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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/300

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  • Publication
    Métadonnées seulement
    Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting
    (2013)
    Stigliano, Egidio
    ;
    Faraco, Marianna
    ;
    Neuhaus, Jean-Marc 
    ;
    Montefusco, Anna
    ;
    Dalessandro, Giuseppe
    ;
    Piro, Gabriella
    ;
    Di Sansebastiano, Gian-Pietro
    Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.
  • Publication
    Métadonnées seulement
    Secretory pathway research: the more experimental systems the better
    (2012)
    Denecke, J.
    ;
    Aniento, F.
    ;
    Frigerio, L.
    ;
    Hawes, C.
    ;
    Hwang, I.
    ;
    Mathur, J.
    ;
    Neuhaus, Jean-Marc 
    ;
    Robinson, D.
    Transient gene expression, in plant protoplasts or specific plant tissues, is a key technique in plant molecular cell biology, aimed at exploring gene products and their modifications to examine functional subdomains, their interactions with other biomolecules, and their subcellular localization. Here, we highlight some of the major advantages and potential pitfalls of the most commonly used transient gene expression models and illustrate how ectopic expression and the use of dominant mutants can provide insights into protein function.
  • Publication
    Métadonnées seulement
    Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium
    (2011)
    Sriranganadane, D.
    ;
    Reichard, U.
    ;
    Salamin, K.
    ;
    Fratti, M.
    ;
    Jousson, O.
    ;
    Waridel, P.
    ;
    Quadroni, M.
    ;
    Neuhaus, Jean-Marc 
    ;
    Monod, M.
    In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepDelta mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low 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. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.
  • Publication
    Métadonnées seulement
    Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH
    (2011)
    Sriranganadane, D.
    ;
    Waridel, P.
    ;
    Salamin, K.
    ;
    Feuermann, M.
    ;
    Mignon, B.
    ;
    Staib, P.
    ;
    Neuhaus, Jean-Marc 
    ;
    Quadroni, M.
    ;
    Monod, M.
    The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.
  • Publication
    Métadonnées seulement
    Plant cell vacuoles
    (Basingstoke: Macmillan Publishers, 2011)
    Neuhaus, Jean-Marc 
    ;
    Martinoia, E.
  • Publication
    Métadonnées seulement
    The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis
    (2010)
    Saint-Jean, B.
    ;
    Seveno-Carpentier, E.
    ;
    Alcon, C.
    ;
    Neuhaus, Jean-Marc 
    ;
    Paris, Nadine
    Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A-sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.
  • Publication
    Métadonnées seulement
    RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens
    (2010)
    Schaefer, Didier 
    ;
    Delacote, F.
    ;
    Charlot, F.
    ;
    Vrielynck, N.
    ;
    Guyon-Debast, A.
    ;
    Le Guin, S.
    ;
    Neuhaus, Jean-Marc 
    ;
    Doutriaux, M. P.
    ;
    Nogue, F.
    Gene targeting (GT) is a major tool for basic and applied research during which the transforming DNA, which shares sequence homology with a chromosomal target, integrates at the corresponding locus by homologous recombination (HR). In eukaryotes, GT recruits enzymes from the HR-mediated double strand break repair pathway. Different mechanisms of HR have been described which depend on the Rad52 epistasis group of genes, but which specific mechanism is used by the cell for GT remains unclear. In Saccharomyces cerevisiae, the RAD52 protein is essential for GT, and the RAD51 protein plays a minor role. In filamentous fungi and animal cells, however, GT depends on RAD51 and is weakly affected by suppression of RAD52. Genetic evidence also indicates that the non-homologous end-joining pathway of DSB repair has a negative impact on GT efficiencies, but how the balance between these two pathways is controlled is poorly understood. Here, we have examined the role of RAD51 in the only plant that exhibits high GT frequencies, the model bryophyte Physcomitrella patens. Our results show that the two RAD51 proteins have partially redundant functions in the maintenance of genome integrity and resistance to ionizing radiation. Furthermore, we demonstrate that loss of function of the two RAD51 proteins completely abolishes GT and strongly increases illegitimate integration rates in this moss. These findings demonstrate for the first time in plant the critical role of RAD51 in controlling the balance between targeted and random integration events observed upon transgenesis, and confirm that P. patens is a particularly interesting tool for studying GT in higher eukaryotes.
  • Publication
    Métadonnées seulement
    Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH
    (2010)
    Sriranganadane, D.
    ;
    Waridel, P.
    ;
    Salamin, K.
    ;
    Reichard, U.
    ;
    Grouzmann, E.
    ;
    Neuhaus, Jean-Marc 
    ;
    Quadroni, M.
    ;
    Monod, M.
    Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, 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 that cannot be removed during sequential digestion by nonspecific exopeptidases.
  • Publication
    Métadonnées seulement
    High-yield production and purification of recombinant T7-tag mature streptavidin in glucose-stressed E. coli
    (2008)
    Humbert, N.
    ;
    Schurmann, P.
    ;
    Zocchi, A.
    ;
    Neuhaus, Jean-Marc 
    ;
    Ward, T. R.
    The overexpression of toxic recombinant proteins is often problematic, leading to either low production levels or inclusion bodies. Streptavidin is no exception and thus the highest production level reported to date for streptavidin is 70 mg/L of functional protein. Herein, we report on the production in Escherichia coli and the purification of a recombinant mature streptavidin bearing a T7-tag. Optimization of critical parameters, including the glucose concentration, the pH and the time of induction as well as the use of BL21(DE3)pLysS cell strain, affords up to 120 mg/L functional streptavidin in soluble form. The yield can be further increased by an osmotic stress during the preculture by adding highly concentrated glucose before the inoculation of the culture medium, thus affording reproducibly 230 mg/L of soluble streptavidin. A single denaturing-renaturing step and affinity chromatography afford highly active tetrameric protein with >3.8/4.0 active sites.
  • Publication
    Métadonnées seulement
    Pharmaceutical proteins in plants. A strategic genetic engineering approach for the production of tuberculosis antigens
    (2008)
    Frutos, R.
    ;
    Denise, H.
    ;
    Vivares, C.
    ;
    Neuhaus, Jean-Marc 
    ;
    Vitale, S.
    ;
    Pedrazzini, E.
    ;
    Ma, J.
    ;
    Dix, P.
    ;
    Gray, J.
    ;
    Pezzotti, M.
    ;
    Conrad, U.
    ;
    Robinson, D.
    Tuberculosis (TB) is a re-emerging disease that is considered a major human health priority as well as an important disease of livestock. TB is also a zoonosis, and Mycobacterium tuberculosis and M. bovis, the human and bovine causative agents, respectively, are very closely related. Protection against TB is essentially achieved through vaccination with the Bacille Calmetle-Guerin (BCG) strain of M. bovis. Protection is, however, incomplete, and novel improved vaccines are currently under investigation. Production of protective antigens in transgenic plants, or "pharming," is a promising emerging approach, and a zoonosis-like TB is a good model for investigating the potential of this approach. Pharma-Planta, a European Commission-funded project and consortium, was set up to address this topic, within which a component is aimed at assessing the production efficacy and stability of the TB antigens in different compartments of the plant cell. This article is meant to introduce this promising approach for veterinary medicine by describing the ongoing project and its specific genetic engineering strategy.