Gly, was identified as the active site serine. A sequence comparison between the active site of PepX and other serine proteases was made, showing only limited sequence homologies in this area. The consensus sequence surrounding the active site serine in the three known X‐prolyl dipeptidyl aminopeptidases (mammalian DPPIV, yeast DPAB and PepX) is G‐X‐S‐Y‐X‐G, where X is a non‐conserved amino acid.

Towards a proteomic map of Lactococcus lactis NCDO 763

Lactococcus lactis is a widely used bacteria in dairy industry, specially in cheese ripening. Numerous lactococcal enzymes and proteins are involved in this process. Proteomics makes it possible to deal with a high number of proteins and identify modification of their patterns in two‐dimensional (2‐D) gels. However, an annotated reference map is necessary prior to analyzing protein variations. We have begun to construct such a map in easily reproducible conditions and identify proteins.

Transcriptome of Dickeya dadantii Infecting Acyrthosiphon pisum Reveals a Strong Defense against Antimicrobial Peptides.

The plant pathogenic bacterium Dickeya dadantii has recently been shown to be able to kill the aphid Acyrthosiphon pisum. While the factors required to cause plant disease are now well characterized, those required for insect pathogeny remain mostly unknown. To identify these factors, we analyzed the transcriptome of the bacteria isolated from infected aphids. More than 150 genes were upregulated and 300 downregulated more than 5-fold at 3 days post infection. No homologue to known toxin genes could be identified in the upregulated genes. The upregulated genes reflect the response of the bacteria to the conditions encountered inside aphids. While only a few genes involved in the response to oxidative stress were induced, a strong defense against antimicrobial peptides (AMP) was induced. Expression of a great number of efflux proteins and transporters was increased. Besides the genes involved in LPS modification by addition of 4-aminoarabinose (the arnBCADTEF operon) and phosphoethanolamine (pmrC, eptB) usually induced in Gram negative bacteria in response to AMPs, dltBAC and pbpG genes, which confer Gram positive bacteria resistance to AMPs by adding alanine to teichoic acids, were also induced. Both types of modification confer D. dadantii resistance to the AMP polymyxin. A. pisum harbors symbiotic bacteria and it is thought that it has a very limited immune system to maintain these populations and do not synthesize AMPs. The arnB mutant was less pathogenic to A. pisum, which suggests that, in contrast to what has been supposed, aphids do synthesize AMP.

Misfolding of the prion protein: linking biophysical and biological approaches

Prion diseases are a group of neurodegenerative diseases that can arise spontaneously, be inherited, or acquired by infection in mammals. The propensity of the prion protein to adopt different structures is a clue to its pathological and perhaps biological role too. While the normal monomeric PrP is well characterized, the misfolded conformations responsible for neurodegeneration remain elusive despite progress in this field. Both structural dynamics and physico-chemical approaches are thus fundamental for a better knowledge of the molecular basis of this pathology. Indeed, multiple misfolding pathways combined with extensive posttranslational modifications of PrP and probable interaction(s) with cofactors call for a combination of approaches. In this review, we outline the current physico-chemical knowledge explaining the conformational diversities of PrP in relation with postulated or putative cellular partners such as proteic or non-proteic ligands.

The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase (X-PDAP) from Lactococcus lactis is a dimeric enzyme catalyzing the removal of Xaa-Pro dipeptides from the N terminus of peptides. The structure of the enzyme was solved at 2.2 A resolution and provides a model for the peptidase family S15. Each monomer is composed of four domains. The larger one presents an alpha/beta hydrolase fold and comprises the active site serine. The specificity pocket is mainly built by residues from a small helical domain which is, together with the N-terminal domain, essential for dimerization. A C-terminal moiety probably plays a role in the tropism of X-PDAP toward the cellular membrane. These results give new insights for further exploration of the role of the enzymes of the SC clan.

Vesicle Permeabilization by Purified Soluble Oligomers of Prion Protein: A Comparative Study of the Interaction of Oligomers and Monomers with Lipid Membranes

The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

Biosensing of lipid-prion interactions: insights on charge effect, Cu(II)-ions binding and prion oligomerization.

The molecular mechanism involved in early stages of prion protein (PrP) conversion has been investigated using the chip based SPR technology, focusing on PrP interactions with membranes, either in its monomeric, oligomeric or Cu(II)-ions bound forms. We observed a strong interaction between PrP and cell membrane models of different lipid compositions. Circular dichroism tests show that membrane-bound, oligomerized or Cu(II)-complexed PrP may adopt a β-sheet-rich conformation. Moreover, upon PrP binding membrane vesicles may aggregate and/or be fragmented depending on vesicle net-charge and their lipid/raft composition. The whole study emphasizes the outstanding performance of the on-a-chip approach for the investigation of prion conversion and could be useful for developing sensor formats for prion assessments in biological samples.

The structural comparison of the bacterial PepX and human DPP-IV reveals sites for the design of inhibitors of PepX activity

X‐prolyl dipeptidyl aminopeptidases (X‐PDAP) are enzymes catalysing the release of dipeptides from the amino termini of polypeptides containing a proline or an alanine at the penultimate position. Involved in various mammalian regulation processes, as well as in chronic human diseases, they have been proposed to play a role in pathogenicity for Streptococci. We compared the structure of X‐PDAP from Lactococcus lactis (PepX) with its human counterpart DPP‐IV. Despite very different overall folds, the residues most implicated for X‐PDAP activity are conserved in the same positions and orientations in both enzymes, thus defining a structural signature for the X‐PDAP specificity that crosses the species frontiers of evolution. Starting from this observation, we tested some inhibitors of DPP‐IV on PepX activity, for which no specific inhibitor is known. We thus found that PepX was highly sensitive to valine‐pyrrolidide with a KI of 9.3 µm, close to that reported in DPP‐IV inhibition. We finally used the structure of PepX from L. lactis as a template for computer‐based homology modeling of PepX from the pathogenic Streptococcus gordonii. Docking simulations of valine‐pyrrolidide into the active site of PepX led to the identification of key residues for a rational drug design against PepX from Streptococci. These results could have applications in human health giving new perspectives to the struggle against pathogens.