RESUMEN
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.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Receptor Toll-Like 9/metabolismo , Secuencias de Aminoácidos , Animales , Femenino , Humanos , Inmunidad Innata , Interferón Tipo I/inmunología , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Lupus Eritematoso Sistémico/metabolismo , Masculino , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Unión Proteica , Transducción de SeñalRESUMEN
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.
Asunto(s)
Transporte Biológico Activo/genética , Biología Computacional/métodos , Proteínas de Transporte de Membrana/metabolismo , Aminoácidos/metabolismo , Aminoácidos/farmacología , Ontologías Biológicas , Línea Celular , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Aprendizaje AutomáticoRESUMEN
Cell-surface proteins are of great biomedical importance, as demonstrated by the fact that 66% of approved human drugs listed in the DrugBank database target a cell-surface protein. Despite this biomedical relevance, there has been no comprehensive assessment of the human surfaceome, and only a fraction of the predicted 5,000 human transmembrane proteins have been shown to be located at the plasma membrane. To enable analysis of the human surfaceome, we developed the surfaceome predictor SURFY, based on machine learning. As a training set, we used experimentally verified high-confidence cell-surface proteins from the Cell Surface Protein Atlas (CSPA) and trained a random forest classifier on 131 features per protein and, specifically, per topological domain. SURFY was used to predict a human surfaceome of 2,886 proteins with an accuracy of 93.5%, which shows excellent overlap with known cell-surface protein classes (i.e., receptors). In deposited mRNA data, we found that between 543 and 1,100 surfaceome genes were expressed in cancer cell lines and maximally 1,700 surfaceome genes were expressed in embryonic stem cells and derivative lines. Thus, the surfaceome diversity depends on cell type and appears to be more dynamic than the nonsurface proteome. To make the predicted surfaceome readily accessible to the research community, we provide visualization tools for intuitive interrogation (wlab.ethz.ch/surfaceome). The in silico surfaceome enables the filtering of data generated by multiomics screens and supports the elucidation of the surfaceome nanoscale organization.
Asunto(s)
Membrana Celular/metabolismo , Predicción/métodos , Proteínas de la Membrana/metabolismo , Membrana Celular/fisiología , Simulación por Computador , Bases de Datos de Compuestos Químicos , Humanos , Aprendizaje Automático , Proteínas de la Membrana/fisiología , Proteoma/metabolismo , Proteómica/métodosRESUMEN
With over 450 genes, solute carriers (SLCs) constitute the largest transporter superfamily responsible for the uptake and efflux of nutrients, metabolites, and xenobiotics in human cells. SLCs are associated with a wide variety of human diseases, including cancer, diabetes, and metabolic and neurological disorders. They represent an important therapeutic target class that remains only partly exploited as therapeutics that target SLCs are scarce. Additionally, many small molecules reported in the literature to target SLCs are poorly characterized. Both features may be due to the difficulty of developing SLC transport assays that fulfill the quality criteria for high-throughput screening. Here, we report one of the main limitations hampering assay development within the RESOLUTE consortium: the lack of a resource providing high-quality information on SLC tool compounds. To address this, we provide a systematic annotation of tool compounds targeting SLCs. We first provide an overview on RESOLUTE assays. Next, we present a list of SLC-targeting compounds collected from the literature and public databases; we found that most data sources lacked specificity data. Finally, we report on experimental tests of 19 selected compounds against a panel of 13 SLCs from seven different families. Except for a few inhibitors, which were active on unrelated SLCs, the tested inhibitors demonstrated high selectivity for their reported targets. To make this knowledge easily accessible to the scientific community, we created an interactive dashboard displaying the collected data in the RESOLUTE web portal (https://re-solute.eu). We anticipate that our open-access resources on assays and compounds will support the development of future drug discovery campaigns for SLCs.
RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Transporte de Nucleósidos/metabolismo , Purinas/metabolismo , Proteínas Transportadoras de Solutos/metabolismo , Factores de Transcripción/metabolismo , Adenina/metabolismo , Vías Biosintéticas , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Cromatina/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Transporte de Membrana , Modelos Biológicos , Proteínas Transportadoras de Solutos/genética , Factores de Transcripción/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Epistasis Genética , Mitocondrias/metabolismo , NAD/metabolismo , Proteína Desacopladora 1/metabolismo , Transporte Biológico , Humanos , Mitocondrias/genética , Oxidación-Reducción , Proteína Desacopladora 1/genéticaRESUMEN
Host factor requirements for different classes of viruses have not been fully unraveled. Replication of the viral genome and synthesis of viral proteins within the human host cell are associated with an increased demand for nutrients and specific metabolites. With more than 400 acknowledged members to date in humans, solute carriers (SLCs) represent the largest family of transmembrane proteins dedicated to the transport of ions and small molecules such as amino acids, sugars and nucleotides. Consistent with their impact on cellular metabolism, several SLCs have been implicated as host factors affecting the viral life cycle and the cellular response to infection. In this study, we aimed at characterizing the role of host SLCs in cell survival upon viral infection by performing unbiased genetic screens using a focused CRISPR knockout library. Genetic screens with the cytolytic vesicular stomatitis virus (VSV) showed that the loss of two SLCs genes, encoding the sialic acid transporter SLC35A1/CST and the zinc transporter SLC30A1/ZnT1, affected cell survival upon infection. Further characterization of these genes suggests a role for both of these transporters in the apoptotic response induced by VSV, offering new insights into the cellular response to oncolytic virus infections.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Transporte de Catión/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Transporte de Nucleótidos/metabolismo , Infecciones por Rhabdoviridae/complicaciones , Replicación Viral , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/virología , Proteínas de Transporte de Catión/genética , Ingeniería Genética , Interacciones Huésped-Patógeno , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/virología , Proteínas de Transporte de Nucleótidos/genética , Infecciones por Rhabdoviridae/virología , Células Tumorales Cultivadas , Vesiculovirus/aislamiento & purificaciónRESUMEN
OBJECTIVE: Cognitive therapy has gained prominence in the treatment of major depression, however, little is known about its long-term benefits when delivered during inpatient treatment or combined with outpatient treatment with severely ill inpatients (HAM-Dâ¯>â¯20). METHOD: To evaluate this question, we conducted a randomized controlled trial investigating the efficacy of extended clinical management (E-CM), psychoeducational cognitive behavioural group therapy (PCBT-G) or PCBT-G and 16 outpatient individual treatment sessions (PCBT-G+I). All patients were treated with pharmacotherapy. 177 inpatients with DSM-IV major depression were randomized either to E-CM or PCBT-G or PCBT-G+I. Outcome measures were collected in the hospital at pre- and posttreatment and following discharge into the community every six months for two years. We compared the study groups on symptom changes, psychosocial functioning, knowledge about depression and rehospitalization. RESULTS: All three treatment interventions are equally effective at reducing depressive symptoms and increasing psychosocial functioning at posttreatment. There was significant group by time interaction for knowledge about depression in favor of PCBT-G and PCBT-G+I over E-CM. We did not find significantly lower rehospitalisation rates at the two-year follow-up for PCBT-G+I compared to E-CM, however, comparing PCBT-G to E-CM. CONCLUSIONS: We conclude that with cognitive psychoeducational group therapy a successful, in the long-term other interventions superior psychological intervention for major depression is available as gains were sustained for two years following discharge from the hospital. More research is needed to evaluate the long-term impact of group treatment starting in inpatient treatment.
Asunto(s)
Terapia Cognitivo-Conductual/métodos , Trastorno Depresivo Mayor/terapia , Psicoterapia de Grupo/métodos , Adulto , Terapia Combinada/métodos , Trastorno Depresivo Mayor/psicología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.