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1.
Semin Immunol ; 70: 101845, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37806032

RESUMO

The gasdermin family of proteins are central effectors of the inflammatory, lytic cell death modality known as pyroptosis. Characterized in 2015, the most well-studied member gasdermin D can be proteolyzed, typically by caspases, to generate an active pore-forming N-terminal domain. At least well-studied three pharmacological inhibitors (necrosulfonamide, disulfiram, dimethyl fumarate) since 2018 have been shown to affect gasdermin D activity either through modulation of processing or interference with pore formation. A multitude of murine in vivo studies have since followed. Here, we discuss the current state of research surrounding these three inhibitors, caveats to their use, and a set of guiding principles that researchers should consider when pursuing further studies of gasdermin D inhibition.


Assuntos
Gasderminas , Animais , Humanos , Camundongos , Caspases/metabolismo , Gasderminas/química , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Piroptose
2.
J Biol Chem ; 299(2): 102908, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36642180

RESUMO

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.


Assuntos
Gasderminas , Piroptose , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Membrana Celular/metabolismo , Inflamassomos/metabolismo , Engenharia de Proteínas
3.
Sci Signal ; 15(765): eabl6781, 2022 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-36538593

RESUMO

Pyroptosis is a mechanism of programmed, necrotic cell death mediated by gasdermins, a family of pore-forming proteins. Caspase-1 activates gasdermin D (GSDMD) under inflammatory conditions, whereas caspase-3 activates GSDME under apoptotic conditions, such as those induced by chemotherapy. These pathways are thought to be separate. However, we found that they are part of an integrated network of gatekeepers that enables pyroptotic cell death. We observed that GSDMD was the primary pyroptotic mediator in cultured blood cells in response to doxorubicin and etoposide, two common chemotherapies for hematopoietic malignancies. Upon treatment, the channel protein pannexin-1 (PANX1), which is stimulated by the initiation of apoptosis, increased membrane permeability to induce K+ efflux-driven activation of the NLRP3 inflammasome and GSDMD. However, either PANX1 or GSDME could also be the primary mediator of chemotherapy-induced pyroptosis when present at higher amounts. The most abundant pore-forming protein in acute myeloid leukemias from patients predicted the cell death pathway in response to chemotherapy. This interconnected network, a multistep switch that converts apoptosis to pyroptosis, could be clinically titratated to modulate cell death with regard to antitumor immunity or tumor lysis syndrome in patients.


Assuntos
Antineoplásicos , Neoplasias Hematológicas , Humanos , Gasderminas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Apoptose , Necrose , Inflamassomos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Conexinas/genética , Conexinas/metabolismo
4.
Cell ; 185(2): 283-298.e17, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35021065

RESUMO

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , Sequência de Bases , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Epiteliais/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Metotrexato/farmacologia , Mutação/genética , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Piroptose/efeitos dos fármacos , Piroptose/genética , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/genética
5.
J Mol Biol ; 434(4): 167183, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-34358546

RESUMO

Pyroptosis, a lytic form of programmed cell death, both stimulates effective immune responses and causes tissue damage. Gasdermin (GSDM) proteins are a family of pore-forming executors of pyroptosis. While the most-studied member, GSDMD, exerts critical functions in inflammasome biology, emerging evidence demonstrates potential broad relevance for GSDM-mediated pyroptosis across diverse pathologies. In this review, we describe GSDM biology, outline conditions where inflammasomes and GSDM-mediated pyroptosis represent rational therapeutic targets, and delineate strategies to manipulate these central immunologic processes for the treatment of human disease.


Assuntos
Inflamassomos , Terapia de Alvo Molecular , Proteínas de Ligação a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Piroptose , Humanos , Inflamassomos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose/efeitos dos fármacos
6.
Cell Rep ; 35(2): 108998, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33852854

RESUMO

Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1ß (IL-1ß) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1ß akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1ß processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1ß release independent of its ability to cause cell death.


Assuntos
Inflamassomos/genética , Interleucina-1beta/genética , Macrófagos/imunologia , Proteínas de Ligação a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptose/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Caspase 1/genética , Caspase 1/imunologia , Caspase 3/genética , Caspase 3/imunologia , Linhagem Celular Transformada , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/imunologia , Proteínas Citotóxicas Formadoras de Poros/deficiência , Proteínas Citotóxicas Formadoras de Poros/imunologia , Piroptose/efeitos dos fármacos , Piroptose/imunologia , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Células THP-1
7.
Nat Commun ; 11(1): 2212, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371889

RESUMO

Gasdermin-D (GSDMD) in inflammasome-activated macrophages is cleaved by caspase-1 to generate N-GSDMD fragments. N-GSDMD then oligomerizes in the plasma membrane (PM) to form pores that increase membrane permeability, leading to pyroptosis and IL-1ß release. In contrast, we report that although N-GSDMD is required for IL-1ß secretion in NLRP3-activated human and murine neutrophils, N-GSDMD does not localize to the PM or increase PM permeability or pyroptosis. Instead, biochemical and microscopy studies reveal that N-GSDMD in neutrophils predominantly associates with azurophilic granules and LC3+ autophagosomes. N-GSDMD trafficking to azurophilic granules causes leakage of neutrophil elastase into the cytosol, resulting in secondary cleavage of GSDMD to an alternatively cleaved N-GSDMD product. Genetic analyses using ATG7-deficient cells indicate that neutrophils secrete IL-1ß via an autophagy-dependent mechanism. These findings reveal fundamental differences in GSDMD trafficking between neutrophils and macrophages that underlie neutrophil-specific functions during inflammasome activation.


Assuntos
Membrana Celular/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neutrófilos/metabolismo , Organelas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Autofagossomos/metabolismo , Autofagia/genética , Caspase 1/metabolismo , Permeabilidade da Membrana Celular/genética , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Elastase de Leucócito/genética , Elastase de Leucócito/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Transporte Proteico , Piroptose/genética
8.
Nat Immunol ; 20(10): 1299-1310, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534238

RESUMO

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.


Assuntos
Infecções por Poxviridae/imunologia , Poxviridae/fisiologia , Domínios Proteicos/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Alelos , Animais , Extinção Biológica , Humanos , Imunidade , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Fosforilação
9.
Immunity ; 51(1): 43-49.e4, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097341

RESUMO

Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their ß1-ß2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Cristalização/métodos , Inflamassomos/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos , Lipídeos/química , Camundongos , Mutagênese Sítio-Dirigida , Mutação/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Ligação a Fosfato , Conformação Proteica , Domínios Proteicos/genética , Multimerização Proteica , Piroptose/genética , Relação Estrutura-Atividade
10.
Cancer Discov ; 9(1): 96-113, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30361447

RESUMO

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44-CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44-PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. SIGNIFICANCE: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44-PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Receptores de Hialuronatos/metabolismo , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Biomarcadores Tumorais , Carcinogênese , Feminino , Humanos , Receptores de Hialuronatos/fisiologia , Camundongos , Neoplasias de Mama Triplo Negativas/fisiopatologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Sci Immunol ; 3(26)2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143556

RESUMO

Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1ß, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.


Assuntos
Acrilamidas/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Sulfonamidas/farmacologia , Acrilamidas/uso terapêutico , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Citocinas/genética , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas de Ligação a Fosfato , Pirina/fisiologia , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/imunologia , Salmonella typhimurium , Sepse/tratamento farmacológico , Sepse/imunologia , Sulfonamidas/uso terapêutico , Células THP-1
12.
Sci Signal ; 11(539)2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018081

RESUMO

The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.


Assuntos
Apoptose , Proteínas Inibidoras de Apoptose/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/química , Camundongos , Domínios Proteicos , Transdução de Sinais
13.
Proc Natl Acad Sci U S A ; 115(26): 6792-6797, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891674

RESUMO

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1ß release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.


Assuntos
Proteínas Reguladoras de Apoptose/química , Inibidores de Caspase/química , Proteínas de Neoplasias/química , Peptídeos/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Inibidores de Caspase/metabolismo , Domínio Catalítico , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células Jurkat , Camundongos , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Proteínas de Ligação a Fosfato , Estrutura Secundária de Proteína , Células RAW 264.7 , Células THP-1
14.
Structure ; 26(5): 778-784.e3, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29576317

RESUMO

Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation.


Assuntos
Proteínas Reguladoras de Apoptose/química , Mutação , Proteínas de Neoplasias/química , Animais , Proteínas Reguladoras de Apoptose/genética , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteínas de Ligação a Fosfato , Domínios Proteicos , Estrutura Secundária de Proteína , Piroptose
15.
Nat Cell Biol ; 20(1): 58-68, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29203883

RESUMO

Ubiquitylation of the TNFR1 signalling complex (TNF-RSC) controls the activation of RIPK1, a kinase critically involved in mediating multiple TNFα-activated deleterious events. However, the molecular mechanism that coordinates different types of ubiquitylation modification to regulate the activation of RIPK1 kinase remains unclear. Here, we show that ABIN-1/NAF-1, a ubiquitin-binding protein, is recruited rapidly into TNF-RSC in a manner dependent on the Met1-ubiquitylating complex LUBAC to regulate the recruitment of A20 to control Lys63 deubiquitylation of RIPK1. ABIN-1 deficiency reduces the recruitment of A20 and licenses cells to die through necroptosis by promoting Lys63 ubiquitylation and activation of RIPK1 with TNFα stimulation under conditions that would otherwise exclusively activate apoptosis in wild-type cells. Inhibition of RIPK1 kinase and RIPK3 deficiency block the embryonic lethality of Abin-1 -/- mice. We propose that ABIN-1 provides a critical link between Met1 ubiquitylation mediated by the LUBAC complex and Lys63 deubiquitylation by phospho-A20 to modulate the activation of RIPK1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fibroblastos/metabolismo , Fosfoproteínas/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Apoptose/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Fibroblastos/citologia , Regulação da Expressão Gênica , Genes Letais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
16.
J Biol Chem ; 292(35): 14649-14658, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28726636

RESUMO

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.


Assuntos
Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Caspases/metabolismo , Inflamassomos/metabolismo , Macrófagos/citologia , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Piroptose , Substituição de Aminoácidos , Animais , Linhagem Celular Transformada , Células HEK293 , Humanos , Inflamassomos/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Microscopia de Vídeo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a Fosfato , Mutação Puntual , Multimerização Proteica , Transporte Proteico , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
17.
J Biol Chem ; 292(23): 9666-9679, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28404814

RESUMO

The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.


Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Transtornos Linfoproliferativos/metabolismo , Mutação , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Linhagem Celular , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Domínios Proteicos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
18.
Science ; 353(6297): 399-403, 2016 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-27463676

RESUMO

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.


Assuntos
Antígeno B7-H1/genética , Neoplasias Cerebelares/imunologia , Quinase 5 Dependente de Ciclina/fisiologia , Regulação Neoplásica da Expressão Gênica , Meduloblastoma/imunologia , Neoplasias Experimentais/imunologia , Evasão Tumoral/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Quinase 5 Dependente de Ciclina/genética , Humanos , Vigilância Imunológica , Fator Regulador 2 de Interferon/genética , Fator Regulador 2 de Interferon/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias Experimentais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
J Immunol ; 197(4): 1353-67, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27385778

RESUMO

Canonical inflammasome activation induces a caspase-1/gasdermin D (Gsdmd)-dependent lytic cell death called pyroptosis that promotes antimicrobial host defense but may contribute to sepsis. The nature of the caspase-1-dependent change in plasma membrane (PM) permeability during pyroptotic progression remains incompletely defined. We assayed propidium(2+) (Pro(2+)) influx kinetics during NLRP3 or Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDMs) as an indicator of this PM permeabilization. BMDMs were characterized by rapid Pro(2+) influx after initiation of NLRP3 or Pyrin inflammasomes by nigericin (NG) or Clostridium difficile toxin B (TcdB), respectively. No Pro(2+) uptake in response to NG or TcdB was observed in Casp1(-/-) or Asc(-/-) BMDMs. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro(2+) influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro(2+) influx and pyroptotic lysis. Extracellular La(3+) and Gd(3+) rapidly and reversibly blocked the induced Pro(2+) influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome assembly and caspase-1 activation. Thus, caspase-1-driven pyroptosis requires induction of initial prelytic pores in the PM that are dependent on Gsdmd expression. These PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca(2+) Although lanthanides and Gsdmd deletion both suppressed PM pore activity and pyroptotic lysis, robust IL-1ß release was observed in lanthanide-treated BMDMs but not in Gsdmd-deficient cells. This suggests roles for Gsdmd in both passive IL-1ß release secondary to pyroptotic lysis and in nonlytic/nonclassical IL-1ß export.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/metabolismo , Piroptose/fisiologia , Animais , Membrana Celular/patologia , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Elementos da Série dos Lantanídeos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato
20.
J Immunol ; 196(10): 4291-7, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27045108

RESUMO

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in that they can all activate NF-κB and induce NF-κB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2's kinase domain with RIPK4's did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction defect in RIPK2(-/-) macrophages. These findings suggest that RIPK2's kinase domain is functionally unique compared with other RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.


Assuntos
Morte Celular , Imunidade Inata , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Biologia Sintética , Expressão Gênica , Células HEK293 , Humanos , Inflamação , Macrófagos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação
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