Biomedical subjects

Philippe Bulet

Publications and source records attributed to Philippe Bulet.

At least 19 recordsLinked to original sources

PubMed2007

Biological and structural characterization of new linear gomesin analogues with improved therapeutic indices.

Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a beta-hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 microM they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3)), Thr(6,11,15), D-Pro(9)]-Gm with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr(2,6,11,15), Pro(8)]-D-Gm and [Thr(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was further analyzed by NMR spectroscopy. (1)H NMR experiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-D-Gm presents a conformation very similar to that of Gm. In our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives.

Amino Acid Sequence↗

PubMed2007

The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

Amino Acid Sequence↗

PubMed2006

Peptidomics and proteomics studies of transformed lymphocytes from patients mutated for the eukaryotic initiation factor 2B.

Mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported in human brain genetic disorders. In order to analyse the functional consequences of the mutations and to find specific biomarkers of eIF2B-related disorders, proteomics and peptidomics studies were performed on lymphoblasts from eIF2B-mutated patients versus healthy patients. Curiously, following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not significantly affect the proteome of the mutated lymphoblasts extracts. However, liquid chromatography based peptidomics studies revealed one apparently instable candidate compound in five out of the six mutated lymphoblastoid cell lines investigated.

Chromatography, High Pressure Liquid↗

PubMed2006

Antimicrobial peptides in the interactions between insects and flagellate parasites.

Innate immunity has a key role in the control of microbial infections in both vertebrates and invertebrates. In insects, including vectors that transmit parasites that cause major human and animal diseases, antimicrobial peptides (AMPs) are important components of innate immunity. AMPs are induced upon parasitic infections and can participate in regulating parasite development in the digestive tract and in the hemolymph. This review presents our current knowledge of a field that is in its infancy: the role of innate immunity in different models of insects infected with flagellate parasites, and in particular the potential role of AMPs in regulating these parasitic infections.

Animals↗

PubMed2006

Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen.

Due to their complexity and diversity, animal venoms represent an extensive source of bioactive compounds such as peptides and proteins. Conventional approaches for their characterization often require large quantities of biological material. Current mass spectrometry (MS) techniques now give access to a wealth of information in a short working time frame with minute amounts of sample. Such MS approaches may now be used for the discovery of novel compounds, and once their structure has been determined they may be synthesized and tested for functional activity. Molecular mass fingerprints of venoms allow the rapid identification of known toxins as well as preliminary structural characterization of new compounds. De novo peptide sequencing by tandem mass spectrometry (MS/MS) offers rapid access to partial or total primary peptide structures. This article, written as a tutorial, also contains new material: molecular mass fingerprint analysis of Orthochirus innesi scorpion venom, and identification of components from bumblebee Bombus lapidarius venom, both collected from one single specimen. The structure of the three major peptides detected in the Bombus venom was fully characterized in one working day by de novo sequencing using an electrospray ionization hybrid quadrupole time-of-flight instrument (ESI-QqTOF) and a matrix-assisted laser desorption ionization time-of-flight instrument (MALDI-LIFT-TOF-TOF). After presenting the MS-based sequence elucidation, perspectives in using MS and MS/MS techniques in toxinology are discussed.

Animals↗

PubMed2006

Solution structures of stomoxyn and spinigerin, two insect antimicrobial peptides with an alpha-helical conformation.

Stomoxyn and spinigerin belong to the class of linear cysteine-free insect antimicrobial peptides that kill a range of microorganisms, parasites, and some viruses but without any lytic activity against mammalian erythrocytes. Stomoxyn is localized in the gut epithelium of the nonvector stable fly that is sympatric with the trypanosome vector tsetse fly. Spinigerin is stored and secreted by hemocytes from the fungus-growing termite. The structure of synthetic stomoxyn and spinigerin in aqueous solution and in TFE/water mixtures was analyzed by CD and NMR spectroscopy combined with molecular modeling calculations. Stomoxyn and spinigerin adopt a flexible random coil structure in water while both assume a stable helical structure in the presence of TFE. In 50% TFE, the structure of stomoxyn is typical of cecropins, including an amphipathic helix at the N-terminus and a hydrophobic C-terminus with helical features that probably fold in a helical conformation at higher TFE concentration. In contrast to stomoxyn, spinigerin acquires very rapidly a helical conformation. In 10% TFE the helix is highly bent and the structure is poorly defined. In 50% TFE, the helical structure is well defined all along its sequence, and the slightly bent alpha-helix displays an amphiphilic character, as observed for magainin 2. The structural similarities between stomoxyn and cecropin A from Hyalophora cecropia and between spinigerin and magainin 2 suggest a similar mode of action on the bacterial membranes of both pairs of peptides. Our results also confirm that TFE induces helix formation and propagation for amino acids showing helical propensity in water but also enhances the helix propagation propensity of nonpolar beta-branched residues.

Amino Acid Sequence↗

PubMed2006

Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability.

Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.

Amino Acid Sequence↗

PubMed2006

PenBase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature.

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.

Amino Acid Sequence↗

PubMed2005

Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression.

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

Amino Acid Sequence↗

PubMed2005

Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases.

The presence of an effective immune response in the hemocoel of arthropods is essential for survival as it prevents the invasion of pathogens throughout the animal body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a novel cysteine-rich AMP has been isolated and characterized from the hemocytes of the cattle tick, Boophilus microplus. In addition to growth inhibition of Escherichia coli and Micrococcus luteus, the newly described AMP, designated ixodidin (derived from the Family Ixodidae), was found to exert proteolytic inhibitory activity against two exogenous serine proteinases, elastase and chymotrypsin. This is the first report of a molecule of an arachnid that has been shown to inhibit bacterial growth and proteinase activity.

Amino Acid Sequence↗

PubMed2005

Antimicrobial peptides in Drosophila: structures, activities and gene regulation.

The production of antimicrobial peptides (AMPs) is an important aspect of host-defence in multicellular organisms. Biochemical analysis of the hemolymph of the fruit-fly Drosophila melanogaster and other Diptera has led to the discovery of eight classes of AMPs. These peptides can be grouped into three families based on their main biological targets, gram-positive bacteria (defensin), gram-negative bacteria (cecropins, drosocin, attacins, diptericin, MPAC), or fungi (drosomycin, metchnikowin). Drosophila AMPs are synthesized by the fat body in response to infection, and secreted into the blood. Most of them can also be induced in surface epithelia in a tissue-specific manner. Finally, some of them are constitutively expressed in defined tissues, such as the salivary glands or the reproductive tract. We review here the structures and activities of these AMPs, as well as the signalling cascades, which lead to their induction upon detection of infectious non-self.

Amino Acid Sequence↗

PubMed2005

Insect antimicrobial peptides: structures, properties and gene regulation.

Antimicrobial peptides (AMPs) are part of the armament that insects have developed to fight off pathogens. Insect AMPs are typically cationic and often made of less than 100 amino acid residues. Although their structures are diverse, most of the AMPs can be assigned to a limited number of families. The most common structures are represented by peptides assuming a alpha-helical conformation in organic solutions or disulfide-stabilized beta-sheets with or without alpha-helical domains present. The diverse activity spectrum of these peptides may indicate different modes of action. Genetic analysis in the Drosophila model evidenced that multiple signal transduction pathways are activating the genes coding AMPs.

Amino Acid Sequence↗

PubMed2004

Solution structure of spheniscin, a beta-defensin from the penguin stomach.

Recently two beta-defensins, named spheniscins, have been isolated from the stomach content of the king penguin (Aptenodytes patagonicus), which is capable of preserving food for several weeks during egg incubation (Thouzeau, C., Le Maho, Y., Froget, G., Sabatier, L., Le Bohec, C., Hoffmann, J. A., and Bulet, P. (2003) J. Biol. Chem. 278, 51053-51058). It has been proposed that, in combination with other antimicrobial peptides, spheniscins may be involved in this long term preservation of food in the bird's stomach. To draw some structure/function features, the three-dimensional structure in aqueous solution of the most abundant spheniscin (Sphe-2) was determined by two-dimensional NMR and molecular modeling techniques. The overall fold of Sphe-2 includes a three-stranded antiparallel beta-sheet stabilized by three disulfide bridges with a pairing typical of beta-defensins. In addition, the N-terminal segment shows helical features on most structures. Sphe-2 is highly cationic, and its surface displays a hydrophobic patch. Comparative modeling revealed that this patch is preserved in avian defensins. The activity of Sphe-2 against a pathogenic Gram-positive strain was retained in vitro in the conditions of osmolarity found in penguin stomach content and also in different salt concentrations and compositions up to those reported for seawater. Comparison with structurally related mammalian beta-defensins showed that the hydrophobic patch is not preserved in mammalian beta-defensins and that the high cationicity of Sphe-2 is presumably the critical factor for its retained activity in high salt concentrations. Such peculiarities, in addition to a broad activity spectrum, suggest that penguin defensins may represent interesting probes for the design of highly efficient antibiotics to fight off pathogens that develop in relatively salt-rich body fluids.

Amino Acid Motifs↗

PubMed2004

Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain.

In Drosophila melanogaster, seven distinct families of antimicrobial peptides with different structures and specificities are synthesized by the fat body and released into the hemolymph during the immune response. Using microscale high performance liquid chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Edman degradation, we have isolated and characterized from immune-challenged Drosophila two novel induced molecules, under the control of the Imd pathway, that correspond to post-translationally modified antimicrobial peptides or peptide fragments. The first molecule is a doubly glycosylated form of drosocin, an O-glycosylated peptide that kills Gram-negative organisms. The second molecule represents a truncated form of the pro-domain of the Drosophila attacin C carrying two post-translational modifications and has significant structural similarities to proline-rich antibacterial peptides including drosocin. We have synthesized this peptide and found that it is active against Gram-negative bacteria. Furthermore, this activity is potentiated when the peptide is used in combination with the Drosophila antimicrobial peptide cecropin A. The synergistic action observed between these two molecules suggests that the truncated post-translationally modified pro-domain of attacin C by itself may play an important role in the antimicrobial defense of Drosophila.

Amino Acid Sequence↗

PubMed2004

Cysteine-rich antimicrobial peptides of the cattle tick Boophilus microplus: isolation, structural characterization and tissue expression profile.

Antimicrobial peptides (AMPs) are components of the immune system of both vertebrate and invertebrate animals. This study describes the isolation, primary structure, cDNA cloning, and tissue expression profile of two cysteine-rich AMPs from the hemolymph of the cattle tick Boophilus microplus. A 10,204 Da polypeptide, with six cysteine residues and no sequence similarity to any known molecule, was isolated from the cell-free hemolymph. Because of its sequence originality, this peptide was named microplusin. The second AMP was isolated from the hemocytes of B. microplus. This peptide, with a molecular mass of 4285 Da and six cysteines, is a defensin with similarity to the insect defensin family members. The cDNA cloning established that microplusin is synthesized as a prepeptide while the tick defensin is synthesized as a prepromolecule. Interestingly, despite the fact that microplusin and defensin have been isolated from different compartments, their gene expression was found to have similar tissue distribution.

Amino Acid Sequence↗

PubMed2004

Anti-microbial peptides: from invertebrates to vertebrates.

Gene-encoded anti-microbial peptides (AMPs) are widespread in nature, as they are synthesized by microorganisms as well as by multicellular organisms from both the vegetal and the animal kingdoms. These naturally occurring AMPs form a first line of host defense against pathogens and are involved in innate immunity. Depending on their tissue distribution, AMPs ensure either a systemic or a local protection of the organism against environmental pathogens. They are classified into three major groups: (i) peptides with an alpha-helical conformation (insect cecropins, magainins, etc.), (ii) cyclic and open-ended cyclic peptides with pairs of cysteine residues (defensins, protegrin, etc.), and (iii) peptides with an over-representation of some amino acids (proline rich, histidine rich, etc.). Most AMPs display hydrophobic and cationic properties, have a molecular mass below 25-30 kDa, and adopt an amphipathic structure (alpha-helix, beta-hairpin-like beta-sheet, beta-sheet, or alpha-helix/beta-sheet mixed structures) that is believed to be essential to their anti-microbial action. Interestingly, in recent years, a series of novel AMPs have been discovered as processed forms of large proteins. Despite the extreme diversity in their primary and secondary structures, all natural AMPs have the in vitro particularity to affect a large number of microorganisms (bacteria, fungi, yeast, virus, etc.) with identical or complementary activity spectra. This review focuses on AMPs forming alpha-helices, beta-hairpin-like beta-sheets, beta-sheets, or alpha-helix/beta-sheet mixed structures from invertebrate and vertebrate origins. These molecules show some promise for therapeutic use.

Amino Acid Sequence↗

PubMed2004

Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.

Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.

Amino Acid Sequence↗

PubMed2003

Proteomic analysis of the systemic immune response of Drosophila.

Improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize proteins involved in a physiological process, such as the immune response of the insect model Drosophila melanogaster. Profiling of the proteins present in the hemolymph (insect blood) of noninfected flies versus flies infected with bacteria or fungi was performed by two-dimensional gel electrophoresis, silver or Coomassie staining, and image analysis. Through this differential analysis, more than 70 out of 160 spots were up- or down-regulated by at least 5-fold after microbial infection. Coomassie staining, in-gel digestion, and database searches yielded the identity of a series of proteins that are directly involved in the Drosophila immune system. This included proteases, protease inhibitors, and recognition molecules such as prophenoloxydase-activating enzymes, serpins, and Gram-negative binding protein-like. Proteins with a potential function in the immune response were also identified, such as an odorant binding protein, peptidylglycine alpha-hydroxylating monooxygenase, and transferrin, affording new candidates for further investigation of innate immune mechanisms. Moreover, several molecules resulting from the cleavage of proteins were detected after the fungal infection. Altogether, this first differential proteomic analysis of the immune response of Drosophila paves the way for the study of proteins affected during innate immunity.

Animals↗