Jérôme Vicogne

Conservation of Epidermal Growth Factor Receptor Function in the Human Parasitic Helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni (Shoemaker, C. B., Ramachandran, H., Landa, A., dos Reis, M. G., and Stein, L. D. (1992) Mol. Biochem. Parasitol. 53, 17–32). Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. 125I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 × 10–9 m) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.

A One-Pot Three-Segment Ligation Strategy for Protein Chemical Synthesis†

Protein chemical synthesis by native peptide ligation of unprotected peptide segments is an interesting complement and potential alternative to the use of living systems for producing proteins. Actually, tremendous efforts are focused on the design of one-pot strategies allowing the assembly of three peptide segments. The goal is to get rapid access to small proteins (less than 150 amino acid residues), while saving intermediate purification steps and obtaining the products in good yield. Such methods are gaining increasing significance for the study of protein function and appear as a potential option for producing various protein-based therapeutics currently under development. To date, proteins were mainly assembled by sequential native chemical ligation (NCL) or extended methodologies in the C-to-N direction (for recent achievements, see Refs. [8, 9]). NCL involves the chemoselective ligation of a Cterminal peptide thioester, usually an alkylthioester, with an N-terminal cysteine (Cys) peptide. The one-pot sequential Cto-N ligation of three peptide segments designed by Kent et al. is increasingly used for synthesizing proteins. Methods that enable the assembly of peptide segments in the reverse N-to-C direction are rare. 11] Fundamentally, the combination of N-to-C and C-to-N assembly techniques is at the basis of the convergent total synthesis of proteins. The general principle of the one-pot assembly of three peptide segments in the N-to-C direction is illustrated in Scheme 1. Ligation of peptide segments A-X and H-Cys-B-Y yields segment A-Cys-B-Y (Scheme 1, ligation 1). Group Y must ideally be inert during ligation 1 or at least be significantly less reactive than group X to avoid oligomerization or cyclization of segment B. Activation of group Y into Y* subsequently allows the ligation with the third segment H-Cys-C (Scheme 1, ligation 2). For designing a one-pot process working in the N-to-C direction, this activation must be carried out in situ after ligation 1 by using reagents compatible with ligation 2. Furthermore, the Y* group must enable an efficient ligation with the Cys segment C. To date only few one-pot strategies have been described that work in the N-to-C direction and enable the coupling of three peptide segments. 5, 12] Fundamentally, these methods, such as kinetically controlled ligation, rely on the differential reactivity of X and Y groups for peptide-bond formation. In other words, the purity of the target polypeptide is highly dependent on the C-terminal residues of A and B segments and more generally on the accessibility of the reactive ends. Clearly, a strategy in which Y is inert during the first ligation step would bypass these limitations and constitute a critical advance. Herein we show that the combination of NCL and SEA ligation (Scheme 1) permitted design of a solution to this important problem. Reaction of a peptide featuring a C-terminal bis(2-sulfanylethyl)amido group, called SEA hereafter (Scheme 1), with a Cys peptide results in the formation of a native peptide bond in water at pH 7. This reaction probably proceeds via Scheme 1. Total protein synthesis by one-pot assembly of three peptide segments in the N-to-C direction. The first step is a native chemical ligation between thioester segment A and Cys segment B, during which the cyclic disulfide SEA acts as a blocked thioester group (SEA = bis(2-sulfanylethyl)amido). Activation of SEA into SEA by reduction with a phosphine and addition of the third Cys segment C triggers the second ligation step.

An unusual receptor tyrosine kinase of Schistosoma mansoni contains a Venus Flytrap module

Previous studies have suggested that successful development of the parasitic helminth Schistosoma mansoni must be dependent on an adaptative molecular dialogue with its hosts and on the existence of receptors for growth factors and hormones. Attempts to identify a homolog of the insulin receptor (IR) have led us to characterize a new receptor tyrosine kinase (RTK) molecule in S. mansoni. SmRTK-1 is an integral membrane protein with a single membrane-spanning sequence separating an extracellular ligand-binding domain and a cytoplasmic TK domain. Structural and phylogenetic analyses of the kinase domain of SmRTK-1 confirmed its similarity to IR catalytic domains. However, sequence analysis of the extracellular domain of SmRTK-1 revealed similarity with various proteins (such as drug receptors) that share a structure known as the Venus Flytrap (VFT) module. Alignment with other VFT modules for which the structure has been solved was used to generate a 3D model of the putative VFT module of SmRTK-1. Phylogenetic analysis indicated that the SmRTK-1 VFT module was closer to that of the GABA(B) receptor. Numerous RTK genes recently discovered in vertebrate and invertebrate species code for large families of modular proteins with diverse structures and ligand-binding specificities. SmRTK-1 probably represents a new class of RTK whose function remains to be determined. RTKs are present in all metazoans and associated with the control of metabolism, growth and development. The preferential localization of SmRTK-1 in sporocyst germinal cells and ovocytes could be in favor of its function in schistosome growth and differentiation.

Diversification of the insulin receptor family in the helminth parasite Schistosoma mansoni

Insulin signalling is a very ancient and well conserved pathway in metazoan cells, dependent on insulin receptors (IR) which are transmembrane proteins with tyrosine kinase activity. A unique IR is usually present in invertebrates whereas two IR members are found with different functions in vertebrates. This work demonstrates the existence of two distinct IR homologs (SmIR‐1 and SmIR‐2) in the parasite trematode Schistosoma mansoni. These two receptors display differences in several structural motifs essential for signalling and are differentially expressed in parasite tissues, suggesting that they could have distinct functions. The gene organization of SmIR‐1 and SmIR‐2 is similar to that of the human IR and to that of the IR homolog from Echinococcus multilocularis (EmIR), another parasitic platyhelminth. SmIR‐1 and SmIR‐2 were shown to interact with human pro‐insulin but not with pro‐insulin‐like growth factor‐1 in two‐hybrid assays. Phylogenetic results indicated that SmIR‐2 and EmIR might be functional orthologs whereas SmIR‐1 would have emerged to fulfil specific functions in schistosomes.

Growth factor receptors in helminth parasites: Signalling and host–parasite relationships

Parasitic helminths remain major pathogens of both humans and animals throughout the world. The success of helminth infections depends on the capacity of the parasite to counteract host immune responses but also to exploit host‐derived signal molecules for its development. Recent progress has been made in the characterization of growth factor receptors of various nematode and flatworm parasites with the demonstration that transforming growth factor beta (TGF‐β), epidermal growth factor (EGF) and insulin receptor signalling pathways are conserved in helminth parasites and potentially implicated in the host–parasite molecular dialogue and parasite development.

The formin-homology protein SmDia interacts with the Src kinase SmTK and the GTPase SmRho1 in the gonads of Schistosoma mansoni.

Background Schistosomiasis (bilharzia) is a parasitic disease of worldwide significance affecting human and animals. As schistosome eggs are responsible for pathogenesis, the understanding of processes controlling gonad development might open new perspectives for intervention. The Src-like tyrosine-kinase SmTK3 of Schistosoma mansoni is expressed in the gonads, and its pharmacological inhibition reduces mitogenic activity and egg production in paired females in vitro. Since Src kinases are important signal transduction proteins it is of interest to unravel the signaling cascades SmTK3 is involved in to understand its cellular role in the gonads. Methodology and Results Towards this end we established and screened a yeast two-hybrid (Y2H) cDNA library of adult S. mansoni with a bait construct encoding the SH3 (src homology) domain and unique site of SmTK3. Among the binding partners found was a diaphanous homolog (SmDia), which was characterized further. SmDia is a single-copy gene transcribed throughout development with a bias towards male transcription. Its deduced amino acid sequence reveals all diaphanous-characteristic functional domains. Binding studies with truncated SmDia clones identified SmTK3 interaction sites demonstrating that maximal binding efficiency depends on the N-terminal part of the FH1 (formin homology) domain and the inter-domain region of SmDia located upstream of FH1 in combination with the unique site and the SH3 domain of SmTK3, respectively. SmDia also directly interacted with the GTPase SmRho1 of S. mansoni. In situ hybridization experiments finally demonstrated that SmDia, SmRho1, and SmTK3 are transcribed in the gonads of both genders. Conclusion These data provide first evidence for the existence of two cooperating pathways involving Rho and Src that bridge at SmDia probably organizing cytoskeletal events in the reproductive organs of a parasite, and beyond that in gonads of eukaryotes. Furthermore, the FH1 and inter domain region of SmDia have been discovered as binding sites for the SH3 and unique site domains of SmTK3, respectively.

One-pot chemical synthesis of small ubiquitin-like modifier protein–peptide conjugates using bis (2-sulfanylethyl)amido peptide latent thioester surrogates

Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO–peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of peptide segments derivatized by thioester or bis(2-sulfanylethyl)amido (SEA) latent thioester functionalities and the one-pot assembly of the SUMO–peptide conjugate by a sequential native chemical ligation (NCL)/SEA native peptide ligation reaction sequence. This protocol also enables the isolation of a SUMO SEA latent thioester, which can be attached to a target peptide or protein in a subsequent step. It is compatible with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, and it gives access to homogeneous, reversible and functional SUMO conjugates that are not easily produced using living systems. The synthesis of SUMO–peptide conjugates on a milligram scale takes 20 working days.

Shedding-generated Met receptor fragments can be routed to either the proteasomal or the lysosomal degradation pathway.

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin‐regulated intramembrane proteolysis (PS‐RIP) is involved in ligand‐independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ‐secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N‐ and C‐terminal fragments are degraded directly in the lysosome, with C‐terminal fragments escaping γ‐secretase cleavage. PS‐RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N‐terminal fragments associate with the high‐affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS‐RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand‐independent downregulation of the receptor.

Gene expression changes in Schistosoma mansoni sporocysts induced by Biomphalaria glabrata embryonic cells.

Abstract.Biomphalaria glabrata embryonic (Bge) cells have been shown to provide favourable environmental conditions for the development of Schistosoma mansoni sporocysts. We investigated the effect of Bge excretory-secretory products on metabolic activity and gene transcription in S. mansoni mother sporocysts. Using the differential-display technique, we identified several sporocyst transcripts regulated by exposure to Bge soluble components. Research in databases indicated that six of the eight differential products analysed were homologous to sequences already present in databases. Two transcripts appeared of interest for schistosome development since they could be associated with cell division and protein synthesis in developing sporocysts. Their up-regulation following contact with cell products was confirmed by semi-quantitative RT-PCR. The first fragment coded for a part of the chaperonin containing T-complex protein gamma subunit-like protein of S. mansoni (SmTCP 1-C). The second one represented a new S. mansoni expressed sequence tag encoding a protein homologous to various glutaminyl-tRNA synthetases (GlnRS). The full-length sequence of SmGlnRS was cloned from adult schistosomes and its primary sequence was compared to other GlnRS. The overexpression of SmTCP-1 and SmGlnRS could be correlated with the metabolic changes observed in Bge-exposed sporocysts.

A novel PEG-based solid support enables the synthesis of >50 amino-acid peptide thioesters and the total synthesis of a functional SUMO-1 peptide conjugate

A bis(2-sulfanylethyl)amino PEG-based resin enabled the synthesis of large (∼50 Aa) SEA or thioester peptides using Fmoc-SPPS. These peptide segments permitted the first total synthesis of a 97 amino-acid long SUMO-1-SEA peptide thioester surrogate and of a functional and reversible SUMO-1 peptide conjugate.