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1.
Microbiol Spectr ; 10(1): e0140221, 2022 02 23.
Article in English | MEDLINE | ID: mdl-35196824

ABSTRACT

Various commercial anti-Spike SARS-CoV-2 antibody tests are used for studies and in clinical settings after vaccination. An international standard for SARS-CoV-2 antibodies has been established to achieve comparability of such tests, allowing conversions to BAU/mL. This study aimed to investigate the comparability of antibody tests regarding the timing of blood collection after vaccination. For this prospective observational study, antibody levels of 50 participants with homologous AZD1222 vaccination were evaluated at 3 and 11 weeks after the first dose and 3 weeks after the second dose using two commercial anti-Spike binding antibody assays (Roche and Abbott) and a surrogate neutralization assay. The correlation between Roche and Abbott changed significantly depending on the time point studied. Although Abbott provided values three times higher than Roche 3 weeks after the first dose, the values for Roche were twice as high as for Abbott 11 weeks after the first dose and 5 to 6 times higher at 3 weeks after the second dose. The comparability of quantitative anti-Spike SARS-CoV-2 antibody tests was highly dependent on the timing of blood collection after vaccination. Therefore, standardization of the timing of blood collection might be necessary for the comparability of different quantitative SARS-COV-2 antibody assays. IMPORTANCE This work showed that the comparability of apparently standardized SARS-CoV-2 antibody assays (Roche, Abbott; both given in BAU/mL) after vaccination depends on the time of blood withdrawal. Initially (3 weeks after the first dose AZD1222), there were 3 times higher values in the Abbott assay, but this relationship inversed before boosting (11 weeks after the first dose) with Roche 2 times greater than Abbott. After the booster, Roche quantified ca. 5 times higher levels than Abbott. This must be considered by clinicians when interpreting SARS-CoV-2 antibody levels.


Subject(s)
Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/diagnosis , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination/trends , Adult , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , Humans , Middle Aged , Prospective Studies , Time Factors , Vaccination/standards
2.
Nat Commun ; 12(1): 7190, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34907165

ABSTRACT

Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.


Subject(s)
Leukemia, Myeloid/metabolism , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Survival/drug effects , Cluster Analysis , Fatty Acids/biosynthesis , Genotype , Humans , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Monocarboxylic Acid Transporters/genetics , Phenotype , Phosphatidylinositol 3-Kinase/metabolism , Phosphoinositide-3 Kinase Inhibitors/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pyrimidinones/metabolism , Pyrimidinones/pharmacology , Pyrrolidines/metabolism , Pyrrolidines/pharmacology , Signal Transduction , Small Molecule Libraries/classification , Symporters/genetics , Systems Analysis , Thiophenes/metabolism , Thiophenes/pharmacology , Triazoles/metabolism , Triazoles/pharmacology , Tumor Cells, Cultured
3.
Nat Metab ; 3(5): 651-664, 2021 05.
Article in English | MEDLINE | ID: mdl-33972798

ABSTRACT

Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.


Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , Nucleoside Transport Proteins/metabolism , Purines/metabolism , Solute Carrier Proteins/metabolism , Transcription Factors/metabolism , Adenine/metabolism , Biosynthetic Pathways , Cell Cycle Proteins/antagonists & inhibitors , Cell Line , Chromatin/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Membrane Transport Proteins , Models, Biological , Solute Carrier Proteins/genetics , Transcription Factors/antagonists & inhibitors , Transcription, Genetic
4.
Nature ; 581(7808): 316-322, 2020 05.
Article in English | MEDLINE | ID: mdl-32433612

ABSTRACT

Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses1-3. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene that is associated with systemic lupus erythematosus4,5-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease4,6-9. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF10,11. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus12-14.


Subject(s)
Interferon Regulatory Factors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lysosomes/metabolism , Membrane Transport Proteins/metabolism , Nerve Tissue Proteins/metabolism , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Toll-Like Receptor 9/metabolism , Amino Acid Motifs , Animals , Female , Humans , Immunity, Innate , Interferon Type I/immunology , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , Lupus Erythematosus, Systemic/metabolism , Male , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Protein Binding , Signal Transduction
5.
Cell Death Differ ; 26(6): 1138-1155, 2019 06.
Article in English | MEDLINE | ID: mdl-30237509

ABSTRACT

Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.


Subject(s)
Cation Transport Proteins/genetics , Chromosome Mapping , Necroptosis/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , CRISPR-Cas Systems/genetics , Cation Transport Proteins/deficiency , Cation Transport Proteins/metabolism , Cell Death , Cell Line , Cell Survival , HEK293 Cells , Humans , Receptors, Tumor Necrosis Factor, Type I/genetics , Tumor Necrosis Factor-alpha/metabolism
6.
Science ; 362(6419): 1171-1177, 2018 12 07.
Article in English | MEDLINE | ID: mdl-30442766

ABSTRACT

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.


Subject(s)
Proto-Oncogene Proteins p21(ras)/metabolism , Transcription Factors/physiology , Ubiquitination , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Drosophila melanogaster , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/antagonists & inhibitors , Gain of Function Mutation , Gene Knockdown Techniques , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/epidemiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Loss of Function Mutation , MAP Kinase Signaling System/genetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridazines/pharmacology , Pyridazines/therapeutic use , Signal Transduction , Transcription Factors/genetics , Ubiquitination/genetics
7.
Curr Opin Pharmacol ; 42: 102-110, 2018 10.
Article in English | MEDLINE | ID: mdl-30193150

ABSTRACT

Treatment of complex diseases such as cancer, cardiovascular disease, diabetes or neurological disorders frequently warrants the utilization of drug combinations for therapeutic intervention. In fact, the most successful example is the current standard of care for HIV patients. However, identification of successful drug cocktails is not a simple task and is hampered by lack of standardization in terminology, experimental protocols and models as well as data analysis. Here we discuss the most recent developments in combinatorial drug screening by covering technological advancements in screening strategies, cellular model systems as well as novel drug classes. We believe the research progress being made provides promising basis to build on and identify, develop and optimize efficacious clinically relevant combinatorial drug treatments.


Subject(s)
Pharmaceutical Preparations/administration & dosage , Pharmaceutical Preparations/chemistry , Animals , Drug Combinations , Drug Evaluation, Preclinical/methods , Humans
8.
Cell Host Microbe ; 23(6): 766-774.e5, 2018 06 13.
Article in English | MEDLINE | ID: mdl-29779931

ABSTRACT

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.


Subject(s)
Bicarbonates/metabolism , Macrophages/metabolism , Phagosomes/metabolism , Sodium-Bicarbonate Symporters/physiology , CRISPR-Cas Systems , Cation Transport Proteins/metabolism , Cytoplasm/metabolism , Gene Knockout Techniques , Homeostasis , Humans , Hydrogen-Ion Concentration , Phagocytosis , Sodium-Bicarbonate Symporters/genetics , THP-1 Cells , Transcriptome , U937 Cells
9.
Lancet Haematol ; 4(12): e595-e606, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29153976

ABSTRACT

BACKGROUND: Patients with refractory or relapsed haematological malignancies have few treatment options and short survival times. Identification of effective therapies with genomic-based precision medicine is hampered by intratumour heterogeneity and incomplete understanding of the contribution of various mutations within specific cancer phenotypes. Ex-vivo drug-response profiling in patient biopsies might aid effective treatment identification; however, proof of its clinical utility is limited. METHODS: We investigated the feasibility and clinical impact of multiparametric, single-cell, drug-response profiling in patient biopsies by immunofluorescence, automated microscopy, and image analysis, an approach we call pharmacoscopy. First, the ability of pharmacoscopy to separate responders from non-responders was evaluated retrospectively for a cohort of 20 newly diagnosed and previously untreated patients with acute myeloid leukaemia. Next, 48 patients with aggressive haematological malignancies were prospectively evaluated for pharmacoscopy-guided treatment, of whom 17 could receive the treatment. The primary endpoint was progression-free survival in pharmacoscopy-treated patients, as compared with their own progression-free survival for the most recent regimen on which they had progressive disease. This trial is ongoing and registered with ClinicalTrials.gov, number NCT03096821. FINDINGS: Pharmacoscopy retrospectively predicted the clinical response of 20 acute myeloid leukaemia patients to initial therapy with 88·1% accuracy. In this interim analysis, 15 (88%) of 17 patients receiving pharmacoscopy-guided treatment had an overall response compared with four (24%) of 17 patients with their most recent regimen (odds ratio 24·38 [95% CI 3·99-125·4], p=0·0013). 12 (71%) of 17 patients had a progression-free survival ratio of 1·3 or higher, and median progression-free survival increased by four times, from 5·7 (95% CI 4·1-12·1) weeks to 22·6 (7·4-34·0) weeks (hazard ratio 3·14 [95% CI 1·37-7·22], p=0·0075). INTERPRETATION: Routine clinical integration of pharmacoscopy for treatment selection is technically feasible, and led to improved treatment of patients with aggressive refractory haematological malignancies in an initial patient cohort, warranting further investigation. FUNDING: Austrian Academy of Sciences; European Research Council; Austrian Science Fund; Austrian Federal Ministry of Science, Research and Economy; National Foundation for Research, Technology and Development; Anniversary Fund of the Austrian National Bank; MPN Research Foundation; European Molecular Biology Organization; and Swiss National Science Foundation.


Subject(s)
Antineoplastic Agents/therapeutic use , Hematologic Neoplasms/drug therapy , Adenine/analogs & derivatives , Adult , Aged , Area Under Curve , Bone Marrow/pathology , Bortezomib/therapeutic use , Cladribine/therapeutic use , Disease-Free Survival , Female , Hematologic Neoplasms/diagnostic imaging , Hematologic Neoplasms/mortality , Hematologic Neoplasms/pathology , Humans , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/drug therapy , Male , Microscopy, Fluorescence , Middle Aged , Odds Ratio , Pilot Projects , Piperidines , Positron Emission Tomography Computed Tomography , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , ROC Curve , Remission Induction , Young Adult
10.
Nat Chem Biol ; 13(6): 681-690, 2017 06.
Article in English | MEDLINE | ID: mdl-28437395

ABSTRACT

Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.


Subject(s)
Immunomodulation/drug effects , Small Molecule Libraries/pharmacology , Cardiac Myosins/metabolism , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Crizotinib , Humans , Myosin Heavy Chains/metabolism , Pyrazoles/pharmacology , Pyridines/pharmacology
11.
Sci Rep ; 6: 28107, 2016 06 27.
Article in English | MEDLINE | ID: mdl-27346722

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphate/analysis , Adenosine Triphosphate/chemistry , Cytoskeletal Proteins/metabolism , Mass Spectrometry , Amino Acid Sequence , Chromatography, High Pressure Liquid , DEAD-box RNA Helicases/chemistry , HEK293 Cells , Humans , Kinetics , Oxygen Isotopes/chemistry , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Kinases/metabolism , Substrate Specificity
12.
Mol Cell Proteomics ; 15(3): 1139-50, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26933192

ABSTRACT

Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.


Subject(s)
Chromatography, Affinity/methods , HSP90 Heat-Shock Proteins/metabolism , Mutation , Protein Kinases/metabolism , Proteomics/methods , Retroviridae/genetics , Tandem Mass Spectrometry/methods , Animals , Cell Line , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Genes, Reporter , HEK293 Cells , HT29 Cells , Humans , K562 Cells , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Protein Kinases/genetics
13.
NPJ Syst Biol Appl ; 2: 16027, 2016.
Article in English | MEDLINE | ID: mdl-28725479

ABSTRACT

Studying the relationship between virus infection and cellular response is paradigmatic for our understanding of how perturbation changes biological systems. Immune response, in this context is a complex yet evolutionarily adapted and robust cellular change, and is experimentally amenable to molecular analysis. To visualize the full cellular response to virus infection, we performed temporal transcriptomics, proteomics, and phosphoproteomics analysis of vesicular stomatitis virus (VSV)-infected mouse macrophages. This enabled the understanding of how infection-induced changes in host gene and protein expression are coordinated with post-translational modifications by cells in time to best measure and control the infection process. The vast and complex molecular changes measured could be decomposed in a limited number of clusters within each category (transcripts, proteins, and protein phosphorylation) each with own kinetic parameter and characteristic pathways/processes, suggesting multiple regulatory options in the overall sensing and homeostatic program. Altogether, the data underscored a prevalent executive function to phosphorylation. Resolution of the molecular events affecting the RIG-I pathway, central to viral recognition, reveals that phosphorylation of the key innate immunity adaptor mitochondrial antiviral-signaling protein (MAVS) on S328/S330 is necessary for activation of type-I interferon and nuclear factor κ B (NFκB) pathways. To further understand the hierarchical relationships, we analyzed kinase-substrate relationships and found RAF1 and, to a lesser extent, ARAF to be inhibiting VSV replication and necessary for NFκB activation, and AKT2, but not AKT1, to be supporting VSV replication. Integrated analysis using the omics data revealed co-regulation of transmembrane transporters including SLC7A11, which was subsequently validated as a host factor in the VSV replication. The data sets are predicted to greatly empower future studies on the functional organization of the response of macrophages to viral challenges.

14.
Science ; 350(6264): 1092-6, 2015 Nov 27.
Article in English | MEDLINE | ID: mdl-26472760

ABSTRACT

Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase ß adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.


Subject(s)
Gene Regulatory Networks , Genes, Essential , Genes, Lethal , Genetic Fitness/genetics , Haploidy , Golgi Apparatus/genetics , Hexosyltransferases/genetics , Humans , Membrane Proteins/genetics , Mutagenesis, Insertional , Mutation , Saccharomyces cerevisiae/genetics
15.
Eur J Immunol ; 45(5): 1500-11, 2015 May.
Article in English | MEDLINE | ID: mdl-25678110

ABSTRACT

Secretion of type I interferon (IFN) is the first cellular reaction to invading pathogens. Despite the protective function of these cytokines, an excessive response to their action can contribute to serious pathologies, such as autoimmune diseases. Transcripts of most cytokines contain adenylate-uridylate (A/U)-rich elements (AREs) that make them highly unstable. RNA-binding proteins (RBPs) are mediators of the regulatory mechanisms that determine the fate of mRNAs containing AREs. Here, we applied an affinity proteomic approach and identified lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR) as the predominant RBP of the IFN-ß mRNA ARE. Reduced expression or chemical inhibition of HuR severely hampered the type I IFN response in various cell lines and fibroblast-like synoviocytes isolated from joints of rheumatoid arthritis patients. These results define a role for HuR as a potent modulator of the type I IFN response. Taken together, HuR could be used as therapeutic target for diseases where type I IFN production is exaggerated.


Subject(s)
ELAV Proteins/immunology , Interferon Type I/biosynthesis , Interferon-beta/genetics , AU Rich Elements , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Base Sequence , ELAV Proteins/antagonists & inhibitors , ELAV Proteins/genetics , ELAV-Like Protein 1 , HeLa Cells , Humans , Interferon Inducers/pharmacology , Molecular Sequence Data , Poly I-C/pharmacology , Protein Multimerization , RNA Processing, Post-Transcriptional/drug effects , RNA Stability/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Synovial Membrane/immunology
16.
Nature ; 519(7544): 477-81, 2015 Mar 26.
Article in English | MEDLINE | ID: mdl-25561175

ABSTRACT

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.


Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cell Line , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/metabolism
17.
Nat Genet ; 46(9): 1021-7, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25129144

ABSTRACT

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.


Subject(s)
Membrane Proteins/deficiency , Membrane Proteins/genetics , Myeloid Cells/metabolism , Neutropenia/congenital , Adolescent , Adult , Apoptosis/genetics , Cell Differentiation/genetics , Cell Survival/genetics , Child , Child, Preschool , Congenital Bone Marrow Failure Syndromes , Female , Glycosylation , Homeostasis/genetics , Humans , Infant , Infant, Newborn , Male , Membrane Proteins/metabolism , Mutation , Neutropenia/genetics , Neutropenia/metabolism , Neutropenia/pathology , Neutrophils/metabolism , Receptors, Granulocyte Colony-Stimulating Factor/genetics , Receptors, Granulocyte Colony-Stimulating Factor/metabolism , Signal Transduction , Young Adult
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