49 research outputs found

    Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network

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    Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity

    Rigorous Polynomial Approximation using Taylor Models in Coq

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    International audienceOne of the most common and practical ways of representing a real function on machines is by using a polynomial approximation. It is then important to properly handle the error introduced by such an approximation. The purpose of this work is to offer guaranteed error bounds for a specific kind of rigorous polynomial approximation called Taylor model. We carry out this work in the Coq proof assistant, with a special focus on genericity and efficiency for our implementation. We give an abstract interface for rigorous polynomial approximations, parameter- ized by the type of coefficients and the implementation of polynomials, and we instantiate this interface to the case of Taylor models with inter- val coefficients, while providing all the machinery for computing them. We compare the performances of our implementation in Coq with those of the Sollya tool, which contains an implementation of Taylor models written in C. This is a milestone in our long-term goal of providing fully formally proved and efficient Taylor models

    Wave Equation Numerical Resolution: a Comprehensive Mechanized Proof of a C Program

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    We formally prove correct a C program that implements a numerical scheme for the resolution of the one-dimensional acoustic wave equation. Such an implementation introduces errors at several levels: the numerical scheme introduces method errors, and floating-point computations lead to round-off errors. We annotate this C program to specify both method error and round-off error. We use Frama-C to generate theorems that guarantee the soundness of the code. We discharge these theorems using SMT solvers, Gappa, and Coq. This involves a large Coq development to prove the adequacy of the C program to the numerical scheme and to bound errors. To our knowledge, this is the first time such a numerical analysis program is fully machine-checked.Comment: No. RR-7826 (2011

    Formation and Growth of Oligomers: A Monte Carlo Study of an Amyloid Tau Fragment

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    Small oligomers formed early in the process of amyloid fibril formation may be the major toxic species in Alzheimer's disease. We investigate the early stages of amyloid aggregation for the tau fragment AcPHF6 (Ac-VQIVYK-NH2) using an implicit solvent all-atom model and extensive Monte Carlo simulations of 12, 24, and 36 chains. A variety of small metastable aggregates form and dissolve until an aggregate of a critical size and conformation arises. However, the stable oligomers, which are β-sheet-rich and feature many hydrophobic contacts, are not always growth-ready. The simulations indicate instead that these supercritical oligomers spend a lengthy period in equilibrium in which considerable reorganization takes place accompanied by exchange of chains with the solution. Growth competence of the stable oligomers correlates with the alignment of the strands in the β-sheets. The larger aggregates seen in our simulations are all composed of two twisted β-sheets, packed against each other with hydrophobic side chains at the sheet–sheet interface. These β-sandwiches show similarities with the proposed steric zipper structure for PHF6 fibrils but have a mixed parallel/antiparallel β-strand organization as opposed to the parallel organization found in experiments on fibrils. Interestingly, we find that the fraction of parallel β-sheet structure increases with aggregate size. We speculate that the reorganization of the β-sheets into parallel ones is an important rate-limiting step in the formation of PHF6 fibrils

    Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing

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    Preservation of cancer biopsies by FFPE introduces DNA fragmentation, hindering analysis of rearrangements. Here the authors introduce FFPE Targeted Locus Capture for identification of translocations in preserved samples.In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.Immunobiology of allogeneic stem cell transplantation and immunotherapy of hematological disease

    Properties of the E2 intra-molecular salt-bridge network.

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    <p><b>A.</b> Atomic view of the UbcH6/UbcH8 intra-molecular salt-bridge network that controls E105/D113 positioning and E3 binding. Images were generated with the PyMOL Molecular Graphics System, Version 1.3 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002754#pcbi.1002754-Schrdinger1" target="_blank">[38]</a>. <b>B.</b> Dynamics of the intra-molecular salt-bridge network. Depicted are E2-specific distances between the indicated side-chains during a 20 ns MD trajectory. Curves were smoothened using a 100 ps running average window.</p

    The highly similar UbcH6 and UbcH8 E2 enzymes interact with different RING E3 domains cohorts.

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    <p><b>A.</b> Sequence alignment of human UbcH6 (UBE2E1) and UbcH8 (UBE2E2). Secondary structures and E3-interface regions (H1, L1 and L2) are indicated. Arrows indicate E2-specific residues; asterisks indicate conserved residues involved in bridging. N-terminal extensions are indicated as shaded. <b>B.</b> E3-interaction profiles of <i>wt</i> UbcH6, UbcH8 and UbcH8 D113E (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002754#s4" target="_blank">Materials and Methods</a> section for details).</p

    Validation of the dynamic salt-bridge network that controls RING E3 interaction selectivity.

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    <p><b>A.</b> Perturbation of critical network components displaces E105/D113 and controls E3 binding. Mutagenesis of strictly conserved residues that mediate the bridging status of D113 in UbcH8 were mutated to challenge the MD predictions (see main text and Material and Methods section for details). <b>B.</b> Schematic representation of the “open” and “bridged” conformations of the intra-molecular network controlling E3 interactions. Indicated are E2-specific residues as well as the conserved network residues that mediate E3 binding.</p
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