Christophe Dhalluin

CUNY Graduate Center, New York City, New York, United States

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Publications (11)119.12 Total impact

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    ABSTRACT: The human recombinant Interferon-α2a (IFN α2a) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFN α2a was developed (PEGASYS). This 40 kDa PEG-conjugated IFNα2a (40PEG-IFNα2a) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNα2a. 40PEG-IFNα2a is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of 40PEG-IFNα2a and its positional isomers, in comparison with IFNα2a, using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNα2a is not modified by the presence of the polymer in all positional isomers constituting 40PEG-IFNα2a. Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of 40PEG-IFNα2a and its positional isomers to its cellular receptors.
    No preview · Article · May 2005 · Bioconjugate Chemistry
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    ABSTRACT: Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.
    No preview · Article · May 2005 · Bioconjugate Chemistry
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    ABSTRACT: MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.
    Full-text · Article · Mar 2001 · Molecular Cell
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    ABSTRACT: MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.
    No preview · Article · Feb 2001 · Molecular Cell
  • C Dhalluin · K S Yan · O Plotnikova · L Zeng · M P Goldfarb · M M Zhou

    No preview · Article · Jan 2001 · Journal of Biomolecular NMR

  • No preview · Article · Nov 2000 · Journal of Biomolecular NMR
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    ABSTRACT: SNT adaptor proteins transduce activation of fibroblast growth factor receptors (FGFRs) and neurotrophin receptors (TRKs) to common signaling targets. The SNT-1 phosphotyrosine binding (PTB) domain recognizes activated TRKs at a canonical NPXpY motif and, atypically, binds to nonphosphorylated FGFRs in a region lacking tyrosine or asparagine. Here, using NMR and mutational analyses, we show that the PTB domain utilizes distinct sets of amino acid residues to interact with FGFRs or TRKs in a mutually exclusive manner. The FGFR1 peptide wraps around the beta sandwich structure of the PTB domain, and its binding is possibly regulated by conformational change of a unique C-terminal beta strand in the protein. Our results suggest mechanisms by which SNTs serve as molecular switches to mediate the essential interplay between FGFR and TRK signaling during neuronal differentiation.
    Full-text · Article · Nov 2000 · Molecular Cell
  • C Dhalluin

    No preview · Article · Oct 2000 · Molecular Cell
  • C Dhalluin · J E Carlson · L Zeng · C He · A K Aggarwal · M M Zhou

    No preview · Article · Aug 1999 · Journal of Biomolecular NMR

  • No preview · Article · Jun 1999 · Journal of Biomolecular NMR
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    ABSTRACT: Histone acetylation is important in chromatin remodelling and gene activation. Nearly all known histone-acetyltransferase (HAT)-associated transcriptional co-activators contain bromodomains, which are ~110-amino- acid modules found in many chromatin-associated proteins. Despite the wide occurrence of these bromodomains, their three-dimensional structure and binding partners remain unknown. Here we report the solution structure of the bromodomain of the HAT co-activator P/CAF (p300/CBP-associated factor). The structure reveals an unusual left-handed up-and-down four-helix bundle. In addition, we show by a combination of structural and site-directed mutagenesis studies that bromodomains can interact specifically with acetylated lysine, making them the first known protein modules to do so. The nature of the recognition of acetyl-lysine by the P/CAF bromodomain is similar to that of acetyl-CoA by histone acetyltransferase. Thus, the bromodomain is functionally linked to the HAT activity of co-activators in the regulation of gene transcription.
    No preview · Article · Jun 1999 · Nature

Publication Stats

1k Citations
119.12 Total Impact Points

Institutions

  • 2001
    • CUNY Graduate Center
      New York City, New York, United States
  • 2000
    • New York Structural Biology Center
      New York, New York, United States
  • 1999
    • Icahn School of Medicine at Mount Sinai
      Manhattan, New York, United States