We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.

Hepatitis C virus (HCV) is a considerable global health and economic burden. The HCV nonstructural protein (NS) 5A is essential for the viral life cycle. The ability of NS5A to interact with different host and viral proteins allow it to manipulate cellular pathways and regulate viral processes, including RNA replication and virus particle assembly. As part of a proteomic screen, we identified several NS5A-binding proteins, including the lysine methyltransferase SET and MYND domain containing protein 3 (SMYD3). We confirmed the interaction in the context of viral replication by co-immunoprecipitation and co-localization studies. Mutational analyses revealed that the MYND-domain of SMYD3 and domain III of NS5A are required for the interaction. Overexpression of SMYD3 resulted in decreased intracellular and extracellular virus titers, whilst viral RNA replication remained unchanged, suggesting that SMYD3 negatively affects HCV particle production in a NS5A-dependent manner.

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[(18)O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates.

The marsupial Tasmanian devil (Sarcophilus harrisii) faces extinction due to transmissible devil facial tumor disease (DFTD). To unveil the molecular underpinnings of this transmissible cancer, we combined pharmacological screens with an integrated systems-biology characterization. Sensitivity to inhibitors of ERBB tyrosine kinases correlated with their overexpression. Proteomic and DNA methylation analyses revealed tumor-specific signatures linked to the evolutionary conserved oncogenic STAT3. ERBB inhibition blocked phosphorylation of STAT3 and arrested cancer cells. Pharmacological blockade of ERBB or STAT3 prevented tumor growth in xenograft models and restored MHC class I expression. This link between the hyperactive ERBB-STAT3 axis and major histocompatibility complex class I-mediated tumor immunosurveillance provides mechanistic insights into horizontal transmissibility and puts forward a dual chemo-immunotherapeutic strategy to save Tasmanian devils from DFTD. VIDEO ABSTRACT.

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFβ complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFβ complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.

TANK-binding kinase 1 (TBK1) is of central importance for the induction of type-I interferon (IFN) in response to pathogens. We identified the DEAD-box helicase DDX3X as an interaction partner of TBK1. TBK1 and DDX3X acted synergistically in their ability to stimulate the IFN promoter, whereas RNAi-mediated reduction of DDX3X expression led to an impairment of IFN production. Chromatin immunoprecipitation indicated that DDX3X is recruited to the IFN promoter upon infection with Listeria monocytogenes, suggesting a transcriptional mechanism of action. DDX3X was found to be a TBK1 substrate in vitro and in vivo. Phosphorylation-deficient mutants of DDX3X failed to synergize with TBK1 in their ability to stimulate the IFN promoter. Overall, our data imply that DDX3X is a critical effector of TBK1 that is necessary for type I IFN induction.

No abstract available

DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.

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.

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.

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.