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Neuhaus, Jean-Marc
Nom
Neuhaus, Jean-Marc
Affiliation principale
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Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
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Voici les éléments 1 - 10 sur 29
- PublicationAccès libreThe functions of RMR proteins in the "Physcomitrella patens" secretory pathway(2017)
Dans le premier chapitre de cette thèse nous avons regroupé les principales données concernant le système sécrétoire et endomembranaire chez les plantes, et nous avons également documenté comment les protéines sont adressées aux vacuoles.
Dans le second chapitre, nous avons étudié le système sécrétoire de la mousse en développant une bibliothèque de marqueurs fluorescents. Différents mécanismes cellulaires semblent conservés entre les mousses et les plantes à fleurs.
Dans le troisième chapitre, nous nous sommes intéressés à la caractérisation des simples et quintuple knock-out mutants RMR. Nous avons finalement obtenu un phénotype de tri vacuolaire: un défaut d’adressage est observé avec le marqueur fluorescent Citrine-Card dans les simples, triple et quintuple KO mutants. Le signal fluorescent a été détecté dans le réticulum endoplasmique chez les mutants, tandis que la fluorescence est observée dans la vacuole centrale chez le WT. Cela montre que l’adressage à la vacuole d’une protéine comportant ce ctVSD est dépendant des RMRs.
Dans la dernière partie de ce travail, nous avons identifié des partenaires interagissant très probablement avec la partie cytosolique de PpRMR2 par des analyses de GST pull-down et de spectrométrie de masse., Plant vacuoles play a wide range of functions within the cell, from the maintenance of turgor pressure and rigidity, to the storage or degradation of various molecules. Two types of vacuoles with distinct pH can coexist in the same single cell. Acidic vacuoles can be considered as homologues to animal lysosomes while neutral vacuoles are involved in proteins and secondary metabolites storage. Targeting of proteins to the lytic vacuole has been extensively studied and the vacuolar receptors involved, the VSRs, are now well characterized in higher plants. However, less is known about the traffic of proteins to the neutral/storage vacuole. RMR proteins are thought to be vacuolar receptors for the neutral/storage vacuole. However, the complete deletion of the five RMR genes in Physcomitrella patens did not lead to any developmental phenotype. This work aimed to investigate the role of RMR proteins in the moss.
In the first chapter, we review the plant secretory pathway system and how proteins are targeted to vacuoles.
In the second chapter, we studied the moss secretory pathway by developing a fluorescent reporter library. Several mechanisms seem to be conserved between the moss and the flowering plants.
In the third chapter, we focused on the characterization of the single and quintuple knock-out RMR mutants. We finally obtained a trafficking phenotype: the fluorescent reporter Citrine-Card was mistargeted in the single, triple and quintuple KO mutants. Fluorescent signal was detected in endoplasmic reticulum in the mutants, while it was observed in the central vacuole in WT. Trafficking to vacuole of a protein carrying this ctVSD was RMR-dependent.
In the last part of this thesis, we identified some putative binding partners of the cytosolic part of PpRMR2 by GST pull-down assay and mass spectrometry analysis. - PublicationAccès libreStudy of moss vacuoles and functional characterization of the putative vacuolar receptors: the RMR proteins(2012)
My work hypothesis is that the vacuolar system of higher plants has evolved from simple ancestors, which might have been preserved in lower plants. This evolution is reflected in the gene families involved in vacuole biogenesis. In a first part, we established the moss P. patens as a model system for the study of the secretory pathways. In a second part, we performed a comparative study of the plant-specific aspects of the vacuolar system. And finally in a third part, we tried to establish the functional role of PpRMR genes by the analysis of the complete RMR deletion mutants. Several strategies were considered to investigate a putative disorder due to RMRs loss of function. So far, no phenotype was detected in the mutants. Nevertheless the absence of the RMR family gene seems not to be necessary for moss development. - PublicationAccès libreComparison of proteolytic system secreted in dermatophytes and 'Aspergillus fumigatus', used as a reference(2011)
Lors de la première étape de ce travail, nous avons montré que deux différents ensembles de protéases étaient sécrétés par Aspergillus fumigatus à pH 4.0 et à pH 7.0. A pH 7.0, cet ensemble comprend une subtilisine et une métalloprotéase qui sont des endoprotéases, des aminopeptidases non spécifiques (Laps pour leucine aminopeptidases) et une dipeptidylpeptidase IV (DppIV) qui est une X-prolyl peptidase. Il était connu que des peptides générés par une activité endoprotéolytique sur des grandes protéines pouvaient être digérés ensuite synergiquement par les Laps et la DppIV. Lors de ce processus les Laps ôtent les acides aminés un par un depuis l’extrémité N-terminale d’un peptide jusqu’à une séquence X-Pro où les laps s’arrêtent. Toutefois, les séquences X-Pro sont enlevées par la DppIV qui génère ainsi un nouveau substrat pour les Laps. A pH 4.0, l’ensemble des protéases sécrétées par Aspergillus fumigatus comprenait une pepsine, une protéase inconnue de la classe des glutamique-protéases (appelée ici AfuGprA), des sédolisines (SED) qui avaient été caractérisées comme étant des tripeptidyl-peptidases non spécifiques, et une nouvelle protéase de la famille S28 (appelée ici AfuS28). Il a été montré dans ce travail que des grands peptides pouvaient être digérés à pH acide par les activités synergiques des Seds et AfuS28. Lors de ce processus les Seds ôtent les acides aminés trois par trois depuis l’extrémité N-terminale d’un peptide jusqu’à une proline en position 3 ou 4. Toutefois, les séquences d’arrêt X-XX-Pro et X-X-XX-Pro sont enlevées par l’activité d’AfuS28 qui génère ainsi un substrat dégradable par les Seds. En conclusion, chacun des eux ensembles des protéases sécrétées par Aspergillus fumigatus comprenait des aminopeptidases non spécifiques butant sur des résidus Pro, et une prolyl peptidase. L’activité de cette dernière enzyme génère un nouveau substrat pour les aminopeptidases non spécifiques.
La deuxième étape a consisté en l’étude d’AfuGprA et son importance dans l’activité endoprotéolytique d’Aspergillus fumigatus. Nous avons montré que soit Pep soit AfuGprA était nécessaire pour la croissance du champignon à pH acide dans un milieu contenant des protéines non dégradées comme seul nutriment.
Et enfin, la dernière partie de cette thèse s’est focalisée sur l’identification des protéases sécrétées par deux espèces de dermatophytes, Microsporum canis et Arthroderma benhamiae dans un milieu ne contenant que des protéines. Comme chez Aspergillus fumigatus, ces deux dermatophytes sécrètent à pH 4.0 et à pH 7.0 un ensemble particulier de protéases. Nombres de ces protéases ne sont pas connues chez ces dernières, mais sont des orthologues probables de protéases caractérisées chez Aspergillus fumigatus. C’est la première fois que des protéases acides sont identifiées à pH acide chez ces champignons. Ces investigations suggèrent des mécanismes communs de dégradation des protéines chez les Aspergillus et chez les dermatophytes., Dermatophytes are highly specialized pathogenic fungi which grow exclusively in the stratum corneum, nails or hair and are the most common agents of superficial mycoses. In a medium containing keratin as the sole nitrogen source they secrete a set of endo- and exoproteases able to digest keratin into amino acids and short peptides to be assimilated. Proteases secreted by dermatophytes are similar to those of Aspergillus spp. Therefore, Aspergillus fumigatus was used as a model to investigate the different steps of protein degradation in acidic and neutral environments by fungi such as dermatohytes.
During growth in a protein medium at neutral pH, Aspergillus fumigatus secretes neutral and alkaline endoproteases, an X-prolyl peptidase (DppIV) and leucine aminopeptidases (Laps) which are non-specific monoaminopeptidases. Laps cannot remove any amino acids from a peptide with a N-terminal X-Pro sequence. However, large peptides generated from protein digestion by endoproteolysis can be further digested into amino acids and X-pro dipeptides by the synergistic action of Laps and DppIV. We have shown that A. fumigatus secretes a distinct set of proteases at acidic pH which includes an aspartic endoprotease of the pepsin family (Pep1), a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II, tripeptidyl-peptidases of the sedolisin family (SedB and SedD) and a novel prolylpeptidase, AfuS28.
The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild type D141 and in a pepΔ mutant. We have shown that either A. fumigatus Pep or AfuGprA is necessary for fungal growth in protein medium at acidic pHIn conclusion, Pep and AfuGprA constitute a pair of endoproteases active at acidic pH in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH.
We have shown that Seds and AfuS28 synergistically digest large peptides generated by exoprotease activity into amino acids, di- and tripeptides. Seds degrade peptides from their N-terminus into tripeptides, however Pro in P1 and P’1 position acts as a stop sequence. In a complementary manner, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues not bypassed by other exoproteases.
In a third part of this thesis we have tested the ability of two dermatophyte species, Microsporum cani and Arthroderma benhamiae, to grow in a protein medium that promotes secretion of proteases. We have shown that at neutral and acidic pH, dermatophytes secreted different proteases. Our investigation revealed new dermatophyte secreted proteases homologous to those secreted by A. fumigatus and suggests common basic mechanisms for extracellular protein digestion in dermatophytes and in Aspergillus spp. at acidic and neutral pH. - PublicationAccès libreThe role of palmitoylation in the secretoy pathway of plants(2011)
In the second part of this thesis, I addressed the more general role of palmitoylation in the secretory pathway through the use of a potent palmitoylation inhibitor: 2BP. This study showed a specific action of the drug in TGN/post-TGN compartments. The drug affected the structural maintenance of macrovesicles of secretion. The macrovesicles of secretion have recently been characterized by cryofixation and by electron tomography. They are structures reminiscent of a bunch of grapes, where each grape is a secretory vesicle. They are associated with the building with TGN-rich secretory vesicles with a diameter of few tens of nanometres. These vesicles are particularly visible in tissues with a high growth rate, such as pollen tubes. 2BP drastically changed the state of aggregation of macrovesicles of secretion. In an imaged way, each grape was released and a diffuse fluorescence was observed. Palmitoylation is therefore important in the formation or stability of this important secretory structure. A possible extension of this work would be the isolation of palmitoylated proteins involved in this stabilization. In addition, palmitoylated Rabs were detected for the first time in a plant. The three plant Rabs for which I detected the effect of 2BP are located in post-Golgi compartments.
In a last part of the thesis, I decided to investigate the route of secretion of GFP-Chi, a vacuolar markerwidely used in the lab that can be followed along the route of secretion. Apparent contradictions have been reported: a vacuolar sorting of this marker by the Golgi-TGN-PVC pathway or an independent-COPII trafficking that can bypass the classic route. Unexpectedly, when GFP-Chi was co-expressed with NtSar1H74L (a dominant-negative mutant blocking the ER-Golgi trafficking by preventing the formation of COPII vesicles), the reporter reached the vacuole. This suggests that the alternative pathway bypassing the Golgi can take place. I also detected the presence of a possible GFP-Chi dimer associated with the membrane fraction upon ultracentrifugation. The nature of this dimer of a soluble protein remains to be investigated although several cases of aggregation of soluble proteins have been reported in the literature (like β-amyloids presents in the of Alzheimer disease). Another matter of great interest is whether the dimer reaches the vacuole or is only transient intermediate during the transport to the vacuole. - PublicationAccès libreIdentification of genes expressed during the compatible interaction of grapevine with Plasmopara viticola through suppression subtractive hybridization (SSH)(2011)
; ; Slaughter, Ana R.Grapevine (Vitis vinifera) is the most widely cultivated and economically important fruit crop, but is susceptible to a large number of diseases. Downy mildew, caused by the obligate biotrophic oomycete pathogen Plasmopara viticola, is a common disease present in all regions where vines are cultivated. We used suppression subtractive hybridization (SSH) to generate two cDNA libraries enriched for transcripts induced and repressed, respectively, in the susceptible grapevine cultivar Chasselas 24 h after inoculation with P. viticola. Differential screening on glass slide microarrays yielded over 800 putative genes that were up-regulated in response to P. viticola infection and over 200 that were down-regulated. One hundred and ninety four of these, were sequenced, identified and functionally categorised. Transcript abundance of twelve genes over a 48 h time course was examined by reverse transcriptase quantitative real-time PCR (RT-qPCR). Ten of these genes were induced/enhanced by P. viticola challenge, confirming the results of the SSH. The vast majority of the genes identified are related to defence. Interestingly, many genes involved in photosynthesis were down-regulated. - PublicationAccès libreLocalization and interaction of AtRMR receptors in the plant secretory pathway(2011)
On toutefois, les RMRs sont beaucoup moins connus que les VSRs. Dans la présente étude, je me suis concentré sur la localisation de différents RMRs présents dans les cellules végétales. Par ailleurs, j'ai étudié la possible dimérisation (homo-dimérisation et hétéro-dimérisation) entre les différents types de récepteurs AtRMR.
Pour la localisation, j'ai généré différents vecteurs d'expression pour les plantes portant différentes protéines fluorescentes fusionnées à différent types de AtRMR. Par la suite, ces protéines de fusion ont été localisées dans les cellules en utilisant le microscope confocal. La localisation a été effectuée chez des plantes d'Arabidopsis thaliana transgéniques et chez des feuilles de Nicotiana benthamiana transformées par agroinfiltration. Dans ces expériences, AtRMR1 et AtRMR2 ont montré des localisations subcellulaires différentes. AtRMR1 est localisé dans TGN, tandis que AtRMR2 est localisé dans la membrane du Reticulum Endoplasmique (ER). Cette différente localisation est due à la présence d'un signal putatif de localisation présent dans la séquence linker de AtRMR1. En fait, quand cette séquence est placée sur AtRMR2, elle est capable de relocaliser la protéine dans le Réseau Trans-Golgien (TGN).
Enfin, pour tester l'éventuelle dimérisation entre différents types de AtRMR, j'ai développé une technique de Complémentation Bimoléculaire Fluorescente (BiFC). En utilisant cette technique, j'ai démontré que AtRMR1 peut former des homo-dimères et peut interagir avec AtRMR2 en formant des hétéro-dimères. De plus, homo-et hétéro-dimères montrent la même localisation dans le TGN. Ce résultat a démontré que AtRMR2 peut quitter l’ER sous forme d’hétéro-dimère grâce à la présence du signal de localisation dans la séquence linker de AtRMR1. Par ailleurs, en utilisant des mutants de délétion de différents domaines, j'ai démontré que le domaine transmembranaire et la séquence linker sont probablement les domaines impliqués dans l'interaction protéine-protéine., In the last few years, it was demonstrated that many vacuolar proteins are sorted to their final destination by cargo receptors. Therefore in this study I focused on RMR proteins (Receptor Membrane Ring-H2), a new family of putative receptors, composed of six genes in Arabidopsis thaliana (AtRMR), probably involved in protein transport to vacuoles (Jiang et al.,, 2000; Park et al., 2005; Park et al., 2007; Hinz et al., 2007). These receptors were identified by their homology to the PA domain (Protease Associated Domain) present in the Vacuolar Sorting Receptors (VSR) that are well known to bind and sort vacuolar proteins (Paris et al., 2002).
Much less is known about these proteins than about VSRs. In the present study I focused on the localization of the different members present in plant cells. Moreover I studied the possible dimerization (homo end/or hetero) between the different types of AtRMR receptors.
For the localization, I have generated different plant expression vectors carrying different fluorescent protein reporters fused to AtRMRs to use in a confocal microscope experiment. The localization was performed in Arabidopsis thaliana transgenic plants and Nicotiana benthamiana leaves transformed by agro-infiltration. In these experiments AtRMR1 and AtRMR2 showed different subcellular localizations. AtRMR1 localizes in TGN while AtRMR2 localizes in the membrane of ER. This different localization is due by the presence of a putative localization signal present in the sequence linker of AtRMR1. In fact this sequence, when is placed on AtRMR2, is able to relocate the protein in the TGN.
To test the possible AtRMR-AtRMR dimerization I developed Bimolecular Fluorescence Complementation (BiFC) reporters. Using this technique I demonstrated that AtRMR1 can make homodimers and can interact with AtRMR2 making heterodimers. Moreover homo- and heterodimers showed the same localization in the TGN. This result demonstrated that AtRMR2 can exit from the ER as a heterodimer thanks to the presence of the localization signal in the sequence linker of AtRMR1. Moreover using AtRMR deletion mutants I demonstrated that the transmembrane domain and the sequence linker are probably the domains involved in protein-protein interaction. - PublicationAccès libreExpression of a glycosylated GFP as a bivalent reporter in exocytosis(2010)
; Di Sansebastiano, Gian PietroThe complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology. - PublicationAccès libreOptimisation and comparison of transient expression methods to express the green fluorescent protein in the obligate biotrophic oomycete Plasmopara viticola(2008)
; Grape downy mildew is caused by Plasmopara viti-cola, an obligate biotrophic oomycete and a major path-ogen of grapevine. Studying obligate biotrophic patho-gens is difficult as they cannot grow without their host. We therefore attempted to develop a method where the pathogen could be visualized and quantified in planta without killing the host plant. To this end P. viticola was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombard-ment and transformation with Agrobacterium tume-faciens were applied. Although some methods yielded positive transformation events, no stable strain of P. viticola expressing gfp could be generated. Using the electroporation method, we obtained transient P. viti-cola transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transform-ing P. viticola. Transformation with A. tumefaciens had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations. - PublicationAccès libreThe knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens(2008)
; - PublicationAccès librePharmaceutical Proteins in Plants: A Strategic Genetic Engineering Approach for the Production of Tuberculosis Antigens(2008)
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