Publications by authors named "Georges Orfanoudakis"

12 Publications

  • Page 1 of 1

Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain.

PLoS One 2011 27;6(10):e25981. Epub 2011 Oct 27.

Oncoprotéines, UMR 7242 CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Université de Strasbourg, Illkirch, France.

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025981PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203139PMC
March 2012

Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b.

Virus Res 2011 Jan 17;155(1):213-20. Epub 2010 Oct 17.

Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas Sophias Ave, 11521 Athens, Greece.

The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.
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http://dx.doi.org/10.1016/j.virusres.2010.10.007DOI Listing
January 2011

Surface plasmon resonance analysis of the binding of high-risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI-1.

J Mol Recognit 2011 Jul-Aug;24(4):511-23. Epub 2010 Sep 14.

Équipe Oncoprotéines, FRE 3211, Institut de Recherche de l'École de Biotechnologie de Strasbourg, Université de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France.

The E6 oncoproteins from high-risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain-containing proteins. Human MAGI-1 is a multi-PDZ domain protein implicated into protein complex assembly at cell-cell contacts. High-risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI-1 via a C-terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione-S-transferase (GST). This approach was applied to measure the binding of MAGI-1 PDZ1 to the C-termini of viral or cellular proteins. Both high-risk mucosal HPV E6 C-terminal peptides and cellular partners of MAGI-1 PDZ1 bind to MAGI-1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI-1 PDZ1 shows a preference for C-termini with a valine at position 0 and a negative charge at position -3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site-directed mutagenesis of the HPV16 C-terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ-binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K(499) residue of MAGI-1 as a novel determinant of binding specificity. Finally, we showed that MAGI-1 PDZ1 also binds to the C-termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI-1.
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http://dx.doi.org/10.1002/jmr.1056DOI Listing
September 2011

Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein.

FEBS J 2010 Feb 7;277(3):774-89. Epub 2010 Jan 7.

Université de Strasbourg, CNRS FRE 3211, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.

The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome. Core+1/S is expressed as a result of internal initiation at AUG codons (85-87) located downstream of the polyprotein initiator codon, and corresponds to the C-terminal part of most ARFPs. Core+1/S is a highly basic polypeptide, and its function still remains unclear. In this work, untagged recombinant Core+1/S was expressed and purified from Escherichia coli in native conditions, and was shown to react with sera of HCV-positive patients. We subsequently undertook the biochemical and biophysical characterization of Core+1/S. The conformation and oligomeric state of Core+1/S were investigated using size exclusion chromatography, dynamic light scattering, fluorescence, CD, and NMR. Consistent with sequence-based disorder predictions, Core+1/S lacks significant secondary structure in vitro, which might be relevant for the recognition of diverse molecular partners and/or for the assembly of Core+1/S. This study is the first reported structural characterization of an HCV ARFP/Core+1 protein, and provides evidence that ARFP/Core+1/S is highly disordered under native conditions, with a tendency for self-association.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07527.xDOI Listing
February 2010

Modification of adenovirus type 5 tropism for a preferential transduction of human papillomavirus-positive cancer cells.

Arch Virol 2008 26;153(10):1921-5. Epub 2008 Aug 26.

UMR 7175, CNRS, Université Louis Pasteur, Oncoprotéines, ESBS, Illkirch Cedex, France.

Human group C adenoviruses can infect many cell types, and this is due to the widespread expression of their receptor, the coxsackievirus and adenovirus receptor (CAR). Adenovirus vectors for cancer gene therapy could be significantly improved if their tropism were restricted to tumor cells. In this work, previously identified peptides that target human papillomaviruses (HPV)-transformed cells were inserted into the HI loop of a non-CAR-binding fiber. These modified fiber proteins were able to assemble into adenovirus particles. We demonstrated that these modifications ablated the native tropism of adenovirus type 5, and these modified adenoviruses were shown to preferentially transduce HPV-transformed cell lines.
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http://dx.doi.org/10.1007/s00705-008-0185-8DOI Listing
November 2008

A novel adenovirus vector for easy cloning in the E3 region downstream of the CMV promoter.

Virol J 2008 Jun 6;5:73. Epub 2008 Jun 6.

Unité Mixte de Recherche 7175, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, France.

The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells.
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http://dx.doi.org/10.1186/1743-422X-5-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427025PMC
June 2008

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies.

Protein Expr Purif 2008 Jul 31;60(1):64-73. Epub 2008 Mar 31.

Equipe Oncoprotéines, UMR CNRS 7175-LC1, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brandt, BP 10413, 67412 Illkirch Cedex, France.

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.
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http://dx.doi.org/10.1016/j.pep.2008.03.022DOI Listing
July 2008

Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells.

Biochem Biophys Res Commun 2007 Sep 18;361(2):487-92. Epub 2007 Jul 18.

UMR 7175-LC1, CNRS, Université Louis Pasteur (Strasbourg I), ESBS, Boulevard Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France.

The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies.
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http://dx.doi.org/10.1016/j.bbrc.2007.07.040DOI Listing
September 2007

Structural and functional analysis of E6 oncoprotein: insights in the molecular pathways of human papillomavirus-mediated pathogenesis.

Mol Cell 2006 Mar;21(5):665-78

Equipe Oncoprotéine, UMR CNRS 7100, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sebastien Brant, BP 10413, 67412 Illkirch Cedex, France.

Oncoprotein E6 is essential for oncogenesis induced by human papillomaviruses (HPVs). The solution structure of HPV16-E6 C-terminal domain reveals a zinc binding fold. A model of full-length E6 is proposed and analyzed in the context of HPV evolution. E6 appears as a chameleon protein combining a conserved structural scaffold with highly variable surfaces participating in generic or specialized HPV functions. We investigated surface residues involved in two specialized activities of high-risk genital HPV E6: p53 tumor suppressor degradation and nucleic acid binding. Screening of E6 surface mutants identified an in vivo p53 degradation-defective mutant that fails to recruit p53 to ubiquitin ligase E6AP and restores high p53 levels in cervical carcinoma cells by competing with endogeneous E6. We also mapped the nucleic acid binding surface of E6, the positive potential of which correlates with genital oncogenicity. E6 structure-function analysis provides new clues for understanding and counteracting the complex pathways of HPV-mediated pathogenesis.
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http://dx.doi.org/10.1016/j.molcel.2006.01.024DOI Listing
March 2006

Binding of human papillomavirus 16 E6 to p53 and E6AP is impaired by monoclonal antibodies directed against the second zinc-binding domain of E6.

J Gen Virol 2005 Apr;86(Pt 4):1001-1007

UMR7100, Ecole Supérieure de Biotechnologie de Strasbourg, Pole API, boulevard Sébastien Brant, 67412 Illkirch Cedex, France.

The E6 protein of cancer-associated human papillomavirus type 16 (16E6) binds to p53 and, in association with E6AP, promotes its degradation through the ubiquitin-proteasome pathway. The aim of this work was to develop monoclonal antibodies against 16E6 and to test their effect on the binding of 16E6 to p53 and E6AP, and on the degradation of p53. It was shown that an antibody directed against the N terminus of 16E6 inhibited E6AP-dependent binding to p53 and degradation of p53, whereas two different antibodies directed to the second zinc-binding domain of 16E6 reduced 16E6 E6AP-independent binding to p53 and binding to E6AP but not degradation of p53.
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http://dx.doi.org/10.1099/vir.0.80607-0DOI Listing
April 2005

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines.

J Mol Recognit 2005 Mar-Apr;18(2):175-82

UMR7100-CNRS Ecole Supérieure de Biotechnologie de Strasbourg, Université Louis Pasteur, boulevard Sébastien Brandt, 67400-Illkirch, France.

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.
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http://dx.doi.org/10.1002/jmr.723DOI Listing
June 2005

Comparative properties of two peptide-antibody interactions as deduced from epitope delineation.

J Immunol Methods 2002 Jan;259(1-2):77-86

FRE2370-CNRS, Biotechnologie des Interactions Mol., Ecole Superieure de Biotech. de Strasbourg (ESBS), Boulevard Sébastien Brandt, 67400 Illkirch Cedex, France.

The linear epitope recognized by three closely related antibodies specific for the E6 oncoprotein of papillomavirus type 16 was delineated by phage display, spot peptide synthesis on cellulose membranes, and kinetic measurements with antigenic variants using a BIACORE. The same approaches, recently applied to an antibody specific for tobacco mosaic virus protein, led to the clear-cut delineation of a functional epitope comprising four key positions with well defined physico-chemical properties. In contrast, the E6 system is characterized by a non-essential contribution to binding of various factors, so that combinations of alternative properties are compatible with measurable binding activity.
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http://dx.doi.org/10.1016/s0022-1759(01)00496-3DOI Listing
January 2002
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