Biomedical subjects

Sebastien Farnaud

Publications and source records attributed to Sebastien Farnaud.

8 recordsLinked to original sources

PubMed2006

Characterisation of recombinant unglycosylated human serum transferrin purified from Saccharomyces cerevisiae.

Structural identity between a recombinant transferrin mutant (N413Q, N611Q) secreted from Saccharomyces cerevisiae and the native protein was shown by CD analysis and immunodiffusion assays against anti-hSTf. The ability of the recombinant protein to bind iron was confirmed by urea-PAGE and EPR analysis of the iron-saturated protein revealed the characteristic holo-transferrin spectrum, indicating conservation of both iron-binding sites. The integrity of the unglycosylated recombinant protein indicates that such protein could be a valuable tool not only for structure-function characterisation but also crystallisation assays. In addition, the recombinant transferrin was found to be as effective as native transferrin as a growth factor in cell culture medium.

Cell Proliferation↗

PubMed2006

Modelling of a metal-containing hepcidin.

Hepcidin was originally identified as a liver-expressed antimicrobial peptide but further studies have shown that it also has a key role in iron homeostasis. The NMR structure of the synthetic peptides reveal a distorted beta-sheet containing 4 disulphide bridges, with an unusual vicinal disulphide bridge which has been suggested to be functionally significant. In this study, we report the presence of co-purified iron with the urine-purified 20 and 25 residue hepcidins. Since the published structure does not allow metal binding, the interaction of hepcidin with metals was investigated for other possible structural conformations by threading its primary sequence onto existing 3D folds. Several alignments were obtained and the best scores were used to build a 3D model of hepcidin containing one atom of iron. The new 3D structure, that contains only reduced Cys residues, is completely different from the solved structure of the synthetic peptide. Although the model presented here shows only one metal bound to the peptide, the binding of several metal atoms cannot be excluded from such a short flexible peptide. The co-purification of iron with both peptides, together with our 3D model, suggest a conformational polymorphism for hepcidin, reminiscent of the iron regulatory proteins IRPs.

Amino Acid Sequence↗

PubMed2006

An overview of molecular aspects of iron metabolism.

Iron (Fe) is an essential, but potentially noxious, metal for almost all organisms. Its precise cellular regulation is necessary to ensure synthesis of numerous iron-containing proteins required for metabolic processes yet at the same time avoiding the build-up of potentially toxic levels of iron. In humans, iron-deficiency results in anemia, while excess iron can lead to organ damage as a result of a build-up of non-transferrin-bound iron (NTBI). In recent years, the cloning of novel proteins has clarified the mechanisms of iron uptake, storage and metabolic regulation. Our current knowledge of the molecular aspects of mammalian iron metabolism and NTBI are presented in this review.

Absorption↗

PubMed2005

Structure/function overview of proteins involved in iron storage and transport.

Iron, the major trace element in the body, is an essential component of many proteins and enzymes. As low-molecular-weight iron is potentially toxic to cells, higher organisms express a number of proteins for the transport and storage of iron. We review our current understanding of the intestinal absorption of iron in the light of recently identified membrane proteins, namely the ferrric reductase, Dcytb, the two iron(II) transport proteins, DMT1 and ferroportin/Ireg1, and hephaestin, the membrane-bound homologue of the ferroxidase ceruloplasmin. Two types of mammalian transferrin receptor, TfR1 and TfR2, are now known to exist. The structure of TfR1 and its role in the process of receptor-mediated cellular uptake of iron are presented together with structural information on the iron storage protein ferritin. Mechanisms for the regulation of levels of TfR1 and ferritin, as well as other proteins involved in iron homeostasis, are discussed. Our current knowledge and understanding of the structure of members of the transferrin family of iron-binding proteins and the nature of the iron-binding centres in transferrins is presented, together with information on the processes of iron-uptake and iron-release by transferrin and a summary of the elements that have been found to bind to transferrins.

Animals↗

PubMed2004

Variation in antimicrobial activity of lactoferricin-derived peptides explained by structure modelling.

Antimicrobial peptides bovine lactoferricin (LfcinB) and human lactoferricin (LfcinH) are produced from the respective lactoferrin, but are more active than their precursors. Despite sequence homology, the bovine peptide and its derivatives are more active than their human homologs. Such differences between not only the peptides and their precursor but also between the bovine and the human peptides could relate to structural differences. Upon sequence alignment of both peptides with their parental proteins, the structural differences observed between the bovine lactoferrin (BLf) and LfcinB were also found between the human lactoferrin (HLf) and the LfcinH. The helical structures in HLf are replaced by beta-strands separated by a strong turn in LfcinH suggesting an antiparallel beta-sheet structure similar to LfcinB. MIC assays with HLP-2 and BLP-2, 11-residue peptides derived from the active core of both Lfcins, against Escherichia coli, showed that the bovine derivative, BLP-2, is more active than its human homolog HLP-2. Both 3D models for HLP-2 and BLP-2 showed that the beta-strand is centred between the aromatic residues giving both side chains the same orientations. The displacement towards the N-terminus observed for the beta-strand in HLP-2, compared with its central location in BLP-2, could be less favourable to membrane interaction and therefore responsible for the decrease in activity. Such a model suggests for LfcinH a mechanism similar to the one observed for LfcinB, where the absence of long-range interaction, present in lactoferrin, destabilises the first alpha helix, as observed in solution and, upon interaction with the membrane, could result in the formation of a beta-strand, as observed in the presence of LPS. The location of the beta-strand in relation to the positive charges, seems to define the efficiency of the activity of the peptide and may explain the difference in activity obtained between HLP-2 and BLP-2.

Amino Acid Sequence↗

PubMed2004

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity.

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.

Amino Acid Sequence↗

PubMed2003

Lactoferrin--a multifunctional protein with antimicrobial properties.

Lactoferrin is a member of the transferrin family of iron-binding proteins. Numerous functions have been reported and continue to be reported for the protein, some of which are related to its iron-binding properties. Its extensive antimicrobial activities were originally attributed to its ability to sequester essential iron, however, it is now established that it possesses bactericidal activities as a result of a direct interaction between the protein or lactoferrin-derived peptides. This article reviews the antimicrobial activities of lactoferrin and discusses the potential mode of action of lactoferrin-derived cationic peptides against Gram-negative bacteria in the light of recent studies.

Animals↗

PubMed2002

Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

Amidohydrolases↗