RESUMEN
BACKGROUND AND OBJECTIVE: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1ß and IL-18 levels were measured by Luminex assay. RESULTS: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance. CONCLUSION: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.
Asunto(s)
Fiebre Mediterránea Familiar/diagnóstico , Inmunofenotipificación/métodos , Pirina/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Preescolar , Colchicina/análisis , Fiebre Mediterránea Familiar/genética , Femenino , Estudios de Asociación Genética , Humanos , Leucocitos Mononucleares , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Pirina/genética , Adulto JovenRESUMEN
This paper describes a workflow towards the reconstruction of the three-dimensional elemental distribution profile within human cervical carcinoma cells (HeLa), at a spatial resolution down to 1 µm, employing state-of-the-art laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) instrumentation. The suspended cells underwent a series of fixation/embedding protocols and were stained with uranyl acetate and an Ir-based DNA intercalator. A priori, laboratory-based absorption micro-computed tomography (µ-CT) was applied to acquire a reference frame of the morphology of the cells and their spatial distribution before sectioning. After CT analysis, a trimmed 300 × 300 × 300 µm3 block was sectioned into a sequential series of 132 sections with a thickness of 2 µm, which were subjected to LA-ICP-MS imaging. A pixel acquisition rate of 250 pixels s-1 was achieved, through a bidirectional scanning strategy. After acquisition, the two-dimensional elemental images were reconstructed using the timestamps in the laser log file. The synchronization of the data required an improved optimization algorithm, which forces the pixels of scans in different ablation directions to be spatially coherent in the direction orthogonal to the scan direction. The volume was reconstructed using multiple registration approaches. Registration using the section outline itself as a fiducial marker resulted into a volume which was in good agreement with the morphology visualized in the µ-CT volume. The 3D µ-CT volume could be registered to the LA-ICP-MS volume, consisting of 2.9 × 107 voxels, and the nucleus dimensions in 3D space could be derived.
Asunto(s)
Espectrometría de Masas/métodos , Análisis de la Célula Individual/métodos , Células HeLa , Humanos , Microtomografía por Rayos XRESUMEN
The authors would like to call the reader's attention to the fact that unfortunately the originally provided affiliation for Dr. Tomoko Asaoka was not correct.
RESUMEN
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/fisiología , Respuesta al Choque Térmico/fisiología , Proteínas de Unión al ARN/genética , Animales , Catálisis , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Proteínas de Drosophila/metabolismo , Mutación , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.
Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , FN-kappa B/metabolismo , Fragmentos de Péptidos/metabolismo , Linfocitos T/metabolismo , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Humanos , Immunoblotting , Interleucina-2/metabolismo , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fragmentos de Péptidos/genética , Proteínas Proto-Oncogénicas c-raf , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Linfocitos T/citología , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
Loss-of-function mutations in the deubiquitinase OTULIN result in an inflammatory pathology termed "OTULIN-related autoinflammatory syndrome" (ORAS). Genetic mouse models revealed essential roles for OTULIN in inflammatory and cell death signaling, but the mechanisms by which OTULIN deficiency connects cell death to inflammation remain unclear. Here, we identify OTULIN deficiency as a cellular condition that licenses RIPK3-mediated cell death in murine macrophages, leading to Nlrp3 inflammasome activation and subsequent IL-1ß secretion. OTULIN deficiency uncoupled Nlrp3 inflammasome activation from gasdermin D-mediated pyroptosis, instead allowing RIPK3-dependent cell death to act as an Nlrp3 inflammasome activator and mechanism for IL-1ß release. Accordingly, elevated serum IL-1ß levels in myeloid-specific OTULIN-deficient mice were diminished by deleting either Ripk3 or Nlrp3. These findings identify OTULIN as an inhibitor of RIPK3-mediated IL-1ß release in mice.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Muerte Celular , Piroptosis , Inflamación/patologíaRESUMEN
Background: Autoinflammation with infantile enterocolitis (AIFEC) is an often fatal disease caused by gain-of-function mutations in the NLRC4 inflammasome. This inflammasomopathy is characterized by macrophage activation syndrome (MAS)-like episodes as well as neonatal-onset enterocolitis. Although elevated IL-18 levels were suggested to take part in driving AIFEC pathology, the triggers for IL-18 production and its ensuing pathogenic effects in these patients are incompletely understood. Methods: Here, we developed and characterized a novel genetic mouse model expressing a murine version of the AIFEC-associated NLRC4V341A mutation from its endogenous Nlrc4 genomic locus. Results: NLRC4V341A expression in mice recapitulated increased circulating IL-18 levels as observed in AIFEC patients. Housing NLRC4V341A-expressing mice in germfree (GF) conditions showed that these systemic IL-18 levels were independent of the microbiota, and unmasked an additional IL-18-inducing effect of NLRC4V341A expression in the intestines. Remarkably, elevated IL-18 levels did not provoke detectable intestinal pathologies in NLRC4V341A-expressing mice, even not upon genetically ablating IL-18 binding protein (IL-18BP), which is an endogenous IL-18 inhibitor that has been used therapeutically in AIFEC. In addition, NLRC4V341A expression did not alter susceptibility to the NLRC4-activating gastrointestinal pathogens Salmonella Typhimurium and Citrobacter rodentium. Conclusion: As observed in AIFEC patients, mice expressing a murine NLRC4V341A mutant show elevated systemic IL-18 levels, suggesting that the molecular mechanisms by which this NLRC4V341A mutant induces excessive IL-18 production are conserved between humans and mice. However, while our GF and infection experiments argue against a role for commensal or pathogenic bacteria, identifying the triggers and mechanisms that synergize with IL-18 to drive NLRC4V341A-associated pathologies will require further research in this NLRC4V341A mouse model.
Asunto(s)
Enterocolitis , Síndrome de Activación Macrofágica , Humanos , Ratones , Recién Nacido , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Mutación , Síndrome de Activación Macrofágica/genética , Enterocolitis/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismoRESUMEN
The expression levels of caspase-8 inhibitory c-FLIP proteins play an important role in regulating death receptor-mediated apoptosis, as their concentration at the moment when the death-inducing signaling complex (DISC) is formed determines the outcome of the DISC signal. Experimental studies have shown that c-FLIP proteins are subject to dynamic turnover and that their stability and expression levels can be rapidly altered. Even though the influence of c-FLIP on the apoptotic behavior of a single cell has been captured in mathematical simulation studies, the effect of c-FLIP turnover and stability has not been investigated. In this study, a mathematical model of apoptosis was developed to analyze how the dynamic turnover and stability of the c-FLIP isoforms regulate apoptotic signaling for both individual cells and cell populations. Intercellular parameter and concentration distributions were used to describe the behavior of cell populations. Monte-Carlo simulations of cell populations showed that c-FLIP turnover is a key determinant of death receptor responses. The fact that the developed model simulates the state of whole cell populations makes it possible to validate it by comparison with empirical data. The proposed modeling approach can be used to further determine limiting factors in the DISC signaling process.
Asunto(s)
Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Comunicación Celular/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Receptor fas/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Línea Celular Tumoral , Humanos , Método de Montecarlo , Receptor fas/genéticaRESUMEN
Vibrio cholerae is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and ~100.000 cholera-associated deaths annually worldwide. Genomic Vibrio cholerae research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of Vibrio cholerae triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor Vibrio cholerae induces IL-1ß maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor Vibrio cholerae engages the canonical Nlrp3 inflammasome for IL-1ß secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1ß secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the V. cholerae El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1ß-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor Vibrio cholerae toxins, and as such increase our understanding of host-pathogen interactions in the context of the Vibrio cholerae biotype associated with the ongoing cholera pandemic.
Asunto(s)
Caspasas Iniciadoras/metabolismo , Cólera/metabolismo , Cólera/microbiología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Pirina/metabolismo , Vibrio cholerae/fisiología , Animales , Caspasas Iniciadoras/genética , Cólera/inmunología , Toxina del Cólera/genética , Toxina del Cólera/inmunología , Toxina del Cólera/metabolismo , Proteínas Hemolisinas , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones NoqueadosAsunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Muerte Celular , Linaje de la Célula , Humanos , Modelos Biológicos , Fosforilación , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Receptores de Muerte Celular/metabolismo , UbiquitinaciónRESUMEN
Fungal infections claim an estimated 1.5 million lives each year. Mechanisms that protect from fungal infections are still elusive. Recognition of fungal pathogens relies on C-type lectin receptors (CLRs) and their downstream signaling kinase SYK. Here we report that the E3 ubiquitin ligase CBLB controls proximal CLR signaling in macrophages and dendritic cells. We show that CBLB associates with SYK and ubiquitinates SYK, dectin-1, and dectin-2 after fungal recognition. Functionally, CBLB deficiency results in increased inflammasome activation, enhanced reactive oxygen species production, and increased fungal killing. Genetic deletion of Cblb protects mice from morbidity caused by cutaneous infection and markedly improves survival after a lethal systemic infection with Candida albicans. On the basis of these findings, we engineered a cell-permeable CBLB inhibitory peptide that protects mice from lethal C. albicans infections. We thus describe a key role for Cblb in the regulation of innate antifungal immunity and establish a novel paradigm for the treatment of fungal sepsis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Candidiasis Invasiva/inmunología , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Péptidos/farmacología , Fagocitosis/inmunología , Proteínas Proto-Oncogénicas c-cbl/inmunología , Especies Reactivas de Oxígeno/inmunología , Sepsis/inmunología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Candida albicans , Caspasa 8 , Citocinas/inmunología , Células Dendríticas/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Inmunoprecipitación , Riñón , Lectinas Tipo C/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-cbl/genética , UbiquitinaciónRESUMEN
Ubiquitin is a small modifier protein that conjugates on lysine (Lys) residues of substrates, and it can be targeted by another ubiquitin molecule to form chains through conjugation on the intrinsic Lys residues and methionine (Met) 1 residue. Ubiquitination of substrates by such chains determines the fate of substrates, thereby influencing various biological processes. In this chapter, we focus on apoptosis with an emphasis on the regulation by ubiquitination. The signal transduction of apoptosis is governed not only by the classical function of ubiquitin, which is proteasome-dependent degradation of substrates, but also by the apoptosis signaling complex formation guided by different types of ubiquitin chains. Ubiquitinations of pro- and antiapoptotic proteins are tightly regulated by particular sets of enzymes, such as ubiquitin E3 ligases and deubiquitinases (DUBs). We further discuss ubiquitination in the tumor necrosis factor (TNF) signaling pathway as an example for the ubiquitin-dependent regulation of apoptosis and cell survival.