A Src-Like Inactive Conformation in the Abl Tyrosine Kinase Domain

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180° with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP–peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR–Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain

Chemical Genetic Control of Protein Levels: Selective in Vivo Targeted Degradation

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin−proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets:  green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create “chemical knockouts,” thus opening new possibilities for the control of protein function

Model for the diffuse gamma-ray and neutrino emission of the Milky Way at multi-TeV energies

International audienceWe are developing new model propagation of cosmic rays in the Galaxy from their sources in three dimensional magnetic field. The model automatically takes into account anisotropic distribution of cosmic rays around their sources at all stages of their propagation in the Galaxy up to their escape. Cosmic rays are interacting with interstellar gas and produce secondary gamma-ray and neutrinos. For this interactions we use recent AAfrag model. We compare predictions of the model for individual sources and for several regions of the Galaxy with existing gamma-ray and neutrino data. We show that in the Galactic Ridge the gamma-ray spectrum measured by Fermi and the neutrino spectrum measured by IceCube and ANTARES are in perfect agreement and are consistent with hadronic origin of diffuse gamma-ray flux corresponding to $1/E^{2.5}$ cosmic ray spectrum

Ranking genomic features using an information-theoretic measure of epigenetic discordance

Abstract Background Establishment and maintenance of DNA methylation throughout the genome is an important epigenetic mechanism that regulates gene expression whose disruption has been implicated in human diseases like cancer. It is therefore crucial to know which genes, or other genomic features of interest, exhibit significant discordance in DNA methylation between two phenotypes. We have previously proposed an approach for ranking genes based on methylation discordance within their promoter regions, determined by centering a window of fixed size at their transcription start sites. However, we cannot use this method to identify statistically significant genomic features and handle features of variable length and with missing data. Results We present a new approach for computing the statistical significance of methylation discordance within genomic features of interest in single and multiple test/reference studies. We base the proposed method on a well-articulated hypothesis testing problem that produces p- and q-values for each genomic feature, which we then use to identify and rank features based on the statistical significance of their epigenetic dysregulation. We employ the information-theoretic concept of mutual information to derive a novel test statistic, which we can evaluate by computing Jensen-Shannon distances between the probability distributions of methylation in a test and a reference sample. We design the proposed methodology to simultaneously handle biological, statistical, and technical variability in the data, as well as variable feature lengths and missing data, thus enabling its wide-spread use on any list of genomic features. This is accomplished by estimating, from reference data, the null distribution of the test statistic as a function of feature length using generalized additive regression models. Differential assessment, using normal/cancer data from healthy fetal tissue and pediatric high-grade glioma patients, illustrates the potential of our approach to greatly facilitate the exploratory phases of clinically and biologically relevant methylation studies. Conclusions The proposed approach provides the first computational tool for statistically testing and ranking genomic features of interest based on observed DNA methylation discordance in comparative studies that accounts, in a rigorous manner, for biological, statistical, and technical variability in methylation data, as well as for variability in feature length and for missing data

A Dysregulated DNA Methylation Landscape Linked to Gene Expression in MLL-Rearranged AML

Translocations of the KMT2A (MLL) gene define a biologically distinct and clinically aggressive subtype of acute myeloid leukaemia (AML), marked by a characteristic gene expression profile and few cooperating mutations. Although dysregulation of the epigenetic landscape in this leukaemia is particularly interesting given the low mutation frequency, its comprehensive analysis using whole genome bisulphite sequencing (WGBS) has not been previously performed. Here we investigated epigenetic dysregulation in nine MLL-rearranged (MLL-r) AML samples by comparing them to six normal myeloid controls, using a computational method that encapsulates mean DNA methylation measurements along with analyses of methylation stochasticity. We discovered a dramatically altered epigenetic profile in MLL-r AML, associated with genome-wide hypomethylation and a markedly increased DNA methylation entropy reflecting an increasingly disordered epigenome. Methylation discordance mapped to key genes and regulatory elements that included bivalent promoters and active enhancers. Genes associated with significant changes in methylation stochasticity recapitulated known MLL-r AML expression signatures, suggesting a role for the altered epigenetic landscape in the transcriptional programme initiated by MLL translocations. Accordingly, we established statistically significant associations between discordances in methylation stochasticity and gene expression in MLL-r AML, thus providing a link between the altered epigenetic landscape and the phenotype

The Structure of Abl in an Intermediate Conformation Suggests a Path for the Transition between the Active and Src-Like Conformations

<div><p>(A) The intermediate structure of Abl (molecule E) is shown, with helix αC shown in blue, the activation loop in red, and the catalytic loop containing Arg 362 in orange.</p> <p>(B) The proposed pathway by which Abl makes the transition to the Src-like conformation.</p></div

The Helical Turn following the DFG motif in Src-Like Inactive Structures

<div><p>(A) The helical turn in the activation loop of molecule B that immediately follows the DFG motif is a characteristic feature of the Src-like conformation and is conserved in four different kinase families.</p> <p>(B and C) The side chain of Arg 386, presented by the helical turn, forms a hydrogen bond to a backbone carbonyl of Ile 360, a salt bridge with Glu 286, and an amino–aromatic interaction with Phe 359 that positions it for interacting with Asp 381 during the DFG flip.</p> <p>(D) An intermediate structure during one of the TMD simulations, showing the capture of Asp 381 by Arg 386.</p></div

Molecule B Helps Explain Mutations in the Kinase Domain of Abl That Confer Resistance to Imatinib

<div><p>(A) The side chains of residues implicated in imatinib resistance in the Azam et al. study [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040144#pbio-0040144-b048" target="_blank">48</a>] are shown in blue in the context of molecule B. A large number of these mutations cluster in the interface between helix αC, the N-lobe, and the helical turn in the activation loop. </p> <p>(B–D) For three of these mutations we have shown the surface for all atoms within 6 Å of the mutated residue in the context of different structures. The Abl:imatinib complex is in green and the Src-like structure (molecule B) in yellow-orange. (B) Asp 276. (C) Leu 387. (D) Met 278.</p></div

Molecule B Closely Resembles the Structure of the Inactive Src Kinases

<div><p>(A) The kinase domain of Abl (molecule B) is shown as a cartoon with the bisubstrate analog inhibitor depicted as a stick model.</p> <p>(B) Structure of the ATP–peptide conjugate at the active site.</p> <p>(C) Comparison between molecule B (left) and the structure of a Src-family kinase (right).</p> <p>(D) Backbone torsion angles of Asp 381 of the DFG motif.</p> <p>(E) The backbone of Asp 381 can start to move from DFG-In to the DFG-Out conformations by three paths in torsion angle space [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040144#pbio-0040144-b032" target="_blank">32</a>]. </p> <p>(F) Illustration of rotations about φ(Asp 381) and ψ(Asp 381) in the Src-like inactive versus the active structures of Abl.</p></div