RIPK1-mediated regulated necrosis (Homo sapiens)
From WikiPathways
Description
Receptor-interacting serine/threonine-kinase protein 1 (RIPK1) and RIPK3-dependent necrosis is called necroptosis or programmed necrosis. The kinase activities of RIPK1 and RIPK3 are essential for the necroptotic cell death in human, mouse cell lines and genetic mice models (Cho YS et al. 2009; He S et al. 2009, 2011; Zhang DW et al. 2009; McQuade T et al. 2013; Newton et al. 2014). The initiation of necroptosis can be stimulated by the same death ligands that activate extrinsic apoptotic signaling pathway, such as tumor necrosis factor (TNF) alpha, Fas ligand (FasL), and TRAIL (TNF-related apoptosis-inducing ligand) or toll like receptors 3 and 4 ligands (Holler N et al. 2000; He S et al. 2009; Feoktistova M et al. 2011; Voigt S et al. 2014). In contrast to apoptosis, necroptosis represents a form of cell death that is optimally induced when caspases are inhibited (Holler N et al. 2000; Hopkins-Donaldson S et al. 2000; Sawai H 2014). Specific inhibitors of caspase-independent necrosis, necrostatins, have recently been identified (Degterev A et al. 2005, 2008). Necrostatins have been shown to inhibit the kinase activity of RIPK1 (Degterev A et al. 2008). Importantly, cell death of apoptotic morphology can be shifted to a necrotic phenotype when caspase 8 activity is compromised, otherwise active caspase 8 blocks necroptosis by the proteolytic cleavage of RIPK1 and RIPK3 (Kalai M et al. 2002; Degterev A et al. 2008; Lin Y et al. 1999; Feng S et al. 2007). When caspase activity is inhibited under certain pathophysiological conditions or by pharmacological agents, deubiquitinated RIPK1 is engaged in physical and functional interactions with the cognate kinase RIPK3 leading to formation of necrosome, a necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; Cho YS et al. 2009, He S et al. 2009, Zhang DW et al. 2009). Within the necrosome RIPK1 and RIPK3 bind to each other through their RIP homotypic interaction motif (RHIM) domains. The RHIMs can facilitate RIPK1:RIPK3 oligomerization, allowing them to form amyloid-like fibrillar structures (Li J et al. 2012; Mompean M et al. 2018). RIPK3 in turn interacts with mixed lineage kinase domain-like protein (MLKL) (Sun L et al. 2012; Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). Mouse MLKL activation relies on transient engagement of RIPK3 to facilitate phosphorylation of the pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2019a), while it appears that stable recruitment of human MLKL by necrosomal RIPK3 is an additional crucial step in human MLKL activation (Davies KA et al. 2018; Petrie EJ et al. 2018, 2019b). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive. The precise oligomeric form of MLKL that mediates plasma membrane disruption has been highly debated (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2017, 2018; Samson AL et al. 2020 ). However, microscopy data revealed that MLKL assembles into higher molecular weight species upon cytoplasmic necrosomes within human cells, and upon phosphorylation by RIPK3, MLKL is trafficked to the plasma membrane (Samson AL et al. 2020). At the plasma membrane, phospho-MLKL forms heterogeneous higher order assemblies, which are thought to permeabilize cells, leading to release of DAMPs to invoke inflammatory responses. While RIPK1, RIPK3 and MLKL are the core signaling components in the necroptosis pathway, many additional molecules have been proposed to positively and negatively tune the signaling pathway. Currently, this picture is evolving rapidly as new modulators continue to be discovered.
The Reactome module describes MLKL-mediated necroptotic events on the plasma membrane. View original pathway at Reactome.</div>
Try the New WikiPathways
View approved pathways at the new wikipathways.org.Quality Tags
Ontology Terms
Bibliography
History
External references
DataNodes
The two principal pathways of apoptosis are (1) the Bcl-2 inhibitable or intrinsic pathway induced by various forms of stress like intracellular damage, developmental cues, and external stimuli and (2) the caspase 8/10 dependent or extrinsic pathway initiated by the engagement of death receptors
The caspase 8/10 dependent or extrinsic pathway is a death receptor mediated mechanism that results in the activation of caspase-8 and caspase-10. Activation of death receptors like Fas/CD95, TNFR1, and the TRAIL receptor is promoted by the TNF family of ligands including FASL (APO1L OR CD95L), TNF, LT-alpha, LT-beta, CD40L, LIGHT, RANKL, BLYS/BAFF, and APO2L/TRAIL. These ligands are released in response to microbial infection, or as part of the cellular, humoral immunity responses during the formation of lymphoid organs, activation of dendritic cells, stimulation or survival of T, B, and natural killer (NK) cells, cytotoxic response to viral infection or oncogenic transformation.
The Bcl-2 inhibitable or intrinsic pathway of apoptosis is a stress-inducible process, and acts through the activation of caspase-9 via Apaf-1 and cytochrome c. The rupture of the mitochondrial membrane, a rapid process involving some of the Bcl-2 family proteins, releases these molecules into the cytoplasm. Examples of cellular processes that may induce the intrinsic pathway in response to various damage signals include: auto reactivity in lymphocytes, cytokine deprivation, calcium flux or cellular damage by cytotoxic drugs like taxol, deprivation of nutrients like glucose and growth factors like EGF, anoikis, transactivation of target genes by tumor suppressors including p53.
In many non-immune cells, death signals initiated by the extrinsic pathway are amplified by connections to the intrinsic pathway. The connecting link appears to be the truncated BID (tBID) protein a proteolytic cleavage product mediated by caspase-8 or other enzymes.
Influenza viruses belong to the family of Orthomyxoviridae; viruses with segmented RNA genomes that are negative sense and single-stranded (Baltimore 1971). Influenza virus strains are named according to their type (A, B, or C), the species from which the virus was isolated (omitted if human), location of isolate, the number of the isolate, the year of isolation, and in the case of influenza A viruses, the hemagglutinin (H) and neuraminidase (N) subtype. For example, the virus of H5N1 subtype isolated from chickens in Hong Kong in 1997 is: influenza A/chicken/Hong Kong/220/97(H5N1) virus. Currently 16 different hemagglutinin (H1 to H16) subtypes and 9 different neuraminidase (N1 to N9) subtypes are known for influenza A viruses. Most human disease is due to influenza viruses of the A type. The events of influenza infection have been annotated in Reactome primarily use protein and genome references to the Influenza A virus A/Puerto Rico/8/1934 H1N1 strain.
The influenza virus particle initially associates with a human host cell by binding to sialic acid receptors on the host cell surface. Sialic acids are found on many vertebrate cells and numerous viruses make use of this ubiquitous receptor. The bound virus is endocytosed by one of four distinct mechanisms. Once endocytosed the low endosomal pH sets in motion a number of steps that lead to viral membrane fusion mediated by the viral hemagglutinin (HA) protein, and the eventual release of the uncoated viral ribonucleoprotein complex into the cytosol of the host cell. The ribonucleoprotein complex is transported through the nuclear pore into the nucleus. Once in the nucleus, the incoming negative-sense viral RNA (vRNA) is transcribed into messenger RNA (mRNA) by a primer-dependent mechanism. Replication occurs via a two step process. A full-length complementary RNA (cRNA), a positive-sense copy of the vRNA, is first made and this in turn is used as a template to produce more vRNA. The viral proteins are expressed and processed and eventually assemble with vRNAs at what will become the budding sites on the host cell membrane. The viral protein and ribonucleoprotein complexes are assembled into complete viral particles and bud from the host cell, enveloped in the host cell's membrane.Infection of a human host cell with influenza virus triggers an array of defensive host processes. This coevolution has driven the development of host processes that interfere with viral replication, notably the production of type I interferon. At the some time the virus counters these responses with the viral NS1 protein playing a central role in the viral response to the host cells defense.
3a:(RIPK1:RIPK3)
oligomercaspase-8:viral
CRMA/SPI-2inhibitors of
RIPK1, RIPK3Annotated Interactions
FLIP(S) is a short-lived protein which is sensitive to ubiquitination and proteasomal degradation (Poukkula M et al. 2005). Protein kinase C (PKC)- mediated phosphorylation of FLIP(S) at Ser193 was shown to prolongs the half-life of FLIP(S) by inhibiting its polyubiquitination (Kaunisto A et al. 2009).
This Reactome event shows RHIM-dependent interaction of RIPK1 and RIPK3.
Reconstitution of RIPK1-deficient human Jurkat cells with mutated kinase-inactive RIPK1 or RIPK1 lacking the N-terminal serine/threonine kinase domain did not trigger FASL-induced necrotic cell death (Holler N et al. 2000). Similarly, mutations in the kinase domain and RIP homotypic interaction motif (RHIM) of RIPK1 also abolished the RIPK1-mediated rescue of tumor necrosis factor (TNF)/zVAD-fmk-induced regulated necrosis in RIPK1-deficient Jurkat cells (Cho YS et al. 2009). Furthermore, the results of structural and mutagenesis studies using necrostatins, which inhibit RIPK1 kinase activity by targeting the kinase domain, revealed that the N-terminal kinase domain of RIPK1 is required for propagating the pronecrotic signal (Degterev A et al. 2008; Cho YS et al. 2009; Xie T et al. 2013). Mass spectroscopy showed that human RIPK1 is phosphorylated within the kinase domain at multiple serine residues, such as Ser14/15, Ser20, Ser161 and Ser166, suggesting that the phosphorylation might regulate RIPK1 kinase activity (Degterev A et al. 2008). Using in vitro cellular systems, two independent studies reported that alanine substitution at Ser161 (S161A) leads to a reduction in RIPK1 kinase activity (Degterev A et al. 2008; McQuade T et al. 2013). RIPK1 autophosphorylation at Ser166 was found to modulate RIPK1 kinase activation (Laurien L et al. 2020). Studies with Ripk1 S166A/S166A knock-in mice revealed that abolishing phosphorylation at S166 prevented the development of RIPK1-mediated inflammatory conditions in vivo in four relevant mouse models of inflammation. Further, abolishing phosphorylation at S166 considerably inhibited RIPK1 kinase activity-dependent cell death downstream of tumor necrosis factor receptor 1 (TNFR1), toll-like receptor 3 (TLR3) and TLR4 in mouse cells isolated from Ripk1 S166A/S166A mice (Laurien L et al. 2020). Phosphorylation of S166 RIPK1 has been established as a biomarker of RIPK1 target engagement (Degterev A et al. 2008; Ofengeim D et al. 2015). The biological role of phosphorylation of individual serine residues in the kinase domain of RIPK1 remains to be further characterized (McQuade T et al. 2013).
RIPK1 is subjected to complex phosphorylation including several events possibly mediated by other kinases such as MAPK-activated protein kinase 2 (MK2) (Dondelinger Y et al. 2016; Jaco I et al. 2017; Delanghe T et al. 2020). S320 and S335 on human RIPK1 (S321 and S336 in mouse RIPK1) were identified as MK2 phosphorylation sites (Jaco I et al. 2017; Menon NB et al. 2017; Dondelinger Y et al. 2017). Transforming growth factor β-activated kinase 1 (TAK1) was also shown to phosphorylate RIPK1 along with TANK binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε) to prevent TNF-induced necroptosis or to dictate the multiple cell death pathways in mammalian cells (Lafont E et al. 2018; Xu D et al. 2018). In addition, IKKα/IKKβ is also able to phosphorylate RIPK1 in order to block RIPK1-dependent cell death in mouse models of infection and inflammation (Dondelinger Y et al. 2015, 2019). RIPK3 might also regulate RIPK1 phosphorylation in mammalian cells. For instance, RIPK3 was shown to directly phosphorylate RIPK1 when kinase-dead RIPK1 and RIPK3 were co-expressed in human embryonic kidney HEK293 cells, immunoprecipitated, and subjected to an in vitro kinase assay (Sun X et al. 2002; Cho et al. 2009). Importantly, mutation within RHIM motif of RIPK3 abrogated RIPK1 phosphorylation by RIPK3, suggesting that RIPK1 phosphorylation by RIPK3 is dependent on the formation of the RIPK1:RIPK3 complex (Sun X et al. 2002).
Several FDA-approved anticancer drugs, including sorafenib, pazopanib and ponatinib showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). RIPK1 has been identified as the main functional target of pazopanib, while sorafenib and ponatinib directly targeted both RIPK1 and RIPK3 (Fauster A et al. 2015; Najjar M et al. 2015; Martens S et al. 2017).
FDA-approved anticancer drugs, including sorafenib and ponatinib, showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). These compounds are tyrosine kinase inhibitors (TKI) that directly targeted RIPK3 and RIPK1 and blocked their kinase activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). Pazopanib, another multi-targeting TKI, was shown to suppress necroptosis preferentially by targeting RIPK1 (Fauster A et al. 2015).
MLKL is composed of an amino-terminal four-helix bundle (4HB) domain, a two-helix “brace� region, and a carboxy-terminal pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2018). The 4HB domain functions as the executioner domain by virtue of its membrane permeabilization activity (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Hildebrand JM et al. 2014; Su L et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016). The 4HB domain enables membrane translocation of MLKL and is responsible for the plasma membrane permeabilization that characterizes necroptotic cell death (Chen X et al. 2014; Cai Z et al. 2014; Dondelinger Y. et al. 2014; Hildebrand JM et al. 2014; Petrie EJ et al. 2020). The 4HB domain executioner function is regulated by the C-terminal pseudokinase domain, which serves as a receiver for upstream signals, such as activation loop phosphorylation by RIPK3 (Hildebrand JM et al. 2014; Sun L et al. 2012; Rodriguez DA et al. 2016; Petrie EJ et al. 2018). Studies using mouse:human MLKL chimeras showed that the first brace helix and the adjacent loop (that connect the 4HB to the pseudokinase domain) of MLKL mediate interdomain communication and oligomerisation upon RIPK3-mediated activation of MLKL (Davies KA et al. 2018). RIPK3-mediated phosphorylation is thought to trigger a conformational change within the pseudokinase of MLKL that promotes 4HB domain exposure, enabling MLKL to form oligomers, which are trafficked to the plasma membrane where cell permeabilization occurs (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Even though the Reactome annotation shows that 4 molecules of MLKL bind to the RIPK1:RIPK3 oligomer, the exact stoichiometry of the binding and the oligomerization of MLKL has been highly debated (Chen X et al. 2014; Cai Z et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; Petrie EJ 2017). While trimers, tetramers, hexamers were reported in studies with the recombinant MLKL protein, single-cell imaging approaches revealed that endogenous human phosphorylated MLKL assembles into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive.
Even though the stoichiometry of the MLKL oligomerization in the Reactome event depicts MLKL homotetramer, the endogenous MLKL was shown to assemble on necrosomes into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020).
Even in the Cai NCB 2013 paper this was not true of all cell types tested, despite this being written into folklore.
In terms of “pores� - this is a term that implies some sort of ordered structure. I prefer membrane perturbations or disruption as a term. If you look at Samson Nat Comm 2020, you can see these membrane structures are irregular in size and form. They also argue against channels as the destination for MLKL. Instead pMLKL coalesces with tight junction proteins in HT29 cells, although whether this is because of the membrane topology at this site or because of other accessory proteins being localized to these junctions, remains unknown.
Also Zargarian Plos Biol 2017. However, the idea of necroptotic bubbles was challenged in Samson Nat Comm 2020 because the bubbles could come and go at sites pMLKL did not accumulate and where membrane damage (“blowout�) did not occur
Various studies showed that the endosomal sorting complexes required for transport (ESCRT) pathway can remove phosphorylated MLKL-containing membrane vesicles from cells undergoing necroptosis, thereby attenuating the cell death process (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). The ESCRT-associated proteins, programmed cell death 6-interacting protein (PDCD6IP or ALG-2-interacting protein X, ALIX) and syntenin-1 (SDCBP), were found to antagonize MLKL-mediated plasma membrane alteration (Fan W et al. 2019). In addition, flotillin-mediated endocytosis was proposed to suppress necroptosis by removing MLKL from the plasma membrane and redirecting it for lysosomal degradation (Fan W et al. 2019).
Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020).
Based on studies showing that the 4HB domain can permeabilize membranes in vitro (Dondelinger et al. 2014; Su et al. 2014; Wang et al. 2014; Tanzer et al. 2016; Petrie et al. 2018), it is thought that MLKL kills cells via direct action on the plasma membrane.
During tumor necrosis factor (TNF)-induced necroptosis, RIPK3 and RIPK1 associate with each other through their RHIM domains into heteromeric RIPK1:RIPK3 complexes that further polymerize into filamentous β-amyloid structures promoting the activation of RIPK3 kinase (Cho Y et al. 2009; Li J et al. 2012). Functionally active RIPK3 activates mixed-lineage kinase domain-like pseudokinase (MLKL), the membrane-disrupting effector of programmed necrosis (Sun L et al. 2012; Murphy JM et al. 2013; Wang H et al. 2014). Other RHIM-containing proteins, such as the TLR3/TLR4 adaptor TRIF (also known as TICAM1) and the DNA sensor DAI/ZBP can form the necroptotic signaling platforms to support activation of RIPK3 and its interaction with MLKL (Kaiser W et al. 2013; Lin J et al. 2016).
This Reactome event shows a scaffolding role of RIPK3 bound to RIPK1 in supporting the formation of SARS-CoV-1 3a oligomers.
MEFs lacking PELI1 have increased RIPK3 and MLKL phosphorylation, and necroptosis in response to necroptotic stimuli. Lung tissues from PELI1 transgenic mice showed a decrease in basal MLKL phosphorylation indicating that upregulated PELI1 may function to preferentially remove activated RIPK3 and reduce MLKL phosphorylation in vivo. In addition, PELI1 reduced endogenous RIPK3 in human lung adenocarcinoma (H2009) and human B lymphoblastoid (Raji) cell lines; conversely, knockdown of PELI1 in HT-29 and RIPK3 (ectopic)-expressing HeLa cell lines led to increased RIPK3 protein without affecting RIPK3 mRNA expression. Proteasome inhibitors (MG132 and BTZ), but not by lysosome inhibitors, prevented PELI1-facilitated degradation of stably expressed RIPK3 in HeLa cells and endogenous RIPK3 in HT-29 cells. In keratinocytes from toxic epidermal necrolysis (TEN) patients, PELI1 expression is low and inversely correlated with RIP3 protein, suggesting that reduction in PELI1 leads to upregulated RIP3 expression, thus contributing to disease progression. Thus, PELI1 is thought to control RIPK3 protein destabilization via ubiquitylation-dependent proteasome-mediated degradation (Choi SW et al. 2018).
Receptor-interacting serine/threonine protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. STUB1, as an E3 ligase, mediates ubiquitylation of RIPK3 at Lys55 and Lys363 and targets it to lysosomal degradation in human non-small cell lung carcinoma (H1299) cells (Seo J et al. 2016). Treatment with geldanamycin (an inhibitor of HSP90) induced the degradation of RIPK3 in mouse fibroblasts L929 cells even under STUB1-depleted conditions, suggesting that HSP90 might not be involved in the STUB1-mediated degradation of RIPK3 (Seo J et al. 2016). Further, RIP3 kinase activity was not required for its interaction with STUB1 in human embryonic kidney 293 (HEK293) cells, suggesting that STUB1-mediated regulation is independent of RIPK3 phosphorylation status (Choi SW et al. 2018). Moreover, Chip(-/-) mouse embryonic fibroblasts, CHIP-depleted L929 and human colorectal adenocarcinoma (HT-29) cells exhibited higher levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNFα (Seo J et al. 2016). Supporting these findings, in vivo studies demonstrated that the inflammatory and lethal phenotypes of Chip−/− mice were rescued by crossing with Ripk3 knockout mice (Seo J et al. 2016). The ubiquitin E3 ligase function of STUB1 was also essential for the degradation of RIPK3 in mouse neuroblastoma N2a cell line. Ansiomycin, an inhibitor of protein synthesis, attenuated necroptosis by upregulating STUB1 in oxygen-glucose deprivation (OGD)-challenged N2a cells and primary cultured mouse hippocampal neurons (Tang MB et al. 2018). These data suggest that STUB1 (CHIP) can negatively regulate necroptosis by ubiquitylation-mediated degradation of RIPK3.
SDCBP (syntenin-1) interacts directly with PDCD6IP (ALIX) through three LYPX(n)L motifs located in its N-terminus and with the conserved cytoplasmic domains of the syndecans, via its PDZ domains (Baietti MF et al. 2012). Syndecans are a family of proteins that by virtue of their extracellular heparan sulfate chains interact with a plethora of signaling and adhesion molecules (Sarrazin S et al. 2011). Since PDCD6IP binds several ESCRT proteins, PDCD6IP:SDCBP adapts syndecans and syndecan cargo to the ESCRT budding machinery, playing a role in membrane budding and scission at the endosome and generating intraluminal vesicles (ILVs) that are released as exosomes when multivesicular endosomes fuse with the plasma membrane (Baietti MF et al. 2012 ). Exocytosis was proposed to counteract the effector phase of necroptosis via ESCRT-, PDCD6IP:SDCBP- or RAB27A/B-mediated expulsion of MLKL-containing bubbles to diminish the MLKL residing at the plasma membrane (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). However, the other study suggests that membrane bubbling during necroptosis is an incredibly dynamic and heterogenous phenomenon with protrusions variously extending, retracting and shedding in a fashion seemingly independent of the primary sites of MLKL accumulation and membrane damage (Samson AL et al. 2020).
3a:(RIPK1:RIPK3)
oligomer3a:(RIPK1:RIPK3)
oligomercaspase-8:viral
CRMA/SPI-2inhibitors of
RIPK1, RIPK3