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Background: Despite sensitivity of MRI to diagnose multiple sclerosis (MS), prognostic biomarkers are still needed for optimized treatment. Objective: The objective of this paper is to identify cerebrospinal fluid (CSF) diagnostic biomarkers of MS using quantitative proteomics and to analyze their expression at different disease stages. Methods: We conducted differential analysis of the CSF proteome from control and relapsing–remitting MS (RRMS) patients followed by verification by ELISA of candidate biomarkers in CSF and serum in control, clinically isolated syndrome (CIS), RRMS and progressive MS (PMS) patients. Results: Twenty-two of the 527 quantified proteins exhibited different abundances in control and RRMS CSF. These include chitinase 3-like protein 1 (CHI3L1) and 2 (CHI3L2), which showed a strong expression in brain of MS patients, especially in astrocytes and microglial cells from white matter plaques. CSF and serum CHI3L1 levels increased with the disease stage and CIS patients with high CSF (>189 ng/ml) and serum (>33 ng/ml) CHI3L1 converted more rapidly to RRMS (log rank test, p < 0.05 and p < 0.001, respectively). In contrast, CSF CHI3L2 levels were lower in PMS than in RRMS patients. Accordingly, CSF CHI3L1/CHI3L2 ratio accurately discriminated PMS from RRMS. Conclusions: CSF CHI3L1 and CHI3L2 and serum CHI3L1 might help to define MS disease stage and have a prognostic value in CIS.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

SRY, a Y chromosome-encoded DNA-binding protein, is required for testis organogenesis in mammals. Expression of the SRY gene in the genital ridge is followed by diverse early cell events leading to Sertoli cell determination/differentiation and subsequent sex cord formation. Little is known about SRY regulation and its mode of action during testis development, and direct gene targets for SRY are still lacking. In this study, we demonstrate that interaction of the human SRY with histone acetyltransferase p300 induces the acetylation of SRY both in vitro and in vivo at a single conserved lysine residue. We show that acetylation participates in the nuclear localisation of SRY by increasing SRY interaction with importin beta, while specific deacetylation by HDAC3 induces a cytoplasmic delocalisation of SRY. Finally, by analysing p300 and HDAC3 expression profiles during both human or mouse gonadal development, we suggest that acetylation and deacetylation of SRY may be important mechanisms for regulating SRY activity during mammalian sex determination.

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.

Dolastatin 10 and dolastatin 15 have been analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. Interestingly, in-source fragmentations could be easily detected for both compounds. Determination of the primary structure of both pseudopeptides could be done by using post-source decay (PSD) analysis. The molecular ion was used as the precursor ion in the case of dolastatin 10, whereas two in-source fragment ions and cationized (Na and K) precursors were selected in the case of dolastatin 15. An analogue of dolastatin 15 containing an amino alkyl linkage (compound 1) was also studied. Despite its close chemical structure its PSD spectrum exhibited a quite different pattern.

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002).

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

Solid phase synthesis of Ψ[CH2NH] pseudopeptide 3 was accomplished by reductive amination of peptidyl aldehyde 1 with resin bound amino peptide 2. No epimerization took place during the aldehyde preparation or the reductive amination step. This convergent strategy should facilitate the synthesis of analogues of the neuropeptide PACAP.

Peptidyl aldehydes are potent transition state analogue inhibitors of cysteine and serine proteinases. The aldehyde function has recently been used for chemoselective peptide ligation. The preparation of peptidyl aldehydes on a solid support requires that the aldehyde be masked during peptide elongation and generated in a final step under mild conditions. We report here the preparation of peptidyl aldehydes by copper salt-mediated neutral hydrolysis of the corresponding C-terminal thiazolidinyl peptides which were elongated on a solid support.

Peptidyl aldehydes were synthesized using thiazolidine peptides as precursors. Hydrolysis of the heterocycles is easily accomplished using cupper salts to furnish the peptidyl aldehydes with very good yield and with no epimerization. © 1997 Published by Elsevier Science Ltd.

Phosphopeptides have been described as protease inhibitors as well as haptens for the preparation of abzymes exhibiting esterase activity. Phosphono- or phosphinopeptides have previously been prepared by solution synthesis, starting from the fully protected phosphoester dipeptide analogue. We have compared the reactivity of phosphonic versus carboxylic acid using BOP- or PyBOP-mediated activation. These reagents have been efficiently used for the preparation of mixed phosphonate diesters in the presence of an alcohol by phosphonic acid activation. We demonstrate their efficiency for solid phase synthesis of phosphonopeptides (or phosphinopeptides) in the presence of an amine by carboxy acid activation, starting from an unprotected phosphodipeptide analogue. In contrast, the efficient BroP-mediated monoesterification of α-amino-phosphonic acid is not suitable for phosphonopeptide preparation by SPPS.

Neurodegenerative diseases often lack early and specific diagnostic and prognostic biomarkers. Many studies are focusing on the cerebrospinal fluid (CSF) proteome to identify relevant biomarkers and therapeutic targets for these disorders. An alternative approach consists in comparing proteins secreted by healthy neurons and neurons degenerating by apoptosis, one of the mechanisms underlying neuronal death in neurodegenerative diseases. Here, we adapted the stable isotope labeling by amino acids in cell culture (SILAC) technology to primary cultures of mouse cerebellar granule neurons (CGNs), a well-characterized in vitro model of neuronal apoptosis, in order to identify variations in protein release by neurons during apoptosis. Using two different heavy isotope labels followed by liquid chromatography coupled with Fourier transform tandem mass spectrometry, we directly compared the secretome of apoptotic and surviving CGNs. A total of 1375 proteins were identified in CGN-conditioned media. Among these proteins, 47 were differentially expressed in the supernatants of apoptotic and surviving neurons. About 50% of them have been previously identified in human CSF and some are involved in neuronal death or neuroprotection. This list of apoptosis-regulated proteins should be considered when using targeted quantitative proteomics approaches to characterize or validate CSF biomarkers of neurodegenerative disorders.

A series of stereoisomeric monosaccharide °-phostones (cyclic phosphonate esters) were prepared and their structures were confirmed by single crystal X-ray analysis. Esterification of the D-gluco phostonic acid with appropriate sugar alcohols gave the first 1→6 and 1→4 disaccharide glycomimetics with phosphorus at the anomeric carbon of the non-reducing unit. Their structures and configuration at phosphorus were established by X-ray analysis.

Dolastatin 15 1(4S,7S) is a potent cytostatic depsipeptide of marine origin. Analysis of the effects of the stereochemistry in the non-peptide moiety on activity revealed that chirality inversion in the aromatic terminal region preserves the antiproliferative activity.

The reduction of (5S)-5-alkyl-2,4-dioxopyrrolidines, so-called tetramic acids, by NaBH 4 gives only partial diastereofacial selectivity in the case of the N-substituted analogues 9f-i, unlike the carbamate derivatives 9a-e which give the reduced cis-pyrrolidinones 10βa-e. Increasing the steric hindrance of either the N- or C-5-substituents enhances the re-face selectivity. On the other hand, reduction of the heterobicyclic compound 9n leads to a dramatic reversal of the stereoselectivity. Preliminary calculations show that the N-atom of the ring is slightly pyramidalized; the direction of hydride addition could be a consequence of this finding

Efficient synthesis of dolastatin 15 (1) was accomplished following a convergent strategy. The pyrrolidinone cycle of 5 was obtained by thermic cyclization of the corresponding Meldrum's adduct 4. The methylation of the enol function was performed under Mitsunobu conditions. On the other hand, the peptide part 10 was elongated by using PyCloP as coupling reagent. The ester linkage between both segments was efficiently performed under conditions we previously described by using a mixed anhydride activation with isopropenyl chlorocarbonate. Final compound 12 was found to exhibit physical properties slightly different from those recently reported by Pettit and co-workers for the natural dolastatin 15 and their synthesis product.

The Mitsunobu reaction was efficiently used to introduce oxazolines and thiazolines in the peptide backbone. The reaction proceeded from β-hydroxy α-amino acid-containing peptides at room temperature in 58-72% isolated yields.

The reduction of (5S)-5-alkyl-2,4-dioxopyrrolidines, so-called tetramic acids, by NaBH 4 gives only partial diastereofacial selectivity in the case of the N-substituted analogues 9f-i, unlike the carbamate derivatives 9a-e which give the reduced cis-pyrrolidinones 10βa-e. Increasing the steric hindrance of either the N- or C-5-substituents enhances the re-face selectivity. On the other hand, reduction of the heterobicyclic compound 9n leads to a dramatic reversal of the stereoselectivity. Preliminary calculations show that the N-atom of the ring is slightly pyramidalized; the direction of hydride addition could be a consequence of this finding

Peptidyl aldehydes were synthesized using thiazolidine peptides as precursors. Hydrolysis of the heterocycles is easily accomplished using cupper salts to furnish the peptidyl aldehydes with very good yield and with no epimerization. © 1997 Published by Elsevier Science Ltd.

The Mitsunobu reaction was efficiently used to introduce oxazolines and thiazolines in the peptide backbone. The reaction proceeded from β-hydroxy α-amino acid-containing peptides at room temperature in 58-72% isolated yields.

Efficient synthesis of dolastatin 15 (1) was accomplished following a convergent strategy. The pyrrolidinone cycle of 5 was obtained by thermic cyclization of the corresponding Meldrum's adduct 4. The methylation of the enol function was performed under Mitsunobu conditions. On the other hand, the peptide part 10 was elongated by using PyCloP as coupling reagent. The ester linkage between both segments was efficiently performed under conditions we previously described by using a mixed anhydride activation with isopropenyl chlorocarbonate. Final compound 12 was found to exhibit physical properties slightly different from those recently reported by Pettit and co-workers for the natural dolastatin 15 and their synthesis product.

Insulin-like growth factor-1 (IGF-1) and pituitary adenylyl cyclase activating polypeptide (PACAP) are both potent neurotrophic and antiapoptotic factors, which exert their effects via phosphorylation cascades initiated by tyrosine kinase and G-protein-coupled receptors, respectively. Here, we have adapted a recently described phosphoproteomic approach to neuronal cultures to characterize the phosphoproteomes generated by these neurotrophic factors. Unexpectedly, IGF-1 and PACAP increased the phosphorylation state of a common set of proteins in neurons. Using PACAP type 1 receptor (PAC1R) null mice, we showed that IGF-1 transactivated PAC1Rs constitutively associated with IGF-1 receptors. This effect was mediated by Src family kinases, which induced PAC1R phosphorylation on tyrosine residues. PAC1R transactivation was responsible for a large fraction of the IGF-1-associated phosphoproteome and played a critical role in the antiapoptotic activity of IGF-1. Hence, in contrast to the general opinion that the trophic activity of IGF-1 is solely mediated by tyrosine kinase receptor-associated signalling, we show that it involves a more complex signalling network dependent on the PAC1 Gs-protein-coupled receptor in neurons.

International audience; Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts

This review paper by Thomas et al. (2005) is timely as many parasitologists are enthusiastic to study the manipulative strategy described. Thomas et al. (2005) present many interesting future prospects for research on parasitic manipulation, including the elucidation of molecular mechanisms causing alteration of host behaviour. Manipulative parasites can change host behaviour by secreting products which act directly and/or i A l m d o I t i a i l W p

International audience; The proteome of most parasite species is currently unknown. Hairworms (Nematomorpha), 300 species distributed around the world, are parasitic in arthropods (mainly terrestrial species) when juveniles, but they are free-living in aquatic environments when adult. Most aspects of their systematics and biology are currently unknown. The aim of this paper was (i) to report a novel and reproducible protocol for the analysis of the proteome of hairworms using two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (matrix laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)) and (ii) to determine the level of proteomic divergence between two sympatric but taxonomically unrelated nematomorph species in the adult stage, Paragordius tricuspidatus Dufour (Nematomorpha, Gordiidae) and Spinochordodes tellinii Camerano (Nematomorpha, Gordiidae). In total, 689 protein spots were observed for P. tricuspidatus, 575 for S. tellinii. Only 36.2% spots were shared between the two species. Quantitative analysis of the proteins which are common to both parasite species reveals substantial differences in the pattern of protein expression. These results suggest a rapid evolutionary divergence between these two nematomorph families. Also, to test the value of our MALDI-TOF protocol, we used Actin-2 (Act-2), a protein highly conserved in the course of evolution. Peptide mass fingerprint (PMF) data obtained for Act-2 of P. tricuspidatus and S. tellinii suggest a very high homology with Act-2 of different worms species belonging to the Bilateria phylum (Annelida and Nematoda) and more specifically to Lumbricus terrestris (Annelida, Lumbricidae) and Caenorhabditis elegans (Nematoda, Rhabditidae). We discuss our results in relationship with current ideas concerning the use of proteomics in systematics

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.

International audience; Chiral N-methyl thiazolidines masked α-amino aldehydes are used for solid phase peptide aldehyde elongation. Contrary to N-Boc-protected α-amino aldehydes, N-trityl protection secures the chiral integrity of the incoming aldehyde chiral C1′ carbon atom during condensation of the amino aldehydes with Image-cysteinyl residues. The Ac-Tyr-Val-Ala-Asp-H caspase inhibitor was prepared on a solid support starting from the N-trityl-amino thiazolidine masked aspartinal as a validation of this process.

The chemical nuclease metalloporphyrin (manganese(III) porphyrin) can cleave DNA irreversibly and can thus constitute a potential antitumor drug. However, these molecules show low permeability to cell surface membranes. We report here the conjugation of an amphipathic carrier peptide to improve considerably its cellular delivery. The metalloporphyrin-peptide conjugate can be internalized by cells within only 5 min of incubation with a yield as high as 80%. Furthermore, the metalloporphyrin-peptide conjugate is able to cleave in vitro high or low molecular weight DNA to the same extend as metalloporphyrin alone without affecting the sequence-specific cleaving activity of the porphyrin. The conjugate is 100-fold more efficient at inducing tumor cells death than the free metalloporphyrin via a mechanism involving genomic DNA cleavage. The results are promising for further therapeutic applications with antitumor drugs such as metalloporphyrin, and also with other existing drugs by using a carrier peptide system in order to improve the cellular uptake of such molecules.

Tert-butyl fluorocarbonate (Boc-F) is efficiently used for the synthesis of tert-butyl esters of N-protected amino acids. The reaction proceeds at room temperature and under mild conditions in the presence of triethylamine and 4-dimethylamino-pyridine.

Monoesters of N-protected α-amino phosphonic acid were prepared from phosphonic acid using BroP or TPyClU as activating agent. This reaction proceeds with good yield, even with hindered alcohols.

Tert-butyl fluorocarbonate (Boc-F) is efficiently used for the synthesis of tert-butyl esters of N-protected amino acids. The reaction proceeds at room temperature and under mild conditions in the presence of triethylamine and 4-dimethylamino-pyridine.