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Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.
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The FEBS Journal publishes research on diverse topics in the molecular life sciences relating to the molecules and mechanisms underpinning biological processes. Editor-in-Chief Seamus Martin discusses the rewards of pursuing a career in basic research, some highlights of the past year at the journal, and what's in store for 2024.
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Disciplinas das Ciências Biológicas , EditoraçãoRESUMO
Apoptosis is a mode of programmed cell death that plays important roles in tissue sculpting during development, in the maintenance of tissue homeostasis in the adult, and in the eradication of injured or infected cells during pathological processes. Numerous physiological as well as pathological stimuli trigger apoptosis, such as engagement of plasma-membrane-associated Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, radiation, and exposure to diverse cytotoxic drugs. Apoptosis is coordinated by members of the caspase family of cysteine proteases, which, upon activation, trigger a series of dramatic morphological and biochemical changes including retraction from the substratum, cell shrinkage, extensive and protracted plasma membrane blebbing, chromatin condensation, DNA hydrolysis, nuclear fragmentation, and proteolytic cleavage of numerous caspase substrates. These dramatic structural and biochemical alterations result not only in the controlled dismantling of the cell, but also in the rapid recognition and removal of apoptotic cells by phagocytes through the cell surface display of phagocytotic triggers such as phosphatidylserine. Necrosis, which is typically nonprogrammed or imposed upon the cell by overwhelming membrane or organelle damage, is characterized by high-amplitude cell swelling, followed by rapid plasma membrane rupture and release of cellular contents into the extracellular space. Necrosis is often provoked by infectious agents or severe departure from physiological conditions due to toxins, temperature extremes, or physical injury. However, forms of programmed necrosis (necroptosis, pyroptosis, ferroptosis) can also occur in specific circumstances. Nonprogrammed and programmed necrosis can be distinguished from apoptosis by morphological features, based on the rapid uptake of vital dyes, and through the application of specific inhibitors of key molecules associated with the latter modes of cell death. This unit describes protocols for the measurement of apoptosis and necrosis and for distinguishing apoptosis from programmed as well as conventional necrosis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of cell morphology by phase-contrast microscopy Alternative Protocol 1: Assessment of morphological changes using eosin-methylene blue staining Alternative Protocol 2: Analysis of nuclear morphology by fluorescence microscopy Support Protocol: Preparation of cytospins Basic Protocol 2: Measurement of plasma membrane composition with annexin V and propidium iodide Basic Protocol 3: Measurement of DNA fragmentation by flow cytometry Alternative Protocol 3: Analysis of DNA fragmentation by the TUNEL assay Basic Protocol 4: Measurement of caspase activation by flow cytometry Basic Protocol 5: Discriminating between apoptosis, necrosis, necroptosis, and ferroptosis.
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Ferroptose , Humanos , Citometria de Fluxo/métodos , Necroptose , Apoptose/fisiologia , Necrose/patologia , Caspases/metabolismo , Microscopia de FluorescênciaRESUMO
TRAIL and FasL are potent inducers of apoptosis but can also promote inflammation through assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) complexes, wherein caspase-8 acts as a scaffold to drive FADD/RIPK1-mediated nuclear factor κB (NF-κB) activation. cFLIP is also recruited to FADDosomes and restricts caspase-8 activity and apoptosis, but whether cFLIP also regulates death receptor-initiated inflammation is unclear. Here, we show that silencing or deletion of cFLIP leads to robustly enhanced Fas-, TRAIL-, or TLR3-induced inflammatory cytokine production, which can be uncoupled from the effects of cFLIP on caspase-8 activation and apoptosis. Mechanistically, cFLIPL suppresses Fas- or TRAIL-initiated NF-κB activation through inhibiting the assembly of caspase-8/FADD/RIPK1 FADDosome complexes, due to the low affinity of cFLIPL for FADD. Consequently, increased cFLIPL occupancy of FADDosomes diminishes recruitment of FADD/RIPK1 to caspase-8, thereby suppressing NF-κB activation and inflammatory cytokine production downstream. Thus, cFLIP acts as a dual suppressor of apoptosis and inflammation via distinct modes of action.
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Proteínas Reguladoras de Apoptose , NF-kappa B , Humanos , NF-kappa B/metabolismo , Caspase 8/metabolismo , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose , Inflamação , Citocinas/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteína de Domínio de Morte Associada a Fas/metabolismoRESUMO
Bcl-2 family proteins serve as key regulators of apoptosis and are frequently overexpressed in cancer. Consequently, small-molecule Bcl-2-antagonists (BH3 mimetics) have emerged as a new class of targeted therapeutics. A recent study by Zhao et al. has unexpectedly found that BH3 mimetics can also activate dendritic cells (DCs) to prime for T cell-mediated tumor clearance.
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Antineoplásicos , Neoplasias , Humanos , Apoptose , Neoplasias/tratamento farmacológico , Células Dendríticas/metabolismo , Antineoplásicos/uso terapêuticoRESUMO
The FEBS Journal publishes high impact research on diverse topics in the life sciences, with a focus on molecular mechanisms underpinning biological processes. Here, Editor-in-Chief Seamus Martin discusses highlights of the journal from the past year and gives a glimpse into what's in store for 2023.
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Disciplinas das Ciências Biológicas , EditoraçãoRESUMO
Interleukin-1 (IL-1) family cytokines are key barrier cytokines that are typically expressed as inactive, or partially active, precursors that require proteolysis within their amino termini for activation. IL-37 is an enigmatic member of the IL-1 family that has been proposed to be activated by caspase-1 and to exert anti-inflammatory activity through engagement of the IL-18R and SIGIRR. However, here we show that the longest IL-37 isoform, IL-37b, exhibits robust proinflammatory activity upon amino-terminal proteolysis by neutrophil elastase or cathepsin S. In sharp contrast, caspase-1 failed to process or activate IL-37 at concentrations that robustly activated its canonical substrate, IL-1ß. IL-37 and IL-36 exhibit high structural homology, and, consistent with this, a K53-truncated form of IL-37, mimicking the cathepsin S-processed form of this cytokine, was found to exert its proinflammatory effects via IL-36 receptor engagement and produced an inflammatory signature practically identical to IL-36. Administration of K53-truncated IL-37b intraperitoneally into wild-type mice also elicited an inflammatory response that was attenuated in IL-36R-/- animals. These data demonstrate that, in common with other IL-1 family members, mature IL-37 can also elicit proinflammatory effects upon processing by specific proteases.
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Interleucina-1 , Peptídeo Hidrolases , Receptores de Interleucina , Animais , Camundongos , Caspases , Catepsinas , Citocinas , Interleucina-1/metabolismo , Células Mieloides , Receptores de Interleucina/metabolismoRESUMO
γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.
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COVID-19 , Células Dendríticas , Proteínas do Nucleocapsídeo , Receptores de Antígenos de Linfócitos T gama-delta , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/imunologia , COVID-19/virologia , Células Dendríticas/imunologia , Humanos , Interferon gama/imunologia , Proteínas do Nucleocapsídeo/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Inflammation driven by environmental allergens is an important source of morbidity in diseases such as asthma and eczema. How common allergens promote inflammation is still poorly understood, but previous studies have implicated the protease activity associated with many allergens as an important component of the pro-inflammatory properties of these agents. The IL-1 family cytokine, IL-33, has recently been shown to undergo processing and activation by proteases associated with multiple common allergens. However, it remains unclear whether the sensing of exogenous protease activity-as a proxy for the detection of invasive microbes, allergens and parasitic worms-is a general property of IL-1 family cytokines. In common with the majority of IL-1 family members, cytokines within the IL-36 sub-family (IL-36α, IL-36ß and IL-36γ) are expressed as inactive precursors that require proteolysis within their N-termini for activation. Here we show that proteases associated with multiple common allergens of plant, insect, fungal and bacterial origin (including: Aspergillus fumigatus, ragweed, rye, house dust mite, cockroach and Bacillus licheniformis) are capable of processing and activating IL-36 family cytokines, with IL-36ß being particularly susceptible to activation by multiple allergens. Furthermore, extracts from several allergens also processed and enhanced IL-1α activity. This suggests that multiple IL-1 family cytokines may serve as sentinels for exogenous proteases, coupling detection of such activity to unleashing the pro-inflammatory activity of these cytokines. Taken together with previous data on the diversity of proteases capable of activating IL-1 family cytokines, this suggests that members of this cytokine family may function as 'activity recognition receptors' for aberrant protease activity associated with infection, tissue injury or programmed necrosis.
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Alérgenos , Peptídeo Hidrolases , Animais , Endopeptidases , Inflamação , PyroglyphidaeRESUMO
Members of the extended IL-1 cytokine family play key roles as instigators of inflammation in numerous infectious and sterile injury contexts and are highly enriched at barrier surfaces such as the skin, lungs and intestinal mucosa. Because IL-1 family cytokines do not possess conventional ER-golgi trafficking and secretory signals, these cytokines are typically released into the extracellular space due to tissue damage resulting in necrosis, or pathogen detection resulting in pyroptosis. The latter feature, in combination with other factors, suggests that IL-1 family cytokines serve as canonical damage-associated molecular patterns (DAMPs), which instigate inflammation in response to tissue damage. However, IL-1 family cytokines also require a proteolytic activation step and diverse intracellular, extracellular and non-self proteases have been identified that are capable of processing and activating members of this family. This suggests that IL-1 family members function as sentinels for aberrant protease activity, which is frequently associated with infection or tissue damage. Here, we overview the diversity of proteases implicated in the activation of IL-1 family cytokines and suggest that this ancient cytokine family may have evolved to complement 'pattern recognition receptors', by serving as 'activity recognition receptors' enabling the detection of aberrant enzyme activity indicative of 'danger'.
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Alarminas , Citocinas , Humanos , Inflamassomos , Inflamação , Interleucina-1 , Peptídeo HidrolasesRESUMO
Professor Dame Janet Thornton has been named as the inaugural recipient of 'The FEBS Journal Open Science Award' for her major role in the development of ELIXIR, the pan-European infrastructure for biological data, as well as the creation of freely available computational tools and knowledge resources.
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Distinções e PrêmiosRESUMO
The FEBS Journal, a leading multidisciplinary journal in the life sciences, publishes high-impact papers on diverse topics relating to molecular mechanisms underpinning biological processes. Here, Editor-in-Chief Seamus Martin discusses the critical importance of data provenance and data integrity to the scientific method and discusses some of the highlights from 2021 at The FEBS Journal.
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Entosis is a form of nonphagocytic cell-in-cell (CIC) interaction where a living cell enters into another. Tumors show evidence of entosis; however, factors controlling entosis remain to be elucidated. Here, we find that besides inducing apoptosis, TRAIL signaling is a potent activator of entosis in colon cancer cells. Initiation of both apoptosis and entosis requires TRAIL receptors DR4 and DR5; however, induction of apoptosis and entosis diverges at caspase-8 as its structural presence is sufficient for induction of entosis but not apoptosis. Although apoptosis and entosis are morphologically and biochemically distinct, knockout of Bax and Bak, or inhibition of caspases, also inhibits entotic cell death and promotes survival and release of inner cells. Analysis of colorectal cancer tumors reveals a significant association between TRAIL signaling and CIC structures. Finally, the presence of CIC structures in the invasive front regions of colorectal tumors shows a strong correlation with adverse patient prognosis.
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Neoplasias do Colo/metabolismo , Entose/fisiologia , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 8/metabolismo , Caspases/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Células HCT116 , Humanos , Glicoproteínas de Membrana/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This 75th birthday tribute to our Editorial Board member Alexander Wlodawer recounts his decades-long service to the community of structural biology researchers. His former and current colleagues tell the story of his upbringing and education, followed by an account of his dedication to quality and rigor in crystallography and structural science. The FEBS Journal Editor-in-Chief Seamus Martin further highlights Alex's outstanding contributions to the journal's success over many years.
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Cristalografia/história , Biologia Molecular/história , História do Século XX , História do Século XXI , HumanosRESUMO
The FEBS Journal, a leading multidisciplinary journal in the life sciences, continues to grow in visibility and impact. Here, the Editor-in-Chief Seamus Martin discusses developments at the journal over the past year and the impact of the COVID-19 crisis on research activities.
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Pesquisa , Publicações Seriadas , Distinções e Prêmios , COVID-19 , Vacinas contra COVID-19 , Congressos como Assunto , HumanosRESUMO
Inflammation triggered by infection or cellular necrosis is initiated by a battery of pattern-recognition receptors, such as Toll-like receptors or IL-1 family receptors. Diverse forms of cell stress, such as ER stress or mitochondrial stress, can also promote inflammatory responses that contribute to the chronic inflammation observed in cancer, obesity, and other conditions. However, the molecular mechanisms of cell-stress-induced inflammation are poorly understood. Here, we show that ER stress initiated NF-κB activation and inflammation through transcriptional upregulation and ligand-independent activation of TRAIL receptors. ER-stress-induced TRAIL receptor activation resulted in caspase-8/FADD/RIPK1-dependent NF-κB activation and inflammatory cytokine production. Silencing or deletion of TRAIL receptors, or their downstream effectors caspase-8, FADD, or RIPK1, suppressed ER-stress-induced inflammation. Furthermore, chemotherapeutic stress-induced inflammatory responses were blunted in DR5/TRAIL-R null animals. We propose that, upon ER stress, TRAIL receptors serve as "stress-associated molecular patterns (SAMPs)" coupling ER stress to NF-κB-dependent inflammation.
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Citocinas/metabolismo , Estresse do Retículo Endoplasmático , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais , Células A549 , Animais , Caspase 8/metabolismo , Células Cultivadas , Citocinas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Células HCT116 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Seamus Martin holds the Smurfit Chair of Medical Genetics at the Smurfit Institute of Genetics, Trinity College Dublin, Ireland. His laboratory works on diverse aspects of cell death control, including the role of 'death receptors' as initiators of inflammation and the role of IL-1 family cytokines as key initiators of inflammation in the context of necrotic cell death. Seamus received the GlaxoSmithKline Award of The Biochemical Society (2006) and The RDS-Irish Times Boyle Medal (2015) for his work on the role of caspases in apoptosis and was elected to the Royal Irish Academy in 2006 and EMBO in 2009. He is the Editor-in-Chief of The FEBS Journal since 2014.
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Acesso à Informação , Pesquisa Biomédica/normas , Publicações Periódicas como Assunto/normas , Editoração/normas , Pesquisa Biomédica/tendências , Humanos , Publicações Periódicas como Assunto/tendências , Editoração/tendênciasRESUMO
In this Editorial, Editor-in-Chief Seamus Martin reviews developments over the past year at The FEBS Journal and highlights some ongoing challenges in the current landscape of scientific publishing.
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Políticas Editoriais , Publicações Periódicas como Assunto , Editoração , HumanosRESUMO
Giving effective and interesting talks are key skills for scientists at every stage of their career. This installment of the Words of Advice series provides some insights into preparing and delivering compelling presentations, as well as alleviating pretalk nerves.