RESUMEN
Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of NF erythroid 2-related factor 2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1ß through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1ß release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Inflamasomas/efectos de los fármacos , Prostaglandina D2/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Bacillus anthracis/química , Toxinas Bacterianas/toxicidad , Western Blotting , Caspasa 1/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inflamasomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Molecular , Proteína con Dominio Pirina 3 de la Familia NLR , Prostaglandina D2/química , Prostaglandina D2/farmacología , Sustancias Protectoras/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacosRESUMEN
Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.
Asunto(s)
Hemo-Oxigenasa 1/sangre , Metaloproteinasa 1 de la Matriz/sangre , Estrés Oxidativo/fisiología , Tuberculosis Pulmonar/patología , Adulto , Anciano , Biomarcadores/sangre , Brasil , Femenino , Hemo-Oxigenasa 1/metabolismo , Humanos , India , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de Unión a TGF-beta Latente/sangre , Pulmón/microbiología , Pulmón/patología , Macrófagos/microbiología , Macrófagos/patología , Masculino , Metaloproteinasa 1 de la Matriz/biosíntesis , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Factor de Transcripción AP-1/metabolismo , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiología , Estados Unidos , Adulto JovenRESUMEN
Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain-like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1ß from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin-mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Arsenicales/farmacología , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Inflamasomas/efectos de los fármacos , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Óxidos/farmacología , Animales , Antígenos Bacterianos/farmacología , Trióxido de Arsénico , Arsenitos/farmacología , Toxinas Bacterianas/farmacología , Caspasa 1/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Flagelina/farmacología , Inmunidad Innata/efectos de los fármacos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Nigericina/farmacología , Óxidos de Nitrógeno/metabolismo , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Proteolisis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Compuestos de Sodio/farmacología , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for ~1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.
RESUMEN
CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ARN Guía de Sistemas CRISPR-Cas , Línea Celular , Sistemas CRISPR-CasRESUMEN
To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Meiosis/fisiología , Separasa/metabolismo , Animales , Animales no Consanguíneos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , División Celular/fisiología , División del Núcleo Celular , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica , Femenino , Células HeLa , Humanos , Cinetocoros/metabolismo , Ratones , Oocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Separasa/fisiología , Quinasa Tipo Polo 1RESUMEN
Anthrax lethal toxin (LT) is a protease that activates the NLRP1b inflammasome sensor in certain rodent strains. Unlike better-studied sensors, relatively little is known about the priming requirements for NLRP1b. In this study, we investigate the rapid and striking priming-independent LT-induced release of IL-1ß in mice within hours of toxin challenge. We find IL-1ß release to be a NLRP1b- and caspase-1-dependent, NLRP3 and caspase-11-independent event that requires both neutrophils and peptidyl arginine deiminiase-4 (PAD4) activity. The simultaneous LT-induced IL-18 response is neutrophil-independent. Bone marrow reconstitution experiments in mice show toxin-induced IL-1ß originates from hematopoietic cells. LT treatment of neutrophils in vitro did not induce IL-1ß, neutrophil extracellular traps (NETs), or pyroptosis. Although platelets interact closely with neutrophils and are also a potential source of IL-1ß, they were unable to bind or endocytose LT and did not secrete IL-1ß in response to the toxin. LT-treated mice had higher levels of cell-free DNA and HMGB1 in circulation than PBS-treated controls, and treatment of mice with recombinant DNase reduced the neutrophil- and NLRP1-dependent IL-1ß release. DNA sensor AIM2 deficiency, however, did not impact IL-1ß release. These data, in combination with the findings on PAD4, suggest a possible role for in vivo NETs or cell-free DNA in cytokine induction in response to LT challenge. Our findings suggest a complex interaction of events and/or mediators in LT-treated mice with the neutrophil as a central player in induction of a profound and rapid inflammatory response to toxin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Antígenos Bacterianos/toxicidad , Proteínas Reguladoras de la Apoptosis/fisiología , Bacillus anthracis/patogenicidad , Toxinas Bacterianas/toxicidad , Trampas Extracelulares/fisiología , Interleucina-1beta/metabolismo , Neutrófilos/metabolismo , Arginina Deiminasa Proteína-Tipo 4/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Carbunco/inmunología , Antígenos Bacterianos/farmacología , Proteínas Reguladoras de la Apoptosis/deficiencia , Bacillus anthracis/fisiología , Toxinas Bacterianas/farmacología , Inflamasomas/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Congénicos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Monocitos/efectos de los fármacos , Monocitos/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/deficiencia , Neutrófilos/efectos de los fármacos , Arginina Deiminasa Proteína-Tipo 4/deficiencia , Piroptosis/efectos de los fármacos , Quimera por Radiación , Especificidad de la Especie , Esporas BacterianasRESUMEN
TorsinA is an ER-resident AAA + ATPase, whose deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is an unusual AAA + ATPase that needs an external activator. Also, it likely does not thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of its still enigmatic function. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 Å structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner channel of ~ 4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA + ATPases. We discuss the implications of these observations for TorsinA function.
Asunto(s)
Adenosina Trifosfatasas/química , Modelos Moleculares , Chaperonas Moleculares/química , Polímeros/química , Conformación Proteica , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Polimerizacion , Polímeros/metabolismoRESUMEN
Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1ß generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1ß. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1ß production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1ß processing by regulating NLRP3-inflammasome assembly in the cytosol.
RESUMEN
The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1ß and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1ß maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1ß secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to our understanding of the beneficial effects of sulforaphane.
Asunto(s)
Inflamasomas/antagonistas & inhibidores , Inflamasomas/metabolismo , Isotiocianatos/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Línea Celular , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Proteína con Dominio Pirina 3 de la Familia NLR , Péptido Hidrolasas/metabolismo , Peritonitis/inmunología , Peritonitis/metabolismo , Peritonitis/microbiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas , Piroptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , SulfóxidosRESUMEN
Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.