RESUMEN
DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities.
Asunto(s)
Ataxina-3 , Roturas del ADN de Doble Cadena , Poli ADP Ribosilación , Ataxina-3/genética , Línea Celular Tumoral , ADN , Daño del ADN , Reparación del ADN/genética , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
The SUMO-targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin-3 counteracts RNF4 activity during the DNA double-strand break (DSB) response. We find that ataxin-3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin-3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co-depletion of RNF4. Ataxin-3 is recruited to DSBs in a SUMOylation-dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO-dependent mechanism for DUB activity toward MDC1. Loss of ataxin-3 results in reduced DNA damage-induced ubiquitylation due to impaired MDC1-dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin-3 is required for efficient MDC1-dependent DSB repair by non-homologous end-joining and homologous recombination. Consequently, loss of ataxin-3 sensitizes cells to ionizing radiation and poly(ADP-ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin-3 consolidate robust MDC1-dependent signaling and repair of DSBs.
Asunto(s)
Ataxina-3/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Proteína SUMO-1/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Ataxina-3/genética , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Represoras/genética , Proteína SUMO-1/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondrial DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol γ) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol γ exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol γ and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol γ reported here could have implications for forms of mtDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTP/dNTP ratios that are expected to prevail in such disease states, Pol γ enters a polymerase/exonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/biosíntesis , Desoxirribonucleósidos/metabolismo , Fosfatos/metabolismo , Animales , ADN Polimerasa gamma/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.
Asunto(s)
Replicación del ADN/fisiología , ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Línea Celular , Células Cultivadas , Medios de Cultivo , ADN Polimerasa Dirigida por ADN/genética , Fibroblastos , Eliminación de Gen , Ratones , Piridinas , Rayos UltravioletaRESUMEN
NIPBL is required to load the cohesin complex on to DNA. While the canonical role of cohesin is to couple replicated sister chromatids together until the onset of mitosis, it also promotes tolerance to DNA damage. Here, we show that NIPBL is recruited to DNA damage throughout the cell cycle via independent mechanisms, influenced by type of damage. First, the heterochromatin protein HP1γ (also known as CBX3) recruits NIPBL to DNA double-strand breaks (DSBs) through the corresponding HP1-binding motif within the N-terminus. By contrast, the C-terminal HEAT repeat domain is unable to recruit NIPBL to DSBs but independently targets NIPBL to laser microirradiation-induced DNA damage. Each mechanism is dependent on the RNF8 and RNF168 ubiquitylation pathway, while the recruitment of the HEAT repeat domain requires further ATM or ATR activity. Thus, NIPBL has evolved a sophisticated response to damaged DNA that is influenced by the form of damage, suggesting a highly dynamic role for NIPBL in maintaining genomic stability.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Proteínas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Modelos Biológicos , Dominios Proteicos , Proteínas/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , CohesinasRESUMEN
Neuraminidase (NA) is one of the two major influenza surface antigens and the main influenza drug target. Although NA has been well characterized and thought to function as a tetramer, the role of the transmembrane domain (TMD) in promoting proper NA assembly has not been systematically studied. Here, we demonstrate that in the absence of the TMD, NA is synthesized and transported in a predominantly inactive state. Substantial activity was rescued by progressive truncations of the stalk domain, suggesting the TMD contributes to NA maturation by tethering the stalk to the membrane. To analyze how the TMD supports NA assembly, the TMD was examined by itself. The NA TMD formed a homotetramer and efficiently trafficked to the plasma membrane, indicating the TMD and enzymatic head domain drive assembly together through matching oligomeric states. In support of this, an unrelated strong oligomeric TMD rescued almost full NA activity, whereas the weak oligomeric mutant of this TMD restored only half of wild type activity. These data illustrate that a large soluble domain can force assembly with a poorly compatible TMD; however, optimal assembly requires coordinated oligomerization between the TMD and the soluble domain.
Asunto(s)
Gripe Humana/enzimología , Neuraminidasa/química , Animales , Dimerización , Perros , Glicoproteínas/química , Células HEK293 , Células HeLa , Humanos , Cinética , Proteínas de la Membrana/química , Modelos Moleculares , Conformación Molecular , Neuraminidasa/metabolismo , Plásmidos , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.
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Enfermedad de Charcot-Marie-Tooth/metabolismo , Glutatión/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas del Tejido Nervioso/metabolismo , Línea Celular , Enfermedad de Charcot-Marie-Tooth/genética , Humanos , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Estrés OxidativoRESUMEN
Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca(2+) and is fueled either by Ca(2+) from the extracellular space when triggered by neuronal activity or by Ca(2+) released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca(2+) pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca(2+) homeostasis in models of ischemia. The proteins include those important for the Ca(2+)-dependent motility of mitochondria and for Ca(2+) transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca(2+) release from the ER, voltage-dependent anion channels that allow Ca(2+) entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca(2+) uniporter together with its regulatory proteins that permit Ca(2+) entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca(2+) from the mitochondria such as the mitochondrial Na(+)/Ca(2+) exchanger or, if the mitochondrial Ca(2+) concentration exceeds a certain threshold, the mitochondrial permeability transition pore.
Asunto(s)
Isquemia Encefálica/metabolismo , Homeostasis , Mitocondrias/metabolismo , Animales , Cationes Bivalentes/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Neuronas/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
The receptor tyrosine kinase (RTK) KIT and its ligand stem cell factor (SCF) are essential for human mast cell (huMC) survival and proliferation. HuMCs expressing oncogenic KIT variants secrete large numbers of extracellular vesicles (EVs). The role KIT plays in regulating EV secretion has not been examined. Here, we investigated the effects of stimulation or inhibition of KIT activity on the secretion of small EVs (sEVs). In huMCs expressing constitutively active KIT, the quantity and quality of secreted sEVs positively correlated with the activity status of KIT. SCF-mediated stimulation of KIT in huMCs or murine MCs, or of transiently expressed KIT in HeLa cells, enhanced the release of sEVs expressing exosome markers. In contrast, ligand-mediated stimulation of the RTK EGFR in HeLa cells did not affect sEV secretion. The release of sEVs induced by either constitutively active or ligand-activated KIT was remarkably decreased when cells were treated with KIT inhibitors, concomitant with reduced exosome markers in sEVs. Similarly, inhibition of oncogenic KIT signalling kinases like PI3K, and MAPK significantly reduced the secretion of sEVs. Thus, activation of KIT and its early signalling cascades stimulate the secretion of exosome-like sEVs in a regulated fashion, which may have implications for KIT-driven functions.
RESUMEN
Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Store-operated Ca(2+) entry over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and is mediated by the sensor STIM1 and the channel ORAI1. We compared cell death susceptibility to oxidative stress in STIM1 knock-out and ORAI1 knockdown mouse embryonic fibroblasts and in knock-out cells with reconstituted wild type and dominant active STIM1. We show that STIM1 and ORAI1 deficiency renders cells more susceptible to oxidative stress, which can be rescued by STIM1 and ORAI1 overexpression. STIM1 knock-out mitochondria are tubular, have a higher Ca(2+) concentration, and are metabolically more active, resulting in constitutive oxidative stress causing increased nuclear translocation of the antioxidant transcription factor NRF2 triggered by increased phosphorylation of the translation initiation factor eIF2α and the protein kinase-like endoplasmic reticulum kinase PERK. This leads to increased transcription of antioxidant genes and a high basal glutathione in STIM1 knock-out cells, which is, however, more rapidly expended upon additional stress, resulting in increased release and nuclear translocation of apoptosis-inducing factor with subsequent cell death. Our data suggest that store-operated Ca(2+) entry and STIM1 are involved in the regulation of mitochondrial shape and bioenergetics and play a role in oxidative stress.
Asunto(s)
Embrión de Mamíferos/metabolismo , Metabolismo Energético/fisiología , Fibroblastos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Apoptosis/fisiología , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Células Cultivadas , Embrión de Mamíferos/citología , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Fibroblastos/citología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Mitocondrias/genética , Proteína ORAI1 , Fosforilación/fisiología , Molécula de Interacción Estromal 1 , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
The HMC-1.2 human mast cell (huMC) line is often employed in the study of attributes of neoplastic huMCs as found in patients with mastocytosis and their sensitivity to interventional drugs in vitro and in vivo. HMC-1.2 cells express constitutively active KIT, an essential growth factor receptor for huMC survival and function, due to the presence of two oncogenic mutations (D816V and V560G). However, systemic mastocytosis is commonly associated with a single D816V-KIT mutation. The functional consequences of the coexisting KIT mutations in HMC-1.2 cells are unknown. We used CRISPR/Cas9-engineering to reverse the V560G mutation in HMC-1.2 cells, resulting in a subline (HMC-1.3) with a single mono-allelic D816V-KIT variant. Transcriptome analyses predicted reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. However, in liquid culture conditions, the growth of HMC-1.2 and HMC-1.3 cells was comparable. Phosphorylation levels of ERK1/2, AKT and STAT5, representing pathways associated with constitutive oncogenic KIT signaling, were also similar between HMC-1.2 and HMC-1.3 cells. Despite these similarities in liquid culture, survival of HMC-1.3 cells was diminished in response to various pharmacological inhibitors, including tyrosine kinase inhibitors used clinically for treatment of advanced systemic mastocytosis, and JAK2 and BCL2 inhibitors, making HMC-1.3 more susceptible to these drugs than HMC-1.2 cells. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, suggesting that engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis.
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Mastocitosis Sistémica , Mastocitosis , Humanos , Animales , Ratones , Mastocitosis Sistémica/tratamiento farmacológico , Mastocitosis Sistémica/genética , Mastocitosis Sistémica/patología , Sistemas CRISPR-Cas , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Mastocitosis/genética , Mutación , Línea CelularRESUMEN
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.
RESUMEN
Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated via the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily via non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells in vitro and in vivo, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.
Asunto(s)
Mastocitos , Factores de Transcripción , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de Zinc , Animales , Proliferación Celular , Humanos , Mastocitos/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Proteína Gli2 con Dedos de Zinc/metabolismoRESUMEN
Activating mutations in the receptor KIT promote the dysregulated proliferation of human mast cells (huMCs). The resulting neoplastic huMCs secrete extracellular vesicles (EVs) that can transfer oncogenic KIT among other cargo into recipient cells. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly investigated. Here, we isolated and characterized KIT-EV subpopulations released by neoplastic huMCs using an immunocapture approach that selectively isolates EVs containing KIT in its proper topology. Immunocapture of EVs on KIT antibody-coated electron microscopy (EM) affinity grids allowed to assess the morphology and size of KIT-EVs. Immunoblot analysis demonstrated KIT-EVs have a distinct protein profile from KIT-depleted EVs, contain exosome and microvesicle markers, and are separated into these subtypes by ultracentrifugation. Cell treatment with sphingomyelinase inhibitors shifted the protein content among KIT-EV subtypes, suggesting different biogenesis routes. Proteomic analysis revealed huMC KIT-EVs are enriched in proteins involved in signalling, immune responses, and cell migration, suggesting diverse biological functions, and indicated neoplastic huMCs disseminate KIT via shuttling in heterogeneous microvesicle- and exosome-like EVs. Further, selective KIT-immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions and potential to serve as biomarkers of specific biological pathologies.
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Exosomas , Vesículas Extracelulares , Biomarcadores/metabolismo , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Mastocitos/metabolismo , Proteómica , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.
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Actinas , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Humanos , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Mast cells are tissue-resident immune cells that play key roles in the initiation and perpetuation of allergic inflammation, usually through IgE-mediated mechanisms. Mast cells are, however, evolutionary ancient immune cells that can be traced back to urochordates and before the emergence of IgE antibodies, suggesting their involvement in antibody-independent biological functions, many of which are still being characterized. Herein, we summarize recent advances in understanding the roles of mast cells in health and disease, partly through the study of emerging non-IgE receptors such as the Mas-related G protein-coupled receptor X2, implicated in pseudo-allergic reactions as well as in innate defense and neuronal sensing; the mechano-sensing adhesion G protein-coupled receptor E2, variants of which are associated with familial vibratory urticaria; and purinergic receptors, which orchestrate tissue damage responses similarly to the IL-33 receptor. Recent evidence also points toward novel mechanisms that contribute to mast cell-mediated pathophysiology. Thus, in addition to releasing preformed mediators contained in granules and synthesizing mediators de novo, mast cells also secrete extracellular vesicles, which convey biological functions. Understanding their release, composition and uptake within a variety of clinical conditions will contribute to the understanding of disease specific pathology and likely lead the way to novel therapeutic approaches. We also discuss recent advances in the development of therapies targeting mast cell activity, including the ligation of inhibitory ITIM-containing receptors, and other strategies that suppress mast cells or responses to mediators for the management of mast cell-related diseases.
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Inflamación , Mastocitos , Humanos , Inmunoglobulina E , Inflamación/tratamiento farmacológico , Mastocitos/patología , Receptores Acoplados a Proteínas GRESUMEN
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from symptomatic patients with COVID-19, but also from asymptomatic individuals. Therefore, robust surveillance and timely interventions are essential for the control of virus spread within the community. In this regard the frequency of testing and speed of reporting, but not the test sensitivity alone, play a crucial role. OBJECTIVES: In order to reduce the costs and meet the expanding demands in real-time RT-PCR (rRT-PCR) testing for SARS-CoV-2, complementary assays, such as rapid antigen tests, have been developed. Rigorous analysis under varying conditions is required to assess the clinical performance of these tests and to ensure reproducible results. RESULTS: We evaluated the sensitivity and specificity of a recently licensed rapid antigen test using 137 clinical samples in two institutions. Test sensitivity was between 88.2-89.6 % when applied to samples with viral loads typically seen in infectious patients. Of 32 rRT-PCR positive samples, 19 demonstrated infectivity in cell culture, and 84 % of these samples were reactive with the antigen test. Seven full-genome sequenced SARS-CoV-2 isolates and SARS-CoV-1 were detected with this antigen test, with no cross-reactivity against other common respiratory viruses. CONCLUSIONS: Numerous antigen tests are available for SARS-CoV-2 testing and their performance to detect infectious individuals may vary. Head-to-head comparison along with cell culture testing for infectivity may prove useful to identify better performing antigen tests. The antigen test analyzed in this study is easy-to-use, inexpensive, and scalable. It can be helpful in monitoring infection trends and thus has potential to reduce transmission.
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Antígenos Virales/genética , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/virología , Células CACO-2 , Línea Celular Tumoral , Células Cultivadas , Genoma Viral/genética , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Carga Viral/inmunologíaRESUMEN
Due to globally rising numbers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resources for real-time reverse-transcription polymerase chain reaction (rRT-PCR)-based testing have been exhausted. In order to meet the demands of testing and reduce transmission, SARS-CoV-2 antigen-detecting rapid diagnostic tests (Ag-RDTs) are being considered. These tests are fast, inexpensive, and simple to use, but whether they detect potentially infectious cases has not been well studied. We evaluated three lateral flow assays (RIDA®QUICK SARS-CoV-2 Antigen (R-Biopharm), SARS-CoV-2 Rapid Antigen Test (Roche)), and NADAL® COVID-19 Ag Test (Nal von Minden GmbH, Regensburg, Germany) and one microfluidic immunofluorescence assay (SARS-CoV-2 Ag Test (LumiraDx GmbH, Cologne, Germany)) using 100 clinical samples. Diagnostic rRT-PCR and cell culture testing as a marker for infectivity were performed in parallel. The overall Ag-RDT sensitivity for rRT-PCR-positive samples ranged from 24.3% to 50%. However, for samples with a viral load of more than 6 log10 RNA copies/mL (22/100), typically seen in infectious individuals, Ag-RDT positivity was between 81.8% and 100%. Only 51.6% (33/64) of the rRT-PCR-positive samples were infectious in cell culture. In contrast, three Ag-RDTs demonstrated a more significant correlation with cell culture infectivity (61.8-82.4%). Our findings suggest that large-scale SARS-CoV-2 Ag-RDT-based testing can be considered for detecting potentially infective individuals and reducing the virus spread.
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The pathology of spinocerebellar ataxia type 3, also known as Machado-Joseph disease, is triggered by aggregation of toxic ataxin-3 (ATXN3) variants containing expanded polyglutamine repeats. The physiological role of this deubiquitylase, however, remains largely unclear. Our recent work showed that ATX-3, the nematode orthologue of ATXN3, together with the ubiquitin-directed segregase CDC-48, regulates longevity in Caenorhabditis elegans. Here, we demonstrate that the long-lived cdc-48.1; atx-3 double mutant displays reduced viability under prolonged starvation conditions that can be attributed to the loss of catalytically active ATX-3. Reducing the levels of the autophagy protein BEC-1 sensitized worms to the effect of ATX-3 deficiency, suggesting a role of ATX-3 in autophagy. In support of this conclusion, the depletion of ATXN3 in human cells caused a reduction in autophagosomal degradation of proteins. Surprisingly, reduced degradation in ATXN3-depleted cells coincided with an increase in the number of autophagosomes while levels of lipidated LC3 remained unaffected. We identified two conserved LIR domains in the catalytic Josephin domain of ATXN3 that directly interacted with the autophagy adaptors LC3C and GABARAP in vitro. While ATXN3 localized to early autophagosomes, it was not subject to lysosomal degradation, suggesting a transient regulatory interaction early in the autophagic pathway. We propose that the deubiquitylase ATX-3/ATXN3 stimulates autophagic degradation by preventing superfluous initiation of autophagosomes, thereby promoting an efficient autophagic flux important to survive starvation.
Asunto(s)
Ataxina-3/metabolismo , Caenorhabditis elegans/metabolismo , Enfermedad de Machado-Joseph/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Autofagia , Humanos , Enfermedad de Machado-Joseph/patologíaRESUMEN
The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S-phase of the cell cycle and induces DNA double-strand breaks (DSBs). Surprisingly, loss of the tRNA-methyltransferase Trm9 - along with the Elongator complex involved in synthesis of 5-methoxy-carbonyl-methyl (mcm(5)) modification in certain tRNAs - conferred resistance against PaT. Overexpression of mcm(5)-modified tRNAs identified tRNA(Gln)((UUG)) as the intracellular target. Consistently, toxin-challenged cells displayed reduced levels of tRNA(Gln) and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA(Gln)((UUG)). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA(Glu)((UUC)), affecting its target in a mcm(5)-dependent manner, PaT exerts activity also on tRNA(Gln) lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNA(Gln)((UUG)), tRNA(Gln)((CUG)) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA(Gln)((CUG)) as an additional TRM9-independent target, overexpression of PaT's tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.