scholarly journals Heterologous expression of human pro-inflammatory Caspase-1 in Saccharomyces cerevisiae and comparison to pro-apoptotic Caspase-8

2021 ◽  
Author(s):  
Marta Valenti ◽  
María Molina ◽  
Víctor J Cid
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Heterologous Expression ◽  
Cysteine Proteases ◽  
Intrinsic Activity ◽  
Caspase 8 ◽  
Caspase 1 ◽  
Death Effector ◽  
The Impact ◽  
Yeast Model

AbstractCaspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human Caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of Caspase-8. Overexpression of both caspases in the heterologous model led to their activation, and caused mitochondrial depolarization, ROS production, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and Caspase-8 was more aggressive than Caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which Caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of Caspase-1 enhanced its activity, while the deletion of the death effector domain (DED) of Caspase-8 diminished it. We propose the yeast model as a useful and manageable tool to explore Caspase-1 structure-function relationships, the impact of mutations or the activity of putative inhibitors or regulators.

scholarly journals Heterologous Expression and Auto-Activation of Human Pro-Inflammatory Caspase-1 in Saccharomyces cerevisiae and Comparison to Caspase-8

2021 ◽  
Vol 12 ◽  
Author(s):  
Marta Valenti ◽  
María Molina ◽  
Víctor J. Cid
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Heterologous Expression ◽  
Cysteine Proteases ◽  
Intrinsic Activity ◽  
Caspase 8 ◽  
Caspase 1 ◽  
Death Effector ◽  
Human Caspase

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure–function relationships.

scholarly journals Caspases: the executioners of apoptosis

1997 ◽  
Vol 326 (1) ◽  
pp. 1-16 ◽  
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.

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

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.

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

2018 ◽  
Vol 11 (546) ◽  
pp. eaao1716 ◽  
Author(s):  
Akshay A. D’Cruz ◽  
Mary Speir ◽  
Meghan Bliss-Moreau ◽  
Sylvia Dietrich ◽  
Shu Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Kinase Domain ◽  
Neutrophil Extracellular Trap ◽  
Human Neutrophils ◽  
Caspase 8 ◽  
Interacting Protein ◽  
Dependent Activity ◽  
Mrsa Infection ◽  
Death Effector ◽  
Net Release

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.

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

2010 ◽  
Vol 427 (1) ◽  
pp. 91-104 ◽  
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.

scholarly journals Pyroptotic Neuronal Cell Death Mediated by the AIM2 Inflammasome

2014 ◽  
Vol 34 (4) ◽  
pp. 621-629 ◽  
Author(s):  
Stephanie E Adamczak ◽  
Juan Pablo de Rivero Vaccari ◽  
Gordon Dale ◽  
Frank J Brand ◽  
Doris Nonner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cortical Neurons ◽  
Interleukin 1 ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Cns Infection ◽  
Cell Death Mechanism ◽  
Caspase 1 ◽  
Brilliant Blue Fcf ◽  
Death Effector

The central nervous system (CNS) is an active participant in the innate immune response to infection and injury. In these studies, we show embryonic cortical neurons express a functional, deoxyribonucleic acid (DNA)-responsive, absent in melanoma 2 (AIM2) inflammasome that activates caspase-1. Neurons undergo pyroptosis, a proinflammatory cell death mechanism characterized by the following: (a) oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC); (b) caspase-1 dependency; (c) formation of discrete pores in the plasma membrane; and (d) release of the inflammatory cytokine interleukin-1 β (IL-1 β). Probenecid and Brilliant Blue FCF, inhibitors of the pannexin1 channel, prevent AIM2 inflammasome-mediated cell death, identifying pannexin1 as a cell death effector during pyroptosis and probenecid as a novel pyroptosis inhibitor. Furthermore, we show activation of the AIM2 inflammasome in neurons by cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients and oligomerization of ASC. These findings suggest neuronal pyroptosis is an important cell death mechanism during CNS infection and injury that may be attenuated by probenecid.

scholarly journals Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients

2021 ◽  
Author(s):  
Tiziano A Schweizer ◽  
Srikanth Mairpady Shambat ◽  
Clement Vulin ◽  
Sylvia Hoeller ◽  
Claudio Acevedo ◽  
...  
Keyword(s):  
Cell Death ◽  
Disease Severity ◽  
Critically Ill ◽  
Cytokine Profile ◽  
Caspase 8 ◽  
Potential Therapeutic Target ◽  
Regulated Cell Death ◽  
Cell Death Program ◽  
Fas Signaling ◽  
The Impact

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

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

2017 ◽  
Vol 114 (6) ◽  
pp. E961-E969 ◽  
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.

scholarly journals The Impact of Autophagy on Cell Death Modalities

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Stefan W. Ryter ◽  
Kenji Mizumura ◽  
Augustine M. K. Choi
Keyword(s):  
Cell Death ◽  
Inflammatory Responses ◽  
Degradation Pathway ◽  
Therapeutic Interventions ◽  
Caspase 1 ◽  
Regulated Cell Death ◽  
Caspase Inhibition ◽  
Autophagic Degradation ◽  
Dependent Cell ◽  
The Impact

Autophagy represents a homeostatic cellular mechanism for the turnover of organelles and proteins, through a lysosome-dependent degradation pathway. During starvation, autophagy facilitates cell survival through the recycling of metabolic precursors. Additionally, autophagy can modulate other vital processes such as programmed cell death (e.g., apoptosis), inflammation, and adaptive immune mechanisms and thereby influence disease pathogenesis. Selective pathways can target distinct cargoes (e.g., mitochondria and proteins) for autophagic degradation. At present, the causal relationship between autophagy and various forms of regulated or nonregulated cell death remains unclear. Autophagy can occur in association with necrosis-like cell death triggered by caspase inhibition. Autophagy and apoptosis have been shown to be coincident or antagonistic, depending on experimental context, and share cross-talk between signal transduction elements. Autophagy may modulate the outcome of other regulated forms of cell death such as necroptosis. Recent advances suggest that autophagy can dampen inflammatory responses, including inflammasome-dependent caspase-1 activation and maturation of proinflammatory cytokines. Autophagy may also act as regulator of caspase-1 dependent cell death (pyroptosis). Strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases in which apoptosis or other forms of regulated cell death may play a cardinal role.

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