ABSTRACT
Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Cyclin-Dependent Kinases/metabolism , Fibroblasts/drug effects , Interferon-gamma/pharmacology , Transcription, Genetic/drug effects , Animals , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinases/genetics , Fibroblasts/enzymology , Fibroblasts/virology , HCT116 Cells , Host-Pathogen Interactions , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , RNA Polymerase II/metabolism , STAT1 Transcription Factor/metabolism , Signal Transduction , Vesiculovirus/pathogenicityABSTRACT
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 TransductionABSTRACT
In search for RNA signals that modulate transcription via direct interaction with RNA polymerase (RNAP), we deep sequenced an E. coli genomic library enriched for RNAP-binding RNAs. Many natural RNAP-binding aptamers, termed RAPs, were mapped to the genome. Over 60% of E. coli genes carry RAPs in their mRNA. Combining in vitro and in vivo approaches, we characterized a subset of inhibitory RAPs (iRAPs) that promote Rho-dependent transcription termination. A representative iRAP within the coding region of the essential gene, nadD, greatly reduces its transcriptional output in stationary phase and under oxidative stress, demonstrating that iRAPs control gene expression in response to changing environment. The mechanism of iRAPs involves active uncoupling of transcription and translation, making nascent RNA accessible to Rho. iRAPs encoded in the antisense strand also promote gene expression by reducing transcriptional interference. In essence, our work uncovers a broad class of cis-acting RNA signals that globally control bacterial transcription.
Subject(s)
Aptamers, Nucleotide/genetics , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , SELEX Aptamer Technique , Transcription Termination, Genetic , Aptamers, Nucleotide/metabolism , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genome, Bacterial , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Open Reading Frames , Ribosomes/metabolism , Time FactorsABSTRACT
Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
Subject(s)
Drug Resistance/genetics , Solute Carrier Proteins/metabolism , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/metabolism , Antineoplastic Agents , Biochemical Phenomena , Biological Transport/genetics , Biological Transport/physiology , CRISPR-Cas Systems , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Drug Resistance/physiology , Genetic Testing , Humans , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Protein Transport/physiology , Solute Carrier Proteins/physiology , Symporters/genetics , Symporters/metabolismABSTRACT
Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.
Subject(s)
Cyclin-Dependent Kinase 8/genetics , Gene Expression Regulation/drug effects , Interferon-gamma/pharmacology , STAT1 Transcription Factor/genetics , Animals , Cyclin-Dependent Kinase 8/immunology , Cyclin-Dependent Kinase 8/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/virology , Hep G2 Cells , Humans , Interferon-gamma/immunology , Interleukin-6/immunology , Interleukin-6/pharmacology , Mice , Oligonucleotide Array Sequence Analysis , Phosphorylation , Promoter Regions, Genetic , RNA Polymerase II/genetics , RNA Polymerase II/immunology , STAT1 Transcription Factor/immunology , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/immunology , Signal Transduction/drug effects , Transcription, Genetic/drug effects , Vesiculovirus/physiologyABSTRACT
Solute carriers (SLCs) are the largest family of transmembrane transporters in the human genome with more than 400 members. Despite the fact that SLCs mediate critical biological functions and several are important pharmacological targets, a large proportion of them is poorly characterized and present no assigned substrate. A major limitation to systems-level de-orphanization campaigns is the absence of a structured, language-controlled chemical annotation. Here we describe a thorough manual annotation of SLCs based on literature. The annotation of substrates, transport mechanism, coupled ions, and subcellular localization for 446 human SLCs confirmed that ~30% of these were still functionally orphan and lacked known substrates. Application of a substrate-based ontology to transcriptomic datasets identified SLC-specific responses to external perturbations, while a machine-learning approach based on the annotation allowed us to identify potential substrates for several orphan SLCs. The annotation is available at https://opendata.cemm.at/gsflab/slcontology/. Given the increasing availability of large biological datasets and the growing interest in transporters, we expect that the effort presented here will be critical to provide novel insights into the functions of SLCs.
Subject(s)
Biological Transport, Active/genetics , Computational Biology/methods , Membrane Transport Proteins/metabolism , Amino Acids/metabolism , Amino Acids/pharmacology , Biological Ontologies , Cell Line , Gene Expression Profiling , Genome, Human , Humans , Machine LearningABSTRACT
AREsite2 represents an update for AREsite, an on-line resource for the investigation of AU-rich elements (ARE) in human and mouse mRNA 3'UTR sequences. The new updated and enhanced version allows detailed investigation of AU, GU and U-rich elements (ARE, GRE, URE) in the transcriptome of Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans and Drosophila melanogaster. It contains information on genomic location, genic context, RNA secondary structure context and conservation of annotated motifs. Improvements include annotation of motifs not only in 3'UTRs but in the whole gene body including introns, additional genomes, and locally stable secondary structures from genome wide scans. Furthermore, we include data from CLIP-Seq experiments in order to highlight motifs with validated protein interaction. Additionally, we provide a REST interface for experienced users to interact with the database in a semi-automated manner. The database is publicly available at: http://rna.tbi.univie.ac.at/AREsite.
Subject(s)
3' Untranslated Regions , Databases, Nucleic Acid , RNA/chemistry , Animals , Genomics , Humans , Mice , Molecular Sequence Annotation , Nucleic Acid Conformation , Nucleotide MotifsABSTRACT
Precise regulation of mRNA decay is fundamental for robust yet not exaggerated inflammatory responses to pathogens. However, a global model integrating regulation and functional consequences of inflammation-associated mRNA decay remains to be established. Using time-resolved high-resolution RNA binding analysis of the mRNA-destabilizing protein tristetraprolin (TTP), an inflammation-limiting factor, we qualitatively and quantitatively characterize TTP binding positions in the transcriptome of immunostimulated macrophages. We identify pervasive destabilizing and non-destabilizing TTP binding, including a robust intronic binding, showing that TTP binding is not sufficient for mRNA destabilization. A low degree of flanking RNA structuredness distinguishes occupied from silent binding motifs. By functionally relating TTP binding sites to mRNA stability and levels, we identify a TTP-controlled switch for the transition from inflammatory into the resolution phase of the macrophage immune response. Mapping of binding positions of the mRNA-stabilizing protein HuR reveals little target and functional overlap with TTP, implying a limited co-regulation of inflammatory mRNA decay by these proteins. Our study establishes a functionally annotated and navigable transcriptome-wide atlas (http://ttp-atlas.univie.ac.at) of cis-acting elements controlling mRNA decay in inflammation.
Subject(s)
Lipopolysaccharides/pharmacology , Macrophages/immunology , RNA, Messenger/chemistry , Tristetraprolin/metabolism , Animals , Binding Sites , Cells, Cultured , Gene Expression Profiling/methods , Gene Expression Regulation , HEK293 Cells , Humans , Macrophages/drug effects , Mice , RNA Stability , RNA, Messenger/metabolism , Sequence Analysis, RNAABSTRACT
Expression of cytokines and chemokines is regulated at multiple steps during the transfer of the genetic information from DNA sequence to the functional protein. The multilayered control of cytokine expression reflects the need of the immune system to precisely and rapidly adjust the magnitude and duration of immune responses to external cues. Common features of the regulation of cytokine expression are temporal and highly dynamic changes in cytokine mRNA stability. Failures in the timing and extent of mRNA decay can result in disease. Recent advances in transcriptome-wide approaches began to shed light into the complex network of cis-acting sequence elements and trans-acting factors controlling mRNA stability. These approaches led to the discovery of novel unexpected paradigms but they also revealed new questions. This review will discuss the control of cytokine mRNA stability both in the context of high content approaches as well as focused mechanistic studies and animal models. The article highlights the need for systems biology approaches as important means to understand how cytokine mRNA decay helps maintain the immune and tissue homeostasis, and to explore options for therapeutical exploitation of mRNA stability regulation.
Subject(s)
Cytokines/immunology , Gene Expression Regulation/immunology , RNA Stability/immunology , RNA, Messenger/immunology , Transcriptome/immunology , Animals , HumansABSTRACT
Phagocytosis, the process by which cells engulf large particles, plays a vital role in driving tissue clearance and host defense. Its dysregulation is connected to autoimmunity, toxic accumulation of proteins, and increased risks for infections. Despite its importance, we lack full understanding of all molecular components involved in the process. To create a functional map in human cells, we performed a genome-wide CRISPRko FACS screen that identified 716 genes. Mapping those hits to a comprehensive protein-protein interaction network annotated for functional cellular processes allowed retrieval of protein complexes identified multiple times and detection of missing phagocytosis regulators. In addition to known components, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, we identified and validated new phagocytosis-relevant functions, including the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) and the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our phagocytosis network comprises elements of cargo uptake, shuffling, and biotransformation through the cell, providing a resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. Our approach of integrating protein-protein interaction offers a broadly applicable way to functionally interpret genome-wide screens.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Hexosyltransferases , Humans , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Proteins , Phagocytosis/genetics , Hexosyltransferases/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
Subject(s)
Failure to Thrive , RNA Nucleotidyltransferases , Animals , Humans , Mice , Mice, Knockout , Muscle Weakness/genetics , Muscles , RNA Nucleotidyltransferases/chemistry , RNA Nucleotidyltransferases/genetics , ZebrafishABSTRACT
For a successful yet controlled immune response, cells need to specifically destabilize inflammatory mRNAs but prevent premature removal of those still used. The regulatory circuits controlling quality and timing in the global inflammatory mRNA decay are not understood. Here, we show that the mRNA-destabilizing function of the AU-rich element-binding protein tristetraprolin (TTP) is inversely regulated by the p38 MAPK activity profile such that after inflammatory stimulus the TTP-dependent decay is initially limited to few mRNAs. With time, the TTP-dependent decay gradually spreads resulting in cumulative elimination of one third of inflammation-induced unstable mRNAs in macrophages in vitro. We confirmed this sequential decay model in vivo since LPS-treated mice with myeloid TTP ablation exhibited similar cytokine dysregulation profile as macrophages. The mice were hypersensitive to LPS but otherwise healthy with no signs of hyperinflammation seen in conventional TTP knockout mice demonstrating the requirement for myeloid TTP in re-installment but not maintenance of immune homeostasis. These findings reveal a TTP- and p38 MAPK-dominated regulatory mechanism that is vital for balancing acute inflammation by a temporally and qualitatively controlled mRNA decay.
Subject(s)
Inflammation/genetics , RNA Stability/immunology , Tristetraprolin/immunology , Animals , Cytokines/immunology , Cytokines/metabolism , Female , Inflammation/immunology , Lipopolysaccharides , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA Stability/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcriptome , Tristetraprolin/genetics , Tristetraprolin/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of SLCs in response to environmental stresses remain poorly elucidated. To infer functional relationships between SLCs and metabolites, we developed a strategy to identify SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from growth-limiting metabolic bottlenecks. Among the transporters identified, we characterized the cationic amino acid transporter SLC7A3 as a gene that, when up-regulated, overcame low availability of arginine and lysine by increasing their uptake, whereas SLC7A5 was able to sustain cellular fitness upon deprivation of several neutral amino acids. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells uncovered functional transporter-nutrient relationships and revealed that transport activity up-regulation may be sufficient to overcome environmental metabolic restrictions.
Subject(s)
Membrane Transport Proteins , Nutrients , Amino Acid Transport Systems, Basic/genetics , Amino Acids/metabolism , Arginine/metabolism , Aspartic Acid/metabolism , Gain of Function Mutation , Glutamates/metabolism , Glutamine/metabolism , Humans , Large Neutral Amino Acid-Transporter 1 , Lysine/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Nutrients/metabolismABSTRACT
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, GeneticABSTRACT
The bioavailability of the major pro-inflammatory cytokines IL-1α and IL-1ß is tightly controlled by transcription and post-translational processing to prevent hyperinflammation. The role of mRNA decay in maintenance of physiological IL-1 amounts remained unknown. Here we show that the down-regulation of Il1a and Il1b mRNA by the mRNA-destabilizing protein TTP (gene Zfp36) is required for immune homeostasis. The TTP deficiency syndrome, a multi organ inflammation in TTP-/- mice, was significantly ameliorated upon deletion of the IL-1 receptor. Il1a and Il1b played non-redundant roles in triggering the pathological IL-1 signaling in TTP-/- mice. Accordingly, tissues from TTP-/- animals contained increased amounts of Il1b mRNA. Unexpectedly, TTP destabilized Il1b mRNA in cell type-specific ways as evident from RNA-Seq and mRNA stability assays. These results demonstrate that TTP-driven mRNA destabilization depends on the cellular context. Moreover, such context-defined mRNA decay is essential for keeping steady state IL-1 levels in the physiological range.
Subject(s)
Gene Expression Regulation , Homeostasis , Immunity/genetics , Interleukin-1/genetics , Tristetraprolin/metabolism , Animals , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Gene Expression Profiling , Inflammation/diagnosis , Inflammation/etiology , Inflammation/metabolism , Interleukin-1/metabolism , Interleukin-1alpha/genetics , Interleukin-1beta/genetics , Mice , Mice, Knockout , RNA Stability , Real-Time Polymerase Chain Reaction , Severity of Illness IndexABSTRACT
About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.
Subject(s)
Epistasis, Genetic , Mitochondria/metabolism , NAD/metabolism , Uncoupling Protein 1/metabolism , Biological Transport , Humans , Mitochondria/genetics , Oxidation-Reduction , Uncoupling Protein 1/geneticsABSTRACT
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 CellsABSTRACT
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/geneticsABSTRACT
Protective responses against pathogens require a rapid mobilization of resting neutrophils and the timely removal of activated ones. Neutrophils are exceptionally short-lived leukocytes, yet it remains unclear whether the lifespan of pathogen-engaged neutrophils is regulated differently from that in the circulating steady-state pool. Here, we have found that under homeostatic conditions, the mRNA-destabilizing protein tristetraprolin (TTP) regulates apoptosis and the numbers of activated infiltrating murine neutrophils but not neutrophil cellularity. Activated TTP-deficient neutrophils exhibited decreased apoptosis and enhanced accumulation at the infection site. In the context of myeloid-specific deletion of Ttp, the potentiation of neutrophil deployment protected mice against lethal soft tissue infection with Streptococcus pyogenes and prevented bacterial dissemination. Neutrophil transcriptome analysis revealed that decreased apoptosis of TTP-deficient neutrophils was specifically associated with elevated expression of myeloid cell leukemia 1 (Mcl1) but not other antiapoptotic B cell leukemia/lymphoma 2 (Bcl2) family members. Higher Mcl1 expression resulted from stabilization of Mcl1 mRNA in the absence of TTP. The low apoptosis rate of infiltrating TTP-deficient neutrophils was comparable to that of transgenic Mcl1-overexpressing neutrophils. Our study demonstrates that posttranscriptional gene regulation by TTP schedules the termination of the antimicrobial engagement of neutrophils. The balancing role of TTP comes at the cost of an increased risk of bacterial infections.