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

2017 ◽  
Vol 114 (36) ◽  
pp. E7450-E7459 ◽  
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.

scholarly journals Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase domain-Like (MLKL) Interaction in Necroptotic Signaling

2013 ◽  
Vol 288 (23) ◽  
pp. 16247-16261 ◽  
Author(s):  
Wanze Chen ◽  
Zhenru Zhou ◽  
Lisheng Li ◽  
Chuan-Qi Zhong ◽  
Xinru Zheng ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Flanking Sequence ◽  
Interacting Protein ◽  
Mixed Lineage Kinase ◽  
Signaling Complex ◽  
Evolutionarily Conserved ◽  
Mouse Cells ◽  
Human And Mouse

Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.

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.

scholarly journals Necroptosis in Cholangiocarcinoma

Cells ◽  
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.

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

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.

scholarly journals A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis

2015 ◽  
Vol 112 (16) ◽  
pp. 5017-5022 ◽  
Author(s):  
Dianrong Li ◽  
Tao Xu ◽  
Yang Cao ◽  
Huayi Wang ◽  
Lin Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Heat Shock Protein 90 ◽  
Kinase Domain ◽  
Hsp90 Inhibitor ◽  
Activation Process ◽  
Interacting Protein ◽  
Tnf Α ◽  
Chemical Inhibitors ◽  
Core Components ◽  
Necrosis Factor

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

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

2017 ◽  
Vol 114 (13) ◽  
pp. E2786-E2795 ◽  
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.

scholarly journals Requirement for antiapoptotic MCL-1 in the survival of BCR-ABL B-lineage acute lymphoblastic leukemia

Blood ◽  
2013 ◽  
Vol 122 (9) ◽  
pp. 1587-1598 ◽  
Author(s):  
Brian Koss ◽  
Jeffrey Morrison ◽  
Rhonda M. Perciavalle ◽  
Harpreet Singh ◽  
Jerold E. Rehg ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Mouse Model ◽  
Lymphoblastic Leukemia ◽  
Genetic Mouse Model ◽  
Key Points ◽  
Mouse Cells ◽  
B Lineage ◽  
Human And Mouse ◽  
Mouse Genetic

Key Points Using a mouse genetic mouse model of Ph+ B-lineage ALL, endogenous antiapoptotic MCL-1 is required for leukemia survival. In BCR-ABL+ B-lineage ALL human and mouse cells, combining TKIs with small-molecule inhibitors of BCL-2 can potentiate sensitivity to cell death.

scholarly journals Necroptosis in Intestinal Inflammation and Cancer: New Concepts and Therapeutic Perspectives

2020 ◽  
Author(s):  
Anna Negroni ◽  
Eleonora Colantoni ◽  
Salvatore Cucchiara ◽  
Laura Stronati
Keyword(s):  
Cell Death ◽  
Intestinal Epithelium ◽  
Inflammatory Cell ◽  
Intestinal Inflammation ◽  
Inflammatory Diseases ◽  
Kinase Domain ◽  
Interacting Protein ◽  
New Concepts ◽  
Independent Form ◽  
Cellular Turnover

Necroptosis is a caspases-independent form of programmed cell death exhibiting intermediate features between necrosis and apoptosis. Albeit some physiological roles during embryonic development, tissue homeostasis and innate immune response are documented, necroptosis is mainly considered a pro-inflammatory cell death. Key actors of necroptosis are the receptor-interacting-protein-kinases, RIPK1 and RIPK3, and their target, the mixed-lineage-kinase-domain-like protein, MLKL. The intestinal epithelium has one of the highest rates of cellular turnover in a process that is tightly regulated. Altered necroptosis at the intestinal epithelium leads to uncontrolled microbial translocation and deleterious inflammation. Indeed, necroptosis has been associated to chronic inflammatory diseases and cancer. Drugs that inhibit necroptosis could, therefore, be used therapeutically for the treatment of these diseases, and researches to develop such inhibitors are already underway. In this Review, we outline pathways for necroptosis and its role in chronic inflammation and cancer. We also discuss current and developing therapies that target necroptosis machinery.

scholarly journals Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding

2018 ◽  
Vol 26 (3) ◽  
pp. 503-513 ◽  
Author(s):  
Sanae Haga ◽  
Akira Kanno ◽  
Takeaki Ozawa ◽  
Naoki Morita ◽  
Mami Asano ◽  
...  
Keyword(s):  
Cell Death ◽  
Specific Inhibitor ◽  
Apoptotic Pathway ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Hepatocyte Injury ◽  
Caspase Inhibition ◽  
Regulated Necrosis ◽  
Tnf Α ◽  
Optic Probe

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-α/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

scholarly journals Kavain inhibition of LPS-induced TNF-α via ERK/LITAF

2016 ◽  
Vol 5 (1) ◽  
pp. 188-196 ◽  
Author(s):  
Xiaoren Tang ◽  
Salomon Amar
Keyword(s):  
Tnf Alpha ◽  
Piper Methysticum ◽  
Tnf Α ◽  
Alpha Factor ◽  
Mouse Cells ◽  
Human And Mouse

Kavain, an extract from the shrub Piper methysticum, was recently reported to modulate TNF-α expression in both human and mouse cells via regulation of LPS-Induced TNF-Alpha Factor (LITAF).

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