Jerome Dupuy

Crystal structure of the apo‐PerR‐Zn protein from Bacillus subtilis

Bacteria adapt to elevated levels of Reactive Oxygen Species (ROS) by increasing the expression of defence and repair proteins, which is regulated by ROS responsive transcription factors. In Bacillus subtilis the zinc protein PerR, a peroxide sensor that binds DNA in the presence of a regulatory metal Mn2+ or Fe2+, mediates the adaptive response to H2O2. This study presents the first crystal structure of apo‐PerR‐Zn which shows that all four cysteine residues of the protein are involved in zinc co‐ordination. The Zn(Cys)4 site locks the dimerization domain and stabilizes the dimer. Sequence alignment of PerR‐like proteins supports that this structural site may constitute a distinctive feature of this class of peroxide stress regulators.

Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography

Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8Å resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.

A Method of Estimating Kinetic Parameters of Thermoset Cures: Application to a Dicyanate Ester Resin

In this work, temperature modulated DSC is used to measure the heat flow evolved during the cure reaction of a dicyanate ester resin. This technique enables the heat capacity contribution to be separated from the heat source term (from the kinetic behavior) in nonisothermal conditions. At any given moment, therefore, the true reaction rate can be calculated without any assumptions about the shape of the baseline of the thermogram. Resulting accurate data are used for the characterization of cure kinetics. The isoconversional analysis shows that the apparent activation energy varies with conversion degree, so that a Kamal & Sourour kinetic model is proposed. Its kinetic parameters are obtained by an inverse method, and such a model is successfully used to simulate nonisothermal, isothermal and multistep cure experiments.

Structural Basis for the Modulation of CDK-Dependent/Independent Activity of Cyclin D1

D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target.Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (i) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (ii) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (iii) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets. Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle.

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP.

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli. We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.

Supported Metallocene Catalysts in Olefin Polymerization: Toward High Performances

In order to get a supported metallocene type catalyst, different methods have been used to link the group IV metal to the surface. The simplest procedure starts with a metal chloride (titanium or zirconium tetrachloride) supported on magnesium chloride or silica. This solid precursors are modified by a chemical treatment in order to replace in one step one of the chlorines by a cyclopentadienyl or a modified cyclopentadienyl ligand. The results are very different when titanium or zirconium are considered: the titanium catalyst is not different from a conventional Ziegler-Natta catalyst. On the contrary, the zirconium catalyst is similar to a metallocene system, producing polyethylene homo and copolymers with narrow molecular weight distribution which can not be fractionated by solvents. Another approach consists of the metal to the surface using a silicium bridging 2 indenyl ligands. In both cases the zirconium catalysts seem to be very close to the definition of a single site system. High activities have been observed in ethylene polymerization but in it was never possible to produce any isotactic polypropylene.

Synthesis of heterogeneous catalysts for gas phase olefin polymerization : Ziegler-Natta catalysts modified with indenyl ligands

A Ziegler-Natta catalyst was first prepared by reacting a silica/magnesium chloride support with Z r Cl 4 in the gas phase, then modified by contacting the supported zirconium with one equivalent of indenyl lithium salt. This new catalyst exhibits high activity when activated with methylaluminoxane both in slurry and gas phase homo- and copolymerizations; the polyethylene produced possesses a narrow molar mass distribution. The behavior of such a heterogeneous catalyst has been investigated under different expiremental conditions, and its behavior in gas phase copolymerization (with butene) has been compared to that of Ind 2 ZrCl 2 .