The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils

Neutrophil extracellular trap (NET) formation can generate short-term, functional anucleate cytoplasts and trigger loss of cell viability. We demonstrated that the necroptotic cell death effector mixed lineage kinase domain–like (MLKL) translocated from the cytoplasm to the plasma membrane and stimulated downstream NADPH oxidase–independent ROS production, loss of cytoplasmic granules, breakdown of the nuclear membrane, chromatin decondensation, histone hypercitrullination, and extrusion of bacteriostatic NETs. This process was coordinated by receptor-interacting protein kinase-1 (RIPK1), which activated the caspase-8–dependent apoptotic or RIPK3/MLKL-dependent necroptotic death of mouse and human neutrophils. Genetic deficiency of RIPK3 and MLKL prevented NET formation but did not prevent cell death, which was because of residual caspase-8–dependent activity. Peptidylarginine deiminase 4 (PAD4) was activated downstream of RIPK1/RIPK3/MLKL and was required for maximal histone hypercitrullination and NET extrusion. This work defines a distinct signaling network that activates PAD4-dependent NET release for the control of methicillin-resistant Staphylococcus aureus (MRSA) infection.

Download Full-text

Mouse cytomegalovirus M36 and M45 death suppressors cooperate to prevent inflammation resulting from antiviral programmed cell death pathways

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616829114 ◽

2017 ◽

Vol 114 (13) ◽

pp. E2786-E2795 ◽

Cited By ~ 22

Author(s):

Lisa P. Daley-Bauer ◽

Linda Roback ◽

Lynsey N. Crosby ◽

A. Louise McCormick ◽

Yanjun Feng ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Double Mutant ◽

Kinase Domain ◽

Murine Cytomegalovirus ◽

Caspase 8 ◽

Interacting Protein ◽

Antiviral Responses ◽

Cell Death Pathways ◽

Infected Cells

The complex interplay between caspase-8 and receptor-interacting protein (RIP) kinase RIP 3 (RIPK3) driving extrinsic apoptosis and necroptosis is not fully understood. Murine cytomegalovirus triggers both apoptosis and necroptosis in infected cells; however, encoded inhibitors of caspase-8 activity (M36) and RIP3 signaling (M45) suppress these antiviral responses. Here, we report that this virus activates caspase-8 in macrophages to trigger apoptosis that gives rise to secondary necroptosis. Infection with double-mutant ΔM36/M45mutRHIM virus reveals a signaling pattern in which caspase-8 activates caspase-3 to drive apoptosis with subsequent RIP3-dependent activation of mixed lineage kinase domain-like (MLKL) leading to necroptosis. This combined cell death signaling is highly inflammatory, greater than either apoptosis induced by ΔM36 or necroptosis induced by M45mutRHIM virus. IL-6 production by macrophages is dramatically increased during double-mutant virus infection and correlates with faster antiviral responses in the host. Collaboratively, M36 and M45 target caspase-8 and RIP3 pathways together to suppress this proinflammatory cell death. This study reveals the effect of antiviral programmed cell death pathways on inflammation, shows that caspase-8 activation may go hand-in-hand with necroptosis in macrophages, and revises current understanding of independent and collaborative functions of M36 and M45 in blocking apoptotic and necroptotic cell death responses.

Download Full-text

Caspases: the executioners of apoptosis

Biochemical Journal ◽

10.1042/bj3260001 ◽

1997 ◽

Vol 326 (1) ◽

pp. 1-16 ◽

Cited By ~ 3045

Author(s):

Gerald M. COHEN

Keyword(s):

Cell Death ◽

Morphological Changes ◽

Cysteine Proteases ◽

Death Receptors ◽

Small Subunit ◽

Caspase 8 ◽

Death Domain ◽

Interacting Protein ◽

Death Effector ◽

Caspase 2

Apoptosis is a major form of cell death, characterized initially by a series of stereotypic morphological changes. In the nematode Caenorhabditis elegans, the gene ced-3 encodes a protein required for developmental cell death. Since the recognition that CED-3 has sequence identity with the mammalian cysteine protease interleukin-1β-converting enzyme (ICE), a family of at least 10 related cysteine proteases has been identified. These proteins are characterized by almost absolute specificity for aspartic acid in the P1 position. All the caspases (ICE-like proteases) contain a conserved QACXG (where X is R, Q or G) pentapeptide active-site motif. Caspases are synthesized as inactive proenzymes comprising an N-terminal peptide (prodomain) together with one large and one small subunit. The crystal structures of both caspase-1 and caspase-3 show that the active enzyme is a heterotetramer, containing two small and two large subunits. Activation of caspases during apoptosis results in the cleavage of critical cellular substrates, including poly(ADP-ribose) polymerase and lamins, so precipitating the dramatic morphological changes of apoptosis. Apoptosis induced by CD95 (Fas/APO-1) and tumour necrosis factor activates caspase-8 (MACH/FLICE/Mch5), which contains an N-terminus with FADD (Fas-associating protein with death domain)-like death effector domains, so providing a direct link between cell death receptors and the caspases. The importance of caspase prodomains in the regulation of apoptosis is further highlighted by the recognition of adapter molecules, such as RAIDD [receptor-interacting protein (RIP)-associated ICH-1/CED-3-homologous protein with a death domain]/CRADD (caspase and RIP adapter with death domain), which binds to the prodomain of caspase-2 and recruits it to the signalling complex. Cells undergoing apoptosis following triggering of death receptors execute the death programme by activating a hierarchy of caspases, with caspase-8 and possibly caspase-10 being at or near the apex of this apoptotic cascade.

Download Full-text

Active MLKL triggers the NLRP3 inflammasome in a cell-intrinsic manner

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613305114 ◽

2017 ◽

Vol 114 (6) ◽

pp. E961-E969 ◽

Cited By ~ 134

Author(s):

Stephanie A. Conos ◽

Kaiwen W. Chen ◽

Dominic De Nardo ◽

Hideki Hara ◽

Lachlan Whitehead ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Adaptor Protein ◽

Cell Lysis ◽

Kinase Domain ◽

Receptor Protein ◽

Interacting Protein ◽

Caspase 1 ◽

Proinflammatory Mediators ◽

Death Effector

Necroptosis is a physiological cell suicide mechanism initiated by receptor-interacting protein kinase-3 (RIPK3) phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which results in disruption of the plasma membrane. Necroptotic cell lysis, and resultant release of proinflammatory mediators, is thought to cause inflammation in necroptotic disease models. However, we previously showed that MLKL signaling can also promote inflammation by activating the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome to recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) and trigger caspase-1 processing of the proinflammatory cytokine IL-1β. Here, we provide evidence that MLKL-induced activation of NLRP3 requires (i) the death effector four-helical bundle of MLKL, (ii) oligomerization and association of MLKL with cellular membranes, and (iii) a reduction in intracellular potassium concentration. Although genetic or pharmacological targeting of NLRP3 or caspase-1 prevented MLKL-induced IL-1β secretion, they did not prevent necroptotic cell death. Gasdermin D (GSDMD), the pore-forming caspase-1 substrate required for efficient NLRP3-triggered pyroptosis and IL-1β release, was not essential for MLKL-dependent death or IL-1β secretion. Imaging of MLKL-dependent ASC speck formation demonstrated that necroptotic stimuli activate NLRP3 cell-intrinsically, indicating that MLKL-induced NLRP3 inflammasome formation and IL-1β cleavage occur before cell lysis. Furthermore, we show that necroptotic activation of NLRP3, but not necroptotic cell death alone, is necessary for the activation of NF-κB in healthy bystander cells. Collectively, these results demonstrate the potential importance of NLRP3 inflammasome activity as a driving force for inflammation in MLKL-dependent diseases.

Download Full-text

Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

Nature Communications ◽

10.1038/s41467-020-20806-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joanna L. Fox ◽

Michelle A. Hughes ◽

Xin Meng ◽

Nikola A. Sarnowska ◽

Ian R. Powley ◽

...

Keyword(s):

Cell Death ◽

Structural Analysis ◽

Cell Fate ◽

Catalytic Domain ◽

Caspase 8 ◽

Type I ◽

Signaling Complex ◽

Catalytic Domains ◽

Regulated Cell Death ◽

Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

Download Full-text

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707531114 ◽

2017 ◽

Vol 114 (36) ◽

pp. E7450-E7459 ◽

Cited By ~ 52

Author(s):

Shuzhen Liu ◽

Hua Liu ◽

Andrea Johnston ◽

Sarah Hanna-Addams ◽

Eduardo Reynoso ◽

...

Keyword(s):

Cell Death ◽

Disulfide Bond ◽

Kinase Domain ◽

Proteinase K ◽

Interacting Protein ◽

Tnf Α ◽

Mouse Cells ◽

Red Dye ◽

Necessary And Sufficient ◽

Human And Mouse

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α–induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Download Full-text

The lysosomal Rag-Ragulator complex licenses RIPK1– and caspase-8–mediated pyroptosis by Yersinia

Science ◽

10.1126/science.abg0269 ◽

2021 ◽

Vol 372 (6549) ◽

pp. eabg0269

Author(s):

Zengzhang Zheng ◽

Wanyan Deng ◽

Yang Bai ◽

Rui Miao ◽

Shenglin Mei ◽

...

Keyword(s):

Cell Death ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Host Cells ◽

Caspase 8 ◽

Death Domain ◽

Interacting Protein ◽

A Genome ◽

Infection Inhibition ◽

Guanosine Triphosphatase

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor–β–activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide CRISPR screen to uncover mediators of caspase-8–dependent pyroptosis identified an unexpected role of the lysosomal folliculin (FLCN)–folliculin-interacting protein 2 (FNIP2)–Rag-Ragulator supercomplex, which regulates metabolic signaling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, Fas-associated death domain (FADD), RIPK1, and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag guanosine triphosphatase activity and lysosomal tethering of Rag-Ragulator but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

Download Full-text

Genetic Regulation of RIPK1 and Necroptosis

Annual Review of Genetics ◽

10.1146/annurev-genet-071719-022748 ◽

2021 ◽

Vol 55 (1) ◽

pp. 235-263

Author(s):

Daichao Xu ◽

Chengyu Zou ◽

Junying Yuan

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Genetic Regulation ◽

Caspase 8 ◽

Inflammatory Signaling ◽

Interacting Protein ◽

Multiple Factors ◽

Cell Death Pathways ◽

Upstream Regulator ◽

Multiple Cell

The receptor-interacting protein kinase 1 (RIPK1) is recognized as a master upstream regulator that controls cell survival and inflammatory signaling as well as multiple cell death pathways, including apoptosis and necroptosis. The activation of RIPK1 kinase is extensively modulated by ubiquitination and phosphorylation, which are mediated by multiple factors that also control the activation of the NF-κB pathway. We discuss current findings regarding the genetic modulation of RIPK1 that controls its activation and interaction with downstream mediators, such as caspase-8 and RIPK3, to promote apoptosis and necroptosis. We also address genetic autoinflammatory human conditions that involve abnormal activation of RIPK1. Leveraging these new genetic and mechanistic insights, we postulate how an improved understanding of RIPK1 biology may support the development of therapeutics that target RIPK1 for the treatment of human inflammatory and neurodegenerative diseases.

Download Full-text

Necroptosis in Cholangiocarcinoma

Cells ◽

10.3390/cells9040982 ◽

2020 ◽

Vol 9 (4) ◽

pp. 982

Author(s):

Samantha Sarcognato ◽

Iris E. M. de Jong ◽

Luca Fabris ◽

Massimiliano Cadamuro ◽

Maria Guido

Keyword(s):

Cell Death ◽

Current Knowledge ◽

Kinase Domain ◽

Alternative Mode ◽

Interacting Protein ◽

Regulated Cell Death ◽

Anti Cancer ◽

Regulatory Complex ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns

Necroptosis is a type of regulated cell death that is increasingly being recognized as a relevant pathway in different pathological conditions. Necroptosis can occur in response to multiple stimuli, is triggered by the activation of death receptors, and is regulated by receptor-interacting protein kinases 1 and 3 and mixed-lineage kinase domain-like, which form a regulatory complex called the necrosome. Accumulating evidence suggests that necroptosis plays a complex role in cancer, which is likely context-dependent and can vary among different types of neoplasms. Necroptosis serves as an alternative mode of programmed cell death overcoming apoptosis and, as a pro-inflammatory death type, it may inhibit tumor progression by releasing damage-associated molecular patterns to elicit robust cross-priming of anti-tumor CD8+ T cells. The development of therapeutic strategies triggering necroptosis shows great potential for anti-cancer therapy. In this review, we summarize the current knowledge on necroptosis and its role in liver biliary neoplasms, underlying the potential of targeting necroptosis components for cancer treatment.

Download Full-text

Interaction with Sug1 enables Ipaf ubiquitination leading to caspase 8 activation and cell death

Biochemical Journal ◽

10.1042/bj20091349 ◽

2010 ◽

Vol 427 (1) ◽

pp. 91-104 ◽

Cited By ~ 26

Author(s):

Yatender Kumar ◽

Vegesna Radha ◽

Ghanshyam Swarup

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Intramolecular Interaction ◽

Dominant Negative ◽

26S Proteasome ◽

Caspase 8 ◽

Interacting Protein ◽

Caspase 1 ◽

Lung Carcinoma Cells ◽

Lrr Domain

Activation of initiator caspases is dependent on interacting proteins, and Ipaf [ICE (interleukin-1β-converting enzyme)-protease activating factor] {NLRC4 [NLR (Nod-like receptor) family CARD (caspase activation and recruitment domain)-containing 4]} an inflammasome component, is involved in caspase 1 activation and apoptosis. Investigating the mechanisms of Ipaf activation, we found that the C-terminal LRR (leucine-rich repeat) domain of Ipaf, through intramolecular interaction, negatively regulates its apoptosis-inducing function. In A549 lung carcinoma cells, expression of Ac-Ipaf (LRR-domain-deleted Ipaf) induced cell death that was dependent on caspase 8, but not on caspase 1. A yeast two-hybrid screen using Ac-Ipaf as bait identified human Sug1 (suppressor of gal 1), a component of the 26S proteasome, as an interacting protein. In mammalian cells Sug1 interacts and co-localizes with Ipaf. Sug1 binds to amino acids 91–253 of Ipaf, which is also the region that the LRR domain binds to. It potentiates cell death induced by Ipaf and Ac-Ipaf, and co-expression of Sug1 and Ipaf induces caspase-8-dependent cell death. Cellular complexes formed by Ipaf and Sug1 contain caspase 8. Expression of Ac-Ipaf or co-expression of Sug1 with Ipaf results in the formation of cytoplasmic aggregates and caspase 8 activation. Sug1 co-expression enabled modification of Ipaf by ubiquitination. Tagging ubiquitin molecules to Ipaf led to aggregate formation, enhanced caspase 8 interaction and activation, resulting in induction of cell death. Using RNAi (RNA interference) and dominant-negative approaches, we have shown that cell death induced by Ac-Ipaf expression or by treatment with TNF-α (tumour necrosis factor α) or doxorubicin is dependent on Sug1. Our results suggest a role for ubiquitination of Ipaf that is enabled by its interaction with Sug1, leading to caspase 8 activation and cell death.

Download Full-text

The novel cyclophilin-D-interacting protein FASTKD1 protects cells against oxidative stress-induced cell death

AJP Cell Physiology ◽

10.1152/ajpcell.00471.2018 ◽

2019 ◽

Vol 317 (3) ◽

pp. C584-C599

Author(s):

Kurt D. Marshall ◽

Paula J. Klutho ◽

Lihui Song ◽

Maike Krenz ◽

Christopher P. Baines

Keyword(s):

Oxidative Stress ◽

Cell Death ◽

Mitochondrial Permeability Transition ◽

Permeability Transition ◽

Kinase Domain ◽

Protective Role ◽

Mitochondrial Morphology ◽

Cyclophilin D ◽

Interacting Protein ◽

Mitochondrial Homeostasis

Opening of the mitochondrial permeability transition (MPT) pore leads to necrotic cell death. Excluding cyclophilin D (CypD), the makeup of the MPT pore remains conjecture. The purpose of these experiments was to identify novel MPT modulators by analyzing proteins that associate with CypD. We identified Fas-activated serine/threonine phosphoprotein kinase domain-containing protein 1 (FASTKD1) as a novel CypD interactor. Overexpression of FASTKD1 protected mouse embryonic fibroblasts (MEFs) against oxidative stress-induced reactive oxygen species (ROS) production and cell death, whereas depletion of FASTKD1 sensitized them. However, manipulation of FASTKD1 levels had no effect on MPT responsiveness, Ca2+-induced cell death, or antioxidant capacity. Moreover, elevated FASTKD1 levels still protected against oxidative stress in CypD-deficient MEFs. FASTKD1 overexpression decreased Complex-I-dependent respiration and ΔΨm in MEFs, effects that were abrogated in CypD-null cells. Additionally, overexpression of FASTKD1 in MEFs induced mitochondrial fragmentation independent of CypD, activation of Drp1, and inhibition of autophagy/mitophagy, whereas knockdown of FASTKD1 had the opposite effect. Manipulation of FASTKD1 expression also modified oxidative stress-induced caspase-3 cleavage yet did not alter apoptotic death. Finally, the effects of FASTKD1 overexpression on oxidative stress-induced cell death and mitochondrial morphology were recapitulated in cultured cardiac myocytes. Together, these data indicate that FASTKD1 supports mitochondrial homeostasis and plays a critical protective role against oxidant-induced death.

Download Full-text