Autoinhibition of c-Abl

AbstractDespite years of investigation, the molecular mechanism responsible for regulation of the c-Abl tyrosine kinase has remained elusive. We now report inhibition of the catalytic activity of purified c-Abl in vitro, demonstrating that regulation is an intrinsic property of the molecule. We show that the interaction of the N-terminal 80 residues with the rest of the protein mediates autoregulation. This N-terminal “cap” is required to achieve and maintain inhibition, and its loss turns c-Abl into an oncogenic protein and contributes to deregulation of BCR-Abl

The structure of the leukemia drug imatinib bound to human quinone reductase 2 (NQO2)

<p>Abstract</p> <p>Background</p> <p>Imatinib represents the first in a class of drugs targeted against chronic myelogenous leukemia to enter the clinic, showing excellent efficacy and specificity for Abl, Kit, and PDGFR kinases. Recent screens carried out to find off-target proteins that bind to imatinib identified the oxidoreductase NQO2, a flavoprotein that is phosphorylated in a chronic myelogenous leukemia cell line.</p> <p>Results</p> <p>We examined the inhibition of NQO2 activity by the Abl kinase inhibitors imatinib, nilotinib, and dasatinib, and obtained IC₅₀values of 80 nM, 380 nM, and >100 μM, respectively. Using electronic absorption spectroscopy, we show that imatinib binding results in a perturbation of the protein environment around the flavin prosthetic group in NQO2. We have determined the crystal structure of the complex of imatinib with human NQO2 at 1.75 Å resolution, which reveals that imatinib binds in the enzyme active site, adjacent to the flavin isoalloxazine ring. We find that phosphorylation of NQO2 has little effect on enzyme activity and is therefore likely to regulate other aspects of NQO2 function.</p> <p>Conclusion</p> <p>The structure of the imatinib-NQO2 complex demonstrates that imatinib inhibits NQO2 activity by competing with substrate for the active site. The overall conformation of imatinib when bound to NQO2 resembles the folded conformation observed in some kinase complexes. Interactions made by imatinib with residues at the rim of the active site provide an explanation for the binding selectivity of NQO2 for imatinib, nilotinib, and dasatinib. These interactions also provide a rationale for the lack of inhibition of the related oxidoreductase NQO1 by these compounds. Taken together, these studies provide insight into the mechanism of NQO2 inhibition by imatinib, with potential implications for drug design and treatment of chronic myelogenous leukemia in patients.</p

In silico Prioritization of Transporter–Drug Relationships From Drug Sensitivity Screens

The interplay between drugs and cell metabolism is a key factor in determining both compound potency and toxicity. In particular, how and to what extent transmembrane transporters affect drug uptake and disposition is currently only partially understood. Most transporter proteins belong to two protein families: the ATP-Binding Cassette (ABC) transporter family, whose members are often involved in xenobiotic efflux and drug resistance, and the large and heterogeneous family of solute carriers (SLCs). We recently argued that SLCs are collectively a rather neglected gene group, with most of its members still poorly characterized, and thus likely to include many yet-to-be-discovered associations with drugs. We searched publicly available resources and literature to define the currently known set of drugs transported by ABCs or SLCs, which involved ∼500 drugs and more than 100 transporters. In order to extend this set, we then mined the largest publicly available pharmacogenomics dataset, which involves approximately 1,000 molecularly annotated cancer cell lines and their response to 265 anti-cancer compounds, and used regularized linear regression models (Elastic Net, LASSO) to predict drug responses based on SLC and ABC data (expression levels, SNVs, CNVs). The most predictive models included both known and previously unidentified associations between drugs and transporters. To our knowledge, this represents the first application of regularized linear regression to this set of genes, providing an extensive prioritization of potentially pharmacologically interesting interactions

Initial characterization of the human central proteome

<p>Abstract</p> <p>Background</p> <p>On the basis of large proteomics datasets measured from seven human cell lines we consider their intersection as an approximation of the human central proteome, which is the set of proteins ubiquitously expressed in all human cells. Composition and properties of the central proteome are investigated through bioinformatics analyses.</p> <p>Results</p> <p>We experimentally identify a central proteome comprising 1,124 proteins that are ubiquitously and abundantly expressed in human cells using state of the art mass spectrometry and protein identification bioinformatics. The main represented functions are proteostasis, primary metabolism and proliferation. We further characterize the central proteome considering gene structures, conservation, interaction networks, pathways, drug targets, and coordination of biological processes. Among other new findings, we show that the central proteome is encoded by exon-rich genes, indicating an increased regulatory flexibility through alternative splicing to adapt to multiple environments, and that the protein interaction network linking the central proteome is very efficient for synchronizing translation with other biological processes. Surprisingly, at least 10% of the central proteome has no or very limited functional annotation.</p> <p>Conclusions</p> <p>Our data and analysis provide a new and deeper description of the human central proteome compared to previous results thereby extending and complementing our knowledge of commonly expressed human proteins. All the data are made publicly available to help other researchers who, for instance, need to compare or link focused datasets to a common background.</p

Internalization of Pseudomonas aeruginosaStrain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction

Identifying the druggable interactome of EWS-FLI1 reveals MCL-1 dependent differential sensitivities of Ewing sarcoma cells to apoptosis inducers

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.(VLID)471264