References of "Twizere, Jean-Claude"
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See detailIdentification of VZV ORF9p potential cellular partners that could be important for the viral egress.
Lebrun, Marielle ULg; riva, laura; Rambout, Xavier ULg et al

Poster (2015, July 26)

ORF9p (homologous to HSV-1 VP22) is a VZV tegument protein essential for the viral replication. During the lytic cycle it is the mostly expressed gene. We have recently demonstrated that it is a substrate ... [more ▼]

ORF9p (homologous to HSV-1 VP22) is a VZV tegument protein essential for the viral replication. During the lytic cycle it is the mostly expressed gene. We have recently demonstrated that it is a substrate of the viral kinase ORF47p and that its ORF47p-dependent phosphorylation is important for the secondary envelopment process. We also have identified an acidic cluster (AC) within the protein that is important for its correct localization in the infected cells and for the interaction with ORF47p. The recombinant VZV expressing ORF9p-ΔAC presents an accumulation of capsids in the perinuclear space. ORF9p seems then to play an important role in several steps of the egress process. In this context, we sought to identify cellular partners of ORF9p that might be important for these functions. We performed a yeast two hybrid screen against the human ORFeome 5.1. and picked out 44 candidates among which 5 proteins playing roles in membrane organization and targeting. We currently are trying to confirm these interactions in infected cells and to assess the role of these interactions for the viral lytic cycle. [less ▲]

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See detailA fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae
Zune, Quentin ULg; Delepierre, Anissa ULg; Gofflot, Sebastien et al

in Applied Microbiology and Biotechnology (2015)

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical ... [more ▼]

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::GFP fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2D-gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein. [less ▲]

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See detailLIGAND-INDEPENDENT IDENTIFICATION OF ORPHAN GPCR ARRESTIN BINDING
Dupuis, Nadine ULg; Gilissen, Julie ULg; Derj, Anouar ULg et al

Poster (2014, June 05)

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See detailScale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according substrate heterogeneities
Brognaux, Alison ULg; Francis, Frédéric ULg; Twizere, Jean-Claude ULg et al

in Bioprocess and Biosystems Engineering (2014)

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane ... [more ▼]

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high-cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64%) confirming the fact that leakage is involved in high-cell density cultures. About 50% of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by 2D-DIGE analysis (2-Dimensional Differential in-Gel Electrophoresis) and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant. [less ▲]

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See detailAn interaction map for HTLV-1 Tax and PDZ-containing proteins
Blibek, Karim ULg; Rambout, Xavier ULg; Beaufays, Jérome et al

in Retrovirology (2014), 11

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See detailPredicting interactome networks perturbations in human cancer: application to gene fusions in acute lymphoblastic leukemia.
Juvenal Hajingabo, Leon; Daakour, Sarah ULg; Martin, Maud ULg et al

in Molecular biology of the cell (2014)

Genomic variations such as point mutations and gene fusions are directly or indirectly associated with human diseases. They are recognized as diagnostic, prognostic markers and therapeutic targets ... [more ▼]

Genomic variations such as point mutations and gene fusions are directly or indirectly associated with human diseases. They are recognized as diagnostic, prognostic markers and therapeutic targets. However, predicting the functional impact of these genetic alterations beyond affected genes and their products is challenging because diseased phenotypes are likely dependent of complex molecular interaction networks. Using as models three different chromosomal translocations ETV6-RUNX1 (TEL-AML1), BCR-ABL1, and TCF3-PBX1 (E2A-PBX1), frequently found in precursor-B cell acute lymphoblastic leukemia (preB-ALL), we develop an approach to extract perturbed molecular interactions from gene expression changes. We show that the MYC and JunD transcriptional circuits are specifically deregulated following ETV6-RUNX1 and TCF3-PBX1 gene fusions, respectively. We also identified the bulk mRNA NXF1-dependent machinery as a direct target for the TCF3-PBX1 fusion protein. Through a novel approach combining gene expression and interactome data analysis, we provide new insight into TCF3-PBX1 and ETV6-RUNX1 acute lymphoblastic leukemia. [less ▲]

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See detailAn interaction map for HTLV-1 Tax and PDZ-containing proteins.
Blibek, Karim ULg; Rambout, Xavier ULg; beaufays, Jérôme et al

Poster (2013, June 29)

Human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes for the Tax protein, which has a transforming capacity in vitro. Tax contains at its C-terminus a binding motif for PDZ domain-containing ... [more ▼]

Human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes for the Tax protein, which has a transforming capacity in vitro. Tax contains at its C-terminus a binding motif for PDZ domain-containing proteins (PSD95-DLG1-ZO1). It has been shown that the C-terminal motif of Tax is involved in Tax oncogenic capacity. Ten different PDZ domain-containing proteins have been reported to interact with Tax, but the specificity of Tax-human PDZome interactions has not been investigated. The objective of this study is to obtain a comprehensive interactome map for Tax and the human PDZome and to determine a global role of Tax-PDZ interactions in HTLV-1 biology. [less ▲]

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See detailInhibition of Tax transformation activity using a small molecule targetting Tax/PDZ domain interactions.
Blibek, Karim ULg; Fujii, Naoaki; Legros, Sebastien et al

Poster (2013, June 29)

Primate T-lymphotropic virus species comprise four members (HTLV-1 to -4) that have been discovered in human. Only the HTLV-1 infection leads to adult T-cell leukemia/lymphoma (ATLL) and tropical spastic ... [more ▼]

Primate T-lymphotropic virus species comprise four members (HTLV-1 to -4) that have been discovered in human. Only the HTLV-1 infection leads to adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis (TSP), an immune degenerative neurologic syndrome. All the four viruses share a similar genomic organization and encode transforming Tax oncoproteins. In contrast to HTLV-2 and 4, HTLV-1 and 3 Tax proteins contain a PSD-95/Drosophila Discs Large/Zona Occludens-I (PDZ) binding motif at their C-terminal that has been shown to play crucial roles in the distinct transforming properties of the Tax proteins. To systematically investigate PDZ-containing proteins roles in HTLV-1 biology, we initiated a global interactome network analysis of Tax and associated human PDZ-containing proteins. This was accomplished through the use of our framework of binary interactome mapping that includes stringent yeast two hybrid and pulldown screening, systematic retesting by protein complementation assay and evaluation of PDZ gene expression in T lymphocytes. [less ▲]

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See detailAn eYFP Reporter Gene for the Yeast Two-hybrid System
Damon, Coralie; Boxus, Mathieu; Twizere, Jean-Claude ULg et al

in Protein Journal (2013), 32(2), 126-130

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See detailInteractomic map of the Ets factors family : Identification of unexpected functions in mRNA processing
Rambout, Xavier ULg; Simonis, Nicolas; Brohée, Sylvain et al

Poster (2013, January 28)

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets ... [more ▼]

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets factors in order to better define their roles and regulations in normal and oncogenic processes. The Ets interactome was built on a high-throughput yeast-two hybrid (Y2H) approach, and a literature and database curation. We identified 431 PPIs and 276 different protein partners. Clustering of the Ets interactome divided it into 24 functional subnetworks classified on their novelty index and their size. Cluster#1 was exclusively composed of newly identified interaction partners and was highly connected to the Erg subfamily of Ets factors. Gene ontology enrichment analysis revealed that it was associated to mRNA processing. In support of this result, we observed in HeLa cells that ERG and the components of cluster#1 localized in p-bodies and stress granules, physically linked cytoplasmic sites of mRNA degradation and silencing. Hence, we hypothesized that Erg proteins might have a role in post-transcriptional gene regulation and be involved in cellular mRNAs degradation. To test this hypothesis, we performed a MS2-based tethering assay and showed that the recruitment of ERG on a mRNA reporter promoted inhibition of its expression via a two-fold decrease of its half-life. ERG controls degradation of target mRNAs via different mechanisms including polysome stability, mRNA deadenylation, and p-bodies aggregation. A microarray-based appraoch identified 321 endogeneous genes whose mRNA decay rate was lowered in ERG silenced cells. Results point out the Nter domain of ERG as the predominant domain required for mRNA degradation. Importantly, oncogenic TET-Erg fusions described in AML and Ewing’s sarcoma exhibited diminished ability to degrade target mRNAs, concomitantly with the loss of the ERG Nter domain. This reinforces the important role of Erg proteins in mRNA degradation in cancer. [less ▲]

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See detailThe homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1.
Bergiers, Isabelle; Bridoux, Laure; Nguyen, Nathan et al

in PloS one (2013), 8(11), 80387

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of ... [more ▼]

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53. [less ▲]

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See detailPP2A regulatory subunit Balpha controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.
Martin, Maud ULg; Geudens, Ilse; Bruyr, Jonathan et al

in EMBO Journal (2013)

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens ... [more ▼]

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Balpha regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Balpha in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Balpha in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Balpha controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Balpha, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Balpha/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion. [less ▲]

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See detailDirect and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: design of milliliter scale-down bioreactors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Biotechnology Progress (2013), 29(1), 48-59

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional ... [more ▼]

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability. [less ▲]

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See detailDevelopment of mini scale-down platform based on the response of GFP microbial biosensors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Poster (2012, May 18)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In our previous works, green fluorescent protein reporters have been used as biosensors of the heterogeneities generated in a two compartment scale-down reactor. As there is a huge variety of available whole cell biosensor to characterize the impact of such heterogeneities at the biological level, there is a need for high-throughput cultivation tools in order to investigate the usefulness of a given microbial biosensor among a library comprising several thousands of clones. This work is based on this statement and aims to investigate the potentialities of a mini scale-down platform. Four green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli: rpoS::gfp, uspA::gfp, csiE::gfp and yciG::gfp. The promoters rpoS and uspA are induced in response to a variety of stresses whereas the two other promoters, csiE and yciG, are supposed to be more specific in front of a glucose limitation. First, the response of these biosensors has been assessed in chemostat reactors. These kinds of experiments allow easier interpretation of responses of stress gene related to a glucose limitation since the extracellular conditions are constants and cells are renewed. Biosensors carrying the csiE and yciG promoters have exhibited an induction in function of the glucose limitation. Secondly, a scale-down platform has been tested with the same biosensors and two kinds of glucose addition mode. This scale-down platform involves high-throughput cultivation tools, i.e. in our case shake flask, equipped with non-invasive optical sensors for the monitoring of the dissolved oxygen profile in front of the glucose addition mode. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. The intermittent addition of glucose induces a slow down at the level of the GFP synthesis, suggesting that temporal accumulation of glucose inhibits the activity of the yciG and csiE promoters. In conclusion, the scale-down platform is able to reproduce the same kind of glucose fluctuations that encounters the cells in large-scale processes but not allows studying the impact of high-cell density culture on gene expression. [less ▲]

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See detailUse of microbial biosensors to detect substrate heterogeneities at the single cell level and assess microbial viability: Validation of a mini-bioreactor platform
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Conference (2012, March 15)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In this work, we have used these biosensors for the elaboration of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e. uspA::gfp, csiE::gfp and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response of substrate limitation and exhibit a strong fluorescence attenuation when cultivated in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini scale-down platform has been proposed as a high throughput tool to investigate rapidly the usefulness of a given microbial biosensor. This platform is composed of shake flask able to operate in fed-batch mode either by using the slow release or the intermittent feeding principle. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. Local heterogeneities have thus been reproduced at the level of these mini-bioreactors and these one have caused a decrease of GFP expression, as in conventional scale-down reactor. The presence of GFP in supernatants has also been noticed and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with the membrane permeability. [less ▲]

Detailed reference viewed: 60 (13 ULg)