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1.
Immunity ; 56(5): 926-943.e7, 2023 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-36948192

RESUMEN

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.


Asunto(s)
Proteínas Reguladoras de la Apoptosis , Inflamasomas , Adenosina Trifosfatasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Flagelina/metabolismo , Inflamasomas/metabolismo , Proteína Inhibidora de la Apoptosis Neuronal/química , Proteína Inhibidora de la Apoptosis Neuronal/metabolismo , Proteínas NLR/metabolismo
3.
Mol Cell ; 80(2): 296-310.e6, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-32979304

RESUMEN

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.


Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Caspasa 8/metabolismo , Necroptosis , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal , Animales , Ciego/lesiones , Ciego/patología , Línea Celular , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones Endogámicos C57BL , Mutación/genética , Necroptosis/efectos de los fármacos , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Fosfotreonina/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología
4.
Nature ; 580(7803): 386-390, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32296174

RESUMEN

The aetiology of inflammatory bowel disease (IBD) is a multifactorial interplay between heredity and environment1,2. Here we report that deficiency in SETDB1, a histone methyltransferase that mediates the trimethylation of histone H3 at lysine 9, participates in the pathogenesis of IBD. We found that levels of SETDB1 are decreased in patients with IBD, and that mice with reduced SETDB1 in intestinal stem cells developed spontaneous terminal ileitis and colitis. SETDB1 safeguards genome stability3, and the loss of SETDB1 in intestinal stem cells released repression of endogenous retroviruses (retrovirus-like elements with long repeats that, in humans, comprise approximately 8% of the genome). Excessive viral mimicry generated by motivated endogenous retroviruses triggered Z-DNA-binding protein 1 (ZBP1)-dependent necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted bowel inflammation. Genome instability, reactive endogenous retroviruses, upregulation of ZBP1 and necroptosis were all seen in patients with IBD. Pharmaceutical inhibition of RIP3 showed a curative effect in SETDB1-deficient mice, which suggests that targeting necroptosis of intestinal stem cells may represent an approach for the treatment of severe IBD.


Asunto(s)
Inestabilidad Genómica , N-Metiltransferasa de Histona-Lisina/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Necroptosis , Células Madre/metabolismo , Animales , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Ratones , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células Madre/citología
5.
Mol Cell Proteomics ; 18(6): 1054-1069, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30850422

RESUMEN

Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.


Asunto(s)
Lipopolisacáridos/metabolismo , Espectrometría de Masas/métodos , Transducción de Señal , Animales , Bases de Datos de Proteínas , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/metabolismo , Fosforilación , Unión Proteica , Células RAW 264.7 , Factor 6 Asociado a Receptor de TNF , Receptor Toll-Like 4/metabolismo
6.
Cell Death Discov ; 10(1): 255, 2024 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-38789425

RESUMEN

Caspase-8 (Casp8) serves as an initiator of apoptosis or a suppressor of necroptosis in context-dependent manner. Members of the p90 RSK family can phosphorylate caspase-8 at threonine-265 (T265), which can inactivate caspase-8 for bypassing caspase-8-mediated blockade of necroptosis and can also decrease caspase-8 level by promoting its degradation. Mutating T265 in caspase-8 to alanine (A) in mice blocked TNF-induced necroptotic cecum damage but resulted in unexpectedly massive injury in the small intestine. Here, we show RSK1, RSK2, and RSK3 redundantly function in caspase-8 phosphorylation, and the duodenum is the most severely affected part of the small intestine when T265 phosphorylation of caspase-8 was prevented. Eliminating caspase-8 phosphorylation resulted in a duodenum-specific increase in basal caspase-8 protein level, which shall be responsible for the increased sensitivity to TNF-induced damage. Apoptosis of intestinal epithelial cells (IECs) was predominant in the duodenum of TNF-treated Rsk1-/-Rsk2-/-Rsk3-/- and Casp8T265A/T265A mice, though necroptosis was also observed. The heightened duodenal injury amplified systemic inflammatory responses, as evidenced by the contribution of hematopoietic cells to the sensitization of TNF-induced animal death. Further analysis revealed that hematopoietic and non-hematopoietic cells contributed differentially to cytokine production in response to the increased cell death. Collectively, RSKs emerges as a previously overlooked regulator that, via tissue/organ-constrained inactivating caspase-8 and/or downregulating caspase-8 protein level, controls the sensitivity to TNF-induced organ injury and animal death.

7.
Cell Rep ; 43(5): 114221, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38748877

RESUMEN

ZBP1 is an interferon (IFN)-induced nucleic acid (NA) sensor that senses unusual Z-form NA (Z-NA) to promote cell death and inflammation. However, the mechanisms that dampen ZBP1 activation to fine-tune inflammatory responses are unclear. Here, we characterize a short isoform of ZBP1 (referred to as ZBP1-S) as an intrinsic suppressor of the inflammatory signaling mediated by full-length ZBP1. Mechanistically, ZBP1-S depresses ZBP1-mediated cell death by competitive binding with Z-NA for Zα domains of ZBP1. Cells from mice (Ripk1D325A/D325A) with cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome are alive but sensitive to IFN-induced and ZBP1-dependent cell death. Intriguingly, Ripk1D325A/D325A cells die spontaneously when ZBP1-S is deleted, indicating that cell death driven by ZBP1 is under the control of ZBP1-S. Thus, our findings reveal that alternative splicing of Zbp1 represents autogenic inhibition for regulating ZBP1 signaling and indicate that uncoupling of Z-NA with ZBP1 could be an effective strategy against autoinflammations.


Asunto(s)
Muerte Celular , Isoformas de Proteínas , Proteínas de Unión al ARN , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Humanos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Ratones Endogámicos C57BL , Empalme Alternativo/genética , Células HEK293 , Inflamación/metabolismo , Inflamación/patología
8.
Research (Wash D C) ; 2022: 9838341, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35958114

RESUMEN

Inflammasomes are essential complexes of innate immune system, which form the first line of host defense against pathogens. Mounting evidence accumulates that inflammasome signaling is highly correlated with coronavirus disease 2019 (COVID-19). However, there remains a significant gap in our understanding of the regulatory mechanism of inflammasome signaling. Combining mathematical modeling with experimental analysis of NLRP1b inflammasome signaling, we found that only the expression levels of caspase-1 and GSDMD have the potential to individually switch cell death modes. Reduction of caspase-1 or GSDMD switches cell death from pyroptosis to apoptosis. Caspase-1 and GSDMD present different thresholds and exert distinct pathway choices in switching death modes. Pyroptosis switches to apoptosis with an extremely low threshold level of caspase-1, but with a high threshold of GSDMD. Caspase-1-impaired cells employ ASC-caspase-8-dependent pathway for apoptosis, while GSDMD-impaired cells primarily utilize caspase-1-dependent pathway. Additionally, caspase-1 and GSDMD can severally ignite the cooccurrence of pyroptosis and apoptosis. Landscape topography unravels that the cooccurrence is dramatically different in caspase-1- and GSDMD-impaired cells. Besides pyroptosis state and apoptosis state, a potential new "coexisting" state in single cells is proposed when GSDMD acts as the driving force of the landscape. The "seesaw model" is therefore proposed, which can well describe the death states that are controlled by caspase-1 or GSDMD in single cells. Our study sheds new light on NLRP1b inflammasome signaling and uncovers the switching mechanisms among various death modes, providing potential clues to guide the development of more rational control strategies for diseases.

9.
STAR Protoc ; 2(1): 100251, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33458710

RESUMEN

TNF-induced necroptosis is involved in many physiological and pathological processes. Phospho-MLKL is a hallmark of necroptosis. Cecum is a sensitive organ with extensive necroptosis responses to TNF in vivo. Here, taking advantage of commercially available mouse TNF and easily accessible reagents and materials, we systematically provide a detailed and highly versatile protocol of detecting necroptosis signaling in mouse cecum by immunohistochemical labeling, which can also be used in other tissues or antibodies in immunohistochemical staining. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020) and Chen et al. (2015).


Asunto(s)
Inmunohistoquímica , Necroptosis , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Animales , Ratones
10.
Protein Cell ; 12(11): 858-876, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-33389663

RESUMEN

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.


Asunto(s)
Apoptosis , Caspasa 8/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Necroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Caspasa 8/genética , Proteínas Activadoras de GTPasa/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética
12.
Nat Commun ; 8: 14329, 2017 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-28176780

RESUMEN

Necroptosis is a type of programmed cell death with great significance in many pathological processes. Tumour necrosis factor-α(TNF), a proinflammatory cytokine, is a prototypic trigger of necroptosis. It is known that mitochondrial reactive oxygen species (ROS) promote necroptosis, and that kinase activity of receptor interacting protein 1 (RIP1) is required for TNF-induced necroptosis. However, how ROS function and what RIP1 phosphorylates to promote necroptosis are largely unknown. Here we show that three crucial cysteines in RIP1 are required for sensing ROS, and ROS subsequently activates RIP1 autophosphorylation on serine residue 161 (S161). The major function of RIP1 kinase activity in TNF-induced necroptosis is to autophosphorylate S161. This specific phosphorylation then enables RIP1 to recruit RIP3 and form a functional necrosome, a central controller of necroptosis. Since ROS induction is known to require necrosomal RIP3, ROS therefore function in a positive feedback circuit that ensures effective induction of necroptosis.


Asunto(s)
Apoptosis/fisiología , Necrosis/patología , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Línea Celular Tumoral , Cisteína/metabolismo , Humanos , Ratones , Mitocondrias/patología , Células 3T3 NIH , Fosforilación/fisiología , Cultivo Primario de Células , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Serina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
13.
Cell Res ; 25(12): 1285-98, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26611636

RESUMEN

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1ß (IL-1ß) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1ß in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1ß secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Interleucina-1beta/metabolismo , Proteínas de Neoplasias/metabolismo , Piroptosis , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Células HEK293 , Humanos , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Unión a Fosfato
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