Nathalie Galéotti

Synaptic multiprotein complexes associated with 5‐HT2C receptors: a proteomic approach

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‐HT2C) 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‐HT2C receptors interact with PSD95 and the Veli3–CASK–Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5‐HT2C receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5‐HT2C receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.

Behavioural manipulation in a grasshopper harbouring hairworm: a proteomics approach.

Abstract The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insects responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshoppers central nervous system (CNS) by producing certain ‘effective’ molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the hosts CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.

Regulation of human SRY subcellular distribution by its acetylation/deacetylation.

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 β, 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.

'Suicide' of crickets harbouring hairworms: a proteomics investigation

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.

PACAP type I receptor transactivation is essential for IGF-1 receptor signalling and antiapoptotic activity in neurons

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.

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

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).

Difference in Mass Analysis Using Labeled Lysines (DIMAL-K) A New, Efficient Proteomic Quantification Method Applied to the Analysis of Astrocytic Secretomes

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 μg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

The proteomics: a new prospect for studying parasitic manipulation

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

Stereochemical control in the preparation of α-amino N-methylthiazolidine masked aldehydes used for peptide aldehydes synthesis

Abstract 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 l -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.

Synthesis and in vitro cytotoxicity of diastereoisomerically modified dolastatin 15 analogues

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.