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Dr. Géoui is a seasoned biotech leader, with a career spanning two decades dedicated to…

Artikel von 🤖 🧠 Thibault GEOUI 🧬 💊

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  • Tech and Drugs - Podcast

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  • Extraction of proteins from formalin-fixed, paraffin-embedded tissue using the Qproteome extraction technique and preparation of tryptic peptides for liquid chromatography/mass spectrometry analysis.

    Curr Protoc Mol Biol.

    This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the…

    This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue.

    Andere Autor:innen
    • H Urlaub
    • U Plessmann
    • Peter Porschewski
    Veröffentlichung anzeigen
  • A bridge crosses the active-site canyon of the Epstein-Barr virus nuclease with DNase and RNase activities.

    J. Mol. Biol.

    Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn(2+), giving a possible explanation for its role in host mRNA degradation. Its crystal…

    Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn(2+), giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.

    Andere Autor:innen
    • Marlyse Buisson
    • David Flot
    • Nicolas Tarbouriech
    • ME Ressing
    • EJ Wiertz
    • Wim Burmeister
    Veröffentlichung anzeigen
  • New insights on the role of the gamma-herpesvirus uracil-DNA glycosylase leucine loop revealed by the structure of the Epstein-Barr virus enzyme in complex with an inhibitor protein.

    J Mol Biol.

    Epstein-Barr virus (EBV) is a human gamma-herpesvirus. Within its 86 open reading frame containing genome, two enzymes avoiding uracil incorporation into DNA can be found: uracil triphosphate hydrolase and uracil-DNA glycosylase (UNG). The latter one excises uracil bases that are due to cytosine deamination or uracil misincorporation from double-stranded DNA substrates. The EBV enzyme belongs to family 1 UNGs. We solved the three-dimensional structure of EBV UNG in complex with the uracil-DNA…

    Epstein-Barr virus (EBV) is a human gamma-herpesvirus. Within its 86 open reading frame containing genome, two enzymes avoiding uracil incorporation into DNA can be found: uracil triphosphate hydrolase and uracil-DNA glycosylase (UNG). The latter one excises uracil bases that are due to cytosine deamination or uracil misincorporation from double-stranded DNA substrates. The EBV enzyme belongs to family 1 UNGs. We solved the three-dimensional structure of EBV UNG in complex with the uracil-DNA glycosylase inhibitor protein (Ugi) from bacteriophage PBS-2 at a resolution of 2.3 A by X-ray crystallography. The structure of EBV UNG encoded by the BKRF3 reading frame shows the excellent global structural conservation within the solved examples of family 1 enzymes. Four out of the five catalytic motifs are completely conserved, whereas the fifth one, the leucine loop, carries a seven residue insertion. Despite this insertion, catalytic constants of EBV UNG are similar to those of other UNGs. Modelling of the EBV UNG-DNA complex shows that the longer leucine loop still contacts DNA and is likely to fulfil its role of DNA binding and deformation differently than the enzymes with previously solved structures. We could show that despite the evolutionary distance of EBV UNG from the natural host protein, bacteriophage Ugi binds with an inhibitory constant of 8 nM to UNG. This is due to an excellent specificity of Ugi for conserved elements of UNG, four of them corresponding to catalytic motifs and a fifth one corresponding to an important beta-turn structuring the catalytic site.

    Andere Autor:innen
    • Marlyse buisson
    • Nicolas Tarbouriech
    • Wim Burmeister
    Veröffentlichung anzeigen
  • Contribution to the Epstein-Barr virus Structural Genomic Project: The Uracil-DNA Glycosylase and the Nucleic-Acid Metabolism Enzyme

    PhD Thesis / Tel Archive

    Epstein-Barr Virus (EBV) is a Human γ –herpesvirus. It is the causative agent of
    diseases such as Infectious Mononucleosis and it is associated with several
    immunoproliferative syndromes. Unlike other herpesviruses such as herpes-simplex,
    there is no treatment against EBV. EBV genome features 86 open-reading frames
    which are potential targets for a structural genomics approach and for the rational
    design of new drugs. The first round of selected targets encode for 23 proteins…

    Epstein-Barr Virus (EBV) is a Human γ –herpesvirus. It is the causative agent of
    diseases such as Infectious Mononucleosis and it is associated with several
    immunoproliferative syndromes. Unlike other herpesviruses such as herpes-simplex,
    there is no treatment against EBV. EBV genome features 86 open-reading frames
    which are potential targets for a structural genomics approach and for the rational
    design of new drugs. The first round of selected targets encode for 23 proteins. The
    main problem we encountered was, unexpectedly, the poor expression and solubility
    of the majority of the selected targets. Among the 8 soluble proteins we obtained, 5
    were crystallized and 4 structures were solved. The main success factor was an
    individual treatment of each target rather than the use of standard protocols. The
    Uracil-DNA Glycosylase (UNG) is a DNA repair enzyme. It was impossible to obtain
    crystals of the protein alone, but crystals diffracting to 2.3 Å grew upon the formation
    of a complex with a phage-encoded inhibitory protein. The analysis of the structure of
    this complex shows that the « leucin-loop », a major domain of the active site,
    features a 7 residue insertion which is common to all γ –herpesvirus. Despite this
    difference, catalytic constants are similar to other organisms UNGs, suggesting a
    similar interaction with DNA. Current work and problems associated with other
    targets is also reported (in particular on the Alkaline Exonuclease).

    Veröffentlichung anzeigen
  • Structural genomics of the Epstein-Barr virus.

    Acta Crystallogr D Biol Crystallogr.

    Epstein-Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The…

    Epstein-Barr virus is a herpesvirus that causes infectious mononucleosis, carcinomas and immunoproliferative disease. Its genome encodes 86 proteins, which provided targets for a structural genomics project. After updating the annotation of the genome, 23 open reading frames were chosen for expression in Escherichia coli, initially selecting for those with known enzyme activity and then supplementing this set based on a series of predicted properties, in particular secondary structure. The major obstacle turned out to be poor expression and low solubility. Surprisingly, this could not be overcome by modifications of the constructs, changes of expression temperature or strain or renaturation. Of the eight soluble proteins, five were crystallized using robotic nanolitre-drop crystallization trials, which led to four solved structures. Although these results depended on individual treatment rather than standardized protocols, a high-throughput miniaturized crystallization screening protocol was a key component of success with these difficult proteins.

    Andere Autor:innen
    • Nicolas Tarbouriech
    • Marlyse Buisson
    • S Daenke
    • Stephen Cusack
    • Wim Burmeister
    Veröffentlichung anzeigen

Projekte

Auszeichnungen/Preise

  • Innovation award

    Elsevier

    For the development and launch of Embiology

  • Lead the Way award

    Elsevier

    "Lead the Way award" for the category Engage and understand the people we serve.
    #1 out of 180 submissions, selected by Elsevier's CEO.

    Challenged to redefine the market position of Reaxys, we partnered with a best-in-class scientific marketing agency to develop the “ChemSearchChallenge.” The mission was to engage chemists worldwide to help solve their real world problems. Based on market research and community engagement, we understood that if chemists cannot get their answers fast…

    "Lead the Way award" for the category Engage and understand the people we serve.
    #1 out of 180 submissions, selected by Elsevier's CEO.

    Challenged to redefine the market position of Reaxys, we partnered with a best-in-class scientific marketing agency to develop the “ChemSearchChallenge.” The mission was to engage chemists worldwide to help solve their real world problems. Based on market research and community engagement, we understood that if chemists cannot get their answers fast, it’s as if the information does not exist. In the first 3 weeks, 900+ participants registered and 1000+ new followers were added to Twitter. It has proven to be an innovative and customer-centric approach to community engagement.

  • Customer Focus Award

    Elsevier

  • MENRT (French Research Ministry) PhD Scholarship

    French Research Ministry

  • Study abroad scholarship

    Région Rhône-Alpes

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  • English

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