Necroptosis (RHIM-dependent RIPK3-MLKL lytic cell death) module

A decomposition of the necroptotic process (GO:0070266): the caspase-8-restrained, RIPK3- and MLKL-dependent form of programmed necrosis. The module follows GO's own partition of the process into a signaling phase (GO:0097527 necroptotic signaling pathway), the assembly of the RIPK1/RIPK3 RHIM platform that licenses the effector kinase (GO:1901026 ripoptosome assembly involved in necroptotic process; complex GO:0097342 ripoptosome), and an execution phase (GO:0097528) carried out by MLKL at the plasma membrane. Design intent: the committing chemistry is a single linear hand-off - an upstream RHIM-bearing sensor or adaptor nucleates a RHIM amyloid core with RIPK3, activated RIPK3 phosphorylates MLKL, and phosphorylated MLKL destabilizes the membrane - so the module is one ordered chain of parts rather than the redundant-suppressor topology used for ferroptosis. The branch point that makes necroptosis a distinct program sits upstream: three alternative initiators converge on the same RIPK3 core and are modelled as a variant set along the axis of which receptor supplies the RHIM (RIPK1 downstream of TNFR1 and other death receptors; TICAM1/TRIF downstream of TLR3 and endosomal TLR4; ZBP1 downstream of Z-form nucleic acid sensing). The caspase-8/FADD/cFLIP brake is modelled as an optional regulatory part that negatively regulates the chain, not as a core step: its proteolysis is what normally keeps the module off, and the same complex is the apoptotic alternative.

MODULE:necroptosisDRAFTCONCRETEBiological Processmodules/necroptosis.yaml

necroptotic processGO:0070266

GO:0070266

necroptotic process

GO defines the necroptotic process as a programmed necrotic cell death characterized by activation of RIPK1 and/or RIPK3 and critical dependence on MLKL, divided into a signaling phase and an execution phase triggered by the former. That division is the top-level decomposition used here.

GO:0097342

ripoptosome

GO's ripoptosome term is defined as a complex whose core components are RIPK1 and RIPK3 and whose formation can induce either extrinsic apoptotic or necroptotic signaling; it carries "necrosome" as a related synonym. This module uses it as the complex term for the RHIM platform (see notes on the ripoptosome/necrosome naming).

PMID:22265413

Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.

Identified MLKL as the RIPK3 substrate and necroptotic effector, fixing the RIPK3 -> MLKL hand-off that separates the signaling phase from the execution phase in this module.

MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis.

PMID:22817896

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

Established that the RHIMs of RIPK1 and RIPK3 assemble a filamentous (amyloid-like) complex and that RHIM mutation abolishes kinase activation and necrosis, which is the basis for modelling RHIM assembly as its own part.

Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo.

PMID:24019532

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.

Showed that TLR3/TLR4 engage RIPK3 directly through a RHIM-dependent TRIF-RIPK3 association, independent of RIPK1, while remaining MLKL-dependent - the evidence for treating the upstream initiator as a variant set over one shared RIPK3-MLKL core.

TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3).

PANTHER:PTN000585372

PAINT node PTN000585372 for RIPK1

Local PAINT IBD row in PTHR44329 propagating P:GO:0097527 necroptotic signaling pathway, seeded by MGI:MGI:108212 and UniProtKB:Q13546; cited as provenance that the RIPK1 necroptotic-signaling role is a PAINT-placed inherited function, not a human-only observation.

PANTHER:PTN008689042

PAINT node PTN008689042 for MLKL

Local PAINT IBD row in PTHR44329 propagating P:GO:0097527 necroptotic signaling pathway, seeded by MGI:MGI:1921818 and UniProtKB:Q8NB16; cited as provenance for the MLKL role being an inherited rather than lineage-specific function.

gomodel:62b4ffe300000504

Poxvirus-OPG065 inhibition of ZBP1-mediated necroptosis (Human-Poxvirus)

Cached production GO-CAM grounding the ZBP1 -> RIPK3 (GO:0097527) -> MLKL (GO:0097528) chain in human cells, used as the GO-CAM anchor for the ZBP1 initiation variant, the RIPK3 licensing step, and the MLKL execution node.

file:genes/human/MLKL/MLKL-ai-review.yaml

Human MLKL AI gene review

The reviewed MLKL core functions - membrane destabilizing activity (GO:0140912), phosphatidylinositol-4,5-bisphosphate binding (GO:0005546) as membrane targeting, and inositol hexakisphosphate binding (GO:0000822) as activation, all in the execution phase - are the source of the three execution-phase annotons here. That review deliberately rejects a pore-forming activity term for MLKL; this module follows it.

file:genes/human/RIPK3/RIPK3-ai-review.yaml

Human RIPK3 AI gene review

The reviewed RIPK3 core functions separate the kinase activity that phosphorylates MLKL (GO:0004674) from the RHIM scaffold activity (GO:0120283, with amyloid fibril formation GO:1990000); this module keeps that separation as two parts.

file:genes/human/RIPK1/RIPK1-ai-review.yaml

Human RIPK1 AI gene review

The reviewed RIPK1 core functions distinguish a kinase-dependent pro-necroptotic role (GO:0004674 in GO:0097527/GO:0070266/GO:1901026) from a kinase-independent scaffold role that restrains necroptosis; only the former is a core part of this module.

Boundary decisions. (1) Scope is the necroptotic process itself (GO:0070266) and its three GO parts. Receptor-proximal biology that merely feeds it is out of scope and is reached by reference, not duplicated: TNFR1 ligation, complex I assembly, cIAP/LUBAC ubiquitination and the NF-kappaB survival output belong to modules/tnf_signaling.yaml; LPS recognition and the TRAM-TRIF endosomal branch belong to modules/tlr4_md2_lps_signaling.yaml; FAS/FADD/DISC apoptotic initiation belongs to modules/death_receptor_apoptotic_signaling.yaml; effector-caspase execution belongs to modules/apoptotic_execution_phase.yaml. The upstream initiator variants therefore model only the RHIM-bearing adaptor that nucleates RIPK3, and the receptor that activates it is named in prose rather than given an annoton. (2) RIPK1 appears only in its kinase-dependent, necrosome-nucleating role. Its kinase-independent scaffold role is an inhibitor of this module (reviewed core function in GO:0060546), and the TRIF and ZBP1 routes can reach RIPK3 without it, which is why RIPK1 is one variant rather than an obligate part. (3) MLKL is placed in the execution phase and RIPK1/RIPK3 in the signaling phase, following the per-gene reviews. MLKL's core activity is taken as GO:0140912 membrane destabilizing activity; the reviews reject GO:0140911 pore-forming activity for MLKL, and this module does not assert it. (4) Caspase-8/FADD/cFLIP are the brake, not the core. They are an optional regulatory part with NEGATIVELY_REGULATES edges, because necroptosis is the output that appears when this proteolytic restraint fails; the same platform is also the apoptotic alternative, modelled in the death-receptor and apoptotic-execution modules. (5) Ripoptosome/necrosome naming. The literature calls the RIPK1-RIPK3 RHIM core the "necrosome" and the RIPK1-FADD-caspase-8-cFLIP platform the "ripoptosome". GO has one term, GO:0097342 ripoptosome, whose definition centres on RIPK1/RIPK3 as core components while carrying "necrosome" as an exact-in-spirit (RELATED) synonym; GO:1901026 is correspondingly named "ripoptosome assembly involved in necroptotic process". This module uses those GO terms as they stand and does not invent a separate necrosome term; the naming mismatch is recorded as an ontology knowledge gap rather than resolved by curator fiat. (6) Parthanatos is excluded. PARP1/AIF-dependent parthanatotic death does not require RIPK3 or MLKL and is not a form of necroptosis, so it is outside this module even though GO:0070266 carries "parthanatos" and "PARP-dependent cell death" as RELATED synonyms. Those synonyms are legacy aggregation, not a scope statement, and nothing in this module should be read as covering parthanatos. (7) No module-level deep research exists for necroptosis: no provider run was available when this module was curated, so there is no necroptosis-deep-research-<provider>.md file. All structural claims are grounded in the per-gene reviews under genes/human/, cached publications, local PAINT rows and the cached GO-CAM named above.

9Nodes

5Parts

1Variant Sets

3Variants

12Annotons

5Connections

Derived QC

Recommended-field compliance

100.0% recommended fields populated

All recommended fields populated.

Module deep research

✗ none found

No MODULE:necroptosis deep-research report alongside the module YAML.

Leaf nodes lacking representative members

✓ every leaf node grounds to a representative protein.

Template conformance

✓ every declared conforms_to bundle matches its template motif.

Function consistency and core coverage

12 gene-function check(s) reflected in core functions · 0 conflict(s) · 0 evidence or coverage gap(s) (12 comparison(s))

Retained noncore functions are reported separately; they do not require promotion to core functions. Support from representative genes does not establish conservation across the whole family. Missing reviews or identifiers leave a claim unverified.

Annoton	Participant	Function	Assessment
ripk1_necroptotic_kinase	RIPK1 UniProtKB:Q13546 Gene	protein serine/threonine kinase activity GO:0004674	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ticam1_rhim_adaptor	TICAM1 (TRIF) UniProtKB:Q8IUC6 Gene	signaling adaptor activity GO:0035591	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
zbp1_z_rna_sensing	ZBP1 (DAI) UniProtKB:Q9H171 Gene	pattern recognition receptor activity GO:0038187	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
zbp1_rhim_adaptor	ZBP1 (DAI) UniProtKB:Q9H171 Gene	signaling adaptor activity GO:0035591	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ripk3_rhim_scaffold	RIPK3 UniProtKB:Q9Y572 Gene	protein serine/threonine kinase binding GO:0120283	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
ripk3_mlkl_kinase	RIPK3 UniProtKB:Q9Y572 Gene	protein serine/threonine kinase activity GO:0004674	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
mlkl_ip6_activation	MLKL UniProtKB:Q8NB16 Gene	inositol hexakisphosphate binding GO:0000822	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
mlkl_pip_membrane_targeting	MLKL UniProtKB:Q8NB16 Gene	phosphatidylinositol-4,5-bisphosphate binding GO:0005546	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
mlkl_membrane_destabilization	MLKL UniProtKB:Q8NB16 Gene	membrane destabilizing activity GO:0140912	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
casp8_ripk1_cleavage	CASP8 UniProtKB:Q14790 Gene	cysteine-type endopeptidase activity GO:0004197	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
fadd_platform_adaptor	FADD UniProtKB:Q13158 Gene	protein-macromolecule adaptor activity GO:0030674	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).
cflar_caspase8_rheostat	CFLAR (c-FLIP) UniProtKB:O15519 Gene	cysteine-type endopeptidase activator activity GO:0140608	Core Supported The GO molecular function is reflected in core_functions (exact term or a reviewed subclass).

Feedback loops (advisory, prose-driven: a regulator counts as "feedback" when its own description says so)

✓ every negative regulator described as feedback has an upstream edge closing the loop.

casp8_fadd_cflip_brake ⊣ necroptotic_signaling [input]
casp8_fadd_cflip_brake is a negative regulator with no upstream edge (not described as feedback)

Gene-review completeness (8/8 grounded genes reviewed)

4 complete review(s) · 1 with deep research · 0 missing review · 7 reviewed but lacking deep research

Gene	Review	Complete	Deep research
CASP8 Q14790	✓	243/264	✗
CFLAR O15519	✓	87/94	✗
FADD Q13158	✓	206/214	✗
MLKL Q8NB16	✓	✓	✗
RIPK1 Q13546	✓	302/304	✗
RIPK3 Q9Y572	✓	✓	✗
TICAM1 Q8IUC6	✓	✓	✓
ZBP1 Q9H171	✓	✓	✗

Tree

NecroptosisBiological Processnecroptosis

Parts
1. 1. necroptotic signaling - a RHIM-bearing sensor or adaptor is activated and engages RIPK3
2. Necroptotic signaling (upstream RHIM-dependent initiation)Signaling Pathwaynecroptotic_signaling
  
  Variants
  1. Upstream RHIM-bearing initiator by which receptor system supplies the RHIM that nucleates RIPK3 (death receptor via RIPK1; TLR3/TLR4 via TRIF; Z-nucleic acid sensing via ZBP1) (One Or More)
    
    RIPK1-dependent initiation downstream of death receptorsRegulatory Stepripk1_death_receptor_initiation
    
    Annotons
    
    RIPK1 kinase-dependent necrosome nucleation Gene Product: RIPK1
    
    TRIF-dependent initiation downstream of TLR3 and endosomal TLR4Regulatory Steptrif_tlr_initiation
    
    Annotons
    
    TICAM1/TRIF RHIM adaptor engaging RIPK3 Gene Product: TICAM1 (TRIF)
    
    ZBP1-dependent initiation downstream of Z-form nucleic acid sensingRegulatory Stepzbp1_z_nucleic_acid_initiation
    
    Annotons
    
    ZBP1 Z-nucleic acid sensing Gene Product: ZBP1 (DAI)
    
    ZBP1 RHIM adaptor engaging the RIPK core Gene Product: ZBP1 (DAI)
3. 2. RHIM amyloid assembly of the necrosome/ripoptosome core, which activates RIPK3
4. Necrosome (ripoptosome) RHIM assemblyProtein Complexnecrosome_rhim_assembly
  
  Annotons
  - RIPK3 RHIM hetero-amyloid scaffold Gene Product: RIPK3
5. 3. committing step - activated RIPK3 phosphorylates MLKL, licensing the executioner
6. RIPK3 phosphorylation of MLKLRegulatory Stepripk3_mlkl_licensing
  
  Annotons
  - RIPK3 necroptotic effector kinase Gene Product: RIPK3
7. 4. execution phase - MLKL destabilizes the plasma membrane and the cell lyses
8. Execution phase of necroptosis (MLKL)Biological Processnecroptosis_execution
  
  Annotons
  - MLKL inositol-phosphate-dependent activation Gene Product: MLKL
  - MLKL phosphatidylinositol-phosphate-dependent membrane targeting Gene Product: MLKL
  - MLKL membrane destabilization (lethal step) Gene Product: MLKL
9. 5. caspase-8/FADD/cFLIP brake - proteolytic restraint whose failure permits the chain above optional
10. Caspase-8/FADD/cFLIP brake on necroptosisRegulatory Stepcasp8_fadd_cflip_brake
  
  Annotons
  - Caspase-8 proteolytic restraint of necroptosis Gene Product: CASP8
  - FADD adaptor assembling the restraining platform Gene Product: FADD
  - cFLIP setting of the caspase-8 rheostat Gene Product: CFLAR (c-FLIP)

Details

Context

MammaliaNCBITaxon:40674

cytosolGO:0005829

NecroptosisBiological Processnecroptosis

necroptotic processGO:0070266

Context

MammaliaNCBITaxon:40674

cytosolGO:0005829

Connections

necroptotic_signaling -> necrosome_rhim_assembly Causes

An activated RHIM-bearing initiator nucleates the RHIM amyloid core with RIPK3.

necrosome_rhim_assembly -> ripk3_mlkl_licensing Causes

RHIM assembly clusters and activates RIPK3; RHIM mutants retain the kinase domain but are not activated.

PMID:22817896

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

RHIM mutation compromises cluster formation, kinase activation and necrosis.

Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo.

ripk3_mlkl_licensing -> necroptosis_execution Causes

MLKL phosphorylation on T357/S358 licenses oligomerization, membrane recruitment and the lethal membrane-destabilizing step.

casp8_fadd_cflip_brake -> necroptotic_signaling Negatively Regulates

Caspase-8 cleavage of RIPK1 terminates kinase-dependent initiation; this is the restraint whose loss permits the pathway.

casp8_fadd_cflip_brake -> necrosome_rhim_assembly Negatively Regulates

Intact caspase-8 activity prevents formation of the RIPK1-RIPK3 RHIM core, including on the TLR-initiated route.

Part 1: necroptotic signaling - a RHIM-bearing sensor or adaptor is activated and engages RIPK3

Necroptotic signaling (upstream RHIM-dependent initiation)Signaling Pathwaynecroptotic_signaling

The signaling phase of necroptosis. An upstream RHIM-containing protein is activated by its own receptor system and presents its RHIM to RIPK3. The three known routes - RIPK1 downstream of ligated death receptors, TICAM1/TRIF downstream of TLR3 and endosomal TLR4, and ZBP1 downstream of Z-form nucleic acid - differ in sensor and in whether RIPK1 is required, but converge on the same RIPK3 core. Receptor ligation itself is modelled in the TNF, TLR4 and death-receptor modules and is not duplicated here.

necroptotic signaling pathwayGO:0097527

Variant set: Upstream RHIM-bearing initiator by which receptor system supplies the RHIM that nucleates RIPK3 (death receptor via RIPK1; TLR3/TLR4 via TRIF; Z-nucleic acid sensing via ZBP1) (One Or More)

PMID:24019532

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.

Demonstrates that RIPK3-dependent programmed necrosis is induced by RIP1-RIP3, DAI(ZBP1)-RIP3 and TRIF-RIP3 complexes alike, which is the basis for treating the initiator as an alternative rather than a fixed step.

Here, we describe two small molecule RIP3 kinase inhibitors and employ them to demonstrate the common requirement for RIP3 kinase in programmed necrosis induced by RIP1-RIP3, DAI-RIP3, and TRIF-RIP3 complexes.

RIPK1-dependent initiation downstream of death receptorsRegulatory Stepripk1_death_receptor_initiation

When caspase-8 activity is absent or blocked, RIPK1 released from ligated TNFR1 (or other death-receptor complexes) dimerizes through its death domain, autophosphorylates, and engages RIPK3 through RHIM-RHIM interaction. TNFR1 ligation, complex I ubiquitination and the NF-kappaB output are upstream and belong to modules/tnf_signaling.yaml.

Annotons

RIPK1 kinase-dependent necrosome nucleation

ripk1_necroptotic_kinase

Participant: Gene Product: RIPK1

Gene Product:

RIPK1UniProtKB:Q13546

Function

protein serine/threonine kinase activityGO:0004674

Targets: RIPK1 (autophosphorylation) and RIPK3

Processes

necroptotic signaling pathwayGO:0097527 necroptotic processGO:0070266

Locations

cytosolGO:0005829

Kinase-dependent activator of the pathway: death-domain dimerization activates RIPK1, which engages RIPK3 via RHIM and undergoes reciprocal phosphorylation with it. RIPK1 does not phosphorylate MLKL; its output is an activated RIPK3. The same protein restrains necroptosis in its kinase-independent scaffold role, which is outside this part.

PMID:29440439

Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis.

Death-domain-mediated dimerization is required for RIPK1 kinase activation in necroptosis.

Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis

PMID:19524513

Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation.

Reciprocal RIP1/RIP3 phosphorylation stabilizes the pronecrotic complex.

The phosphorylation of RIP1 and RIP3 stabilizes their association within the pronecrotic complex

PANTHER:PTN000585372

IBD propagation of GO:0097527 at PAINT node PTN000585372 in PTHR44329, seeded by UniProtKB:Q13546 and MGI:MGI:108212.

TRIF-dependent initiation downstream of TLR3 and endosomal TLR4Regulatory Steptrif_tlr_initiation

TLR3 and endosomal TLR4 can drive necroptosis without RIPK1 by a direct RHIM-dependent association between TICAM1/TRIF and RIPK3. LPS recognition and TRIF recruitment to the receptor are modelled in modules/tlr4_md2_lps_signaling.yaml.

Annotons

TICAM1/TRIF RHIM adaptor engaging RIPK3

ticam1_rhim_adaptor

Participant: Gene Product: TICAM1 (TRIF)

Gene Product:

TICAM1 (TRIF)UniProtKB:Q8IUC6

Function

signaling adaptor activityGO:0035591

Targets: RIPK3

Processes

necroptotic signaling pathwayGO:0097527 toll-like receptor 3 signaling pathwayGO:0034138

Locations

cytosolGO:0005829

Non-catalytic adaptor whose C-terminal RHIM binds RIPK3 directly, allowing TLR3/TLR4 to initiate necroptosis independently of RIPK1 while remaining MLKL-dependent. The reviewed human TICAM1 annotations carry no necroptotic process term, so this role is asserted from the primary literature and recorded as an open question in knowledge_gaps.

PMID:24019532

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.

TRIF associates with RIPK3 through a RHIM-dependent interaction to activate programmed necrosis.

TLR3 or TLR4 directly activates programmed necrosis through a RIP homotypic interaction motif-dependent association of TRIF with RIP3 kinase (also called RIPK3).

PMID:24019532

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.

The TRIF route proceeds without RIPK1 but still requires MLKL downstream of RIPK3, which is why it is a variant of initiation rather than a separate module.

it remains dependent on mixed lineage kinase domain-like protein (MLKL) downstream of RIP3 kinase

ZBP1-dependent initiation downstream of Z-form nucleic acid sensingRegulatory Stepzbp1_z_nucleic_acid_initiation

ZBP1 senses Z-form RNA (and Z-DNA) through its Zalpha domains and presents its RHIMs to RIPK3 (mouse) or RIPK1 (human), initiating necroptosis during influenza A and herpesvirus infection. Nuclear initiation is documented for influenza A, where MLKL is activated in the nucleus.

Annotons

ZBP1 Z-nucleic acid sensing

zbp1_z_rna_sensing

Participant: Gene Product: ZBP1 (DAI)

Gene Product:

ZBP1 (DAI)UniProtKB:Q9H171

Function

pattern recognition receptor activityGO:0038187

Processes

antiviral innate immune responseGO:0140374

Locations

cytosolGO:0005829 nucleusGO:0005634

Sensing step that licenses the adaptor role below; the reviewed gene review notes that a more specific Z-RNA immune receptor term postdates the validated ontology release, so the parent pattern-recognition term is used.

PMID:32200799

Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis.

Replicating influenza A generates Z-RNA that activates ZBP1 in the nucleus.

Here, we show that replicating IAV generates Z-RNAs, which activate ZBP1 in the nucleus of infected cells.

ZBP1 RHIM adaptor engaging the RIPK core

zbp1_rhim_adaptor

Participant: Gene Product: ZBP1 (DAI)

Gene Product:

ZBP1 (DAI)UniProtKB:Q9H171

Function

signaling adaptor activityGO:0035591

Targets: RIPK3 (mouse) / RIPK1 (human)

Processes

necroptotic signaling pathwayGO:0097527 positive regulation of necroptotic processGO:0060545

Locations

cytosolGO:0005829

Contributes no catalytic activity; its RHIMs hold the RIP kinases in a productive arrangement. Human and mouse ZBP1 differ in which kinase they require, which is recorded as a knowledge gap rather than resolved here.

PMID:19590578

DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB.

ZBP1 RHIMs recruit the RHIM-containing kinases RIPK1 and RIPK3.

we report the identification of two receptor-interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI-induced NF-kappaB signals through the recruitment of the RHIM-containing kinases RIP1 and RIP3

PMID:42436309

Human ZBP1 is a potent inducer of cell death through mechanisms divergent from mouse ZBP1.

Human ZBP1-induced death depends on RIPK1 rather than RIPK3, unlike mouse ZBP1.

In contrast to mZBP1, which signals primarily through RIPK3, hZBP1-induced cell death depends on RIPK1 in a RIPK3-independent manner.

Part 2: RHIM amyloid assembly of the necrosome/ripoptosome core, which activates RIPK3

Necrosome (ripoptosome) RHIM assemblyProtein Complexnecrosome_rhim_assembly

The RHIM of RIPK3 polymerizes with the RHIM of the upstream initiator (RIPK1, TRIF or ZBP1) into hetero-amyloid filaments that form the core of the necrosome. This assembly is not merely an interaction: RHIM mutation abolishes cluster formation, RIPK3 kinase activation and necroptosis, so assembly is the step that switches the effector kinase on.

ripoptosome assembly involved in necroptotic processGO:1901026 ripoptosome (necrosome core)GO:0097342

Annotons

RIPK3 RHIM hetero-amyloid scaffold

ripk3_rhim_scaffold

Participant: Gene Product: RIPK3

Gene Product:

RIPK3UniProtKB:Q9Y572

Function

protein serine/threonine kinase bindingGO:0120283

Targets: RIPK1 RHIM (and the RHIMs of ZBP1 and TRIF)

Processes

ripoptosome assembly involved in necroptotic processGO:1901026 amyloid fibril formationGO:1990000

Locations

cytosolGO:0005829

Non-catalytic, RHIM-mediated role of RIPK3, kept separate from its kinase activity because the two are distinct core functions in the gene review and because RHIM mutants retain kinase domains while failing to be activated. The reciprocal RIPK1 RHIM contribution is described at this node rather than asserted as a separate RIPK1 molecular function, since no GO molecular function for RHIM polymerization is available.

PMID:22817896

The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.

The RHIMs of RIPK1 and RIPK3 assemble heterodimeric filamentous structures.

Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures.

PMID:29681455

The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex.

Structural evidence that the RIPK1-RIPK3 core is a hetero-amyloid signaling complex.

The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis

PMID:19524512

Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.

RIPK3 is recruited to RIPK1 to form the necrosis-inducing complex.

Upon induction of necrosis, RIP3 is recruited to RIPK1 to form a necrosis-inducing complex

Part 3: committing step - activated RIPK3 phosphorylates MLKL, licensing the executioner

RIPK3 phosphorylation of MLKLRegulatory Stepripk3_mlkl_licensing

Within the assembled necrosome, RIPK3 phosphorylates MLKL on T357/S358 in the pseudokinase activation loop. This is the committing step of the pathway: it is required for MLKL oligomerization and membrane translocation, and activating mutations in the MLKL pseudoactive site cause RIPK3-independent necroptosis.

necroptotic signaling pathwayGO:0097527

Annotons

RIPK3 necroptotic effector kinase

ripk3_mlkl_kinase

Participant: Gene Product: RIPK3

Gene Product:

RIPK3UniProtKB:Q9Y572

Function

protein serine/threonine kinase activityGO:0004674

Substrates: MLKL (T357/S358)

Targets: MLKL

Processes

necroptotic signaling pathwayGO:0097527 necroptotic processGO:0070266

Locations

cytosolGO:0005829

The one kinase step that is required by all three initiation variants; RIPK3 kinase-dead cells are necroptosis-resistant, and the substrate that matters for execution is MLKL rather than RIPK1.

PMID:22265413

Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.

RIPK3 phosphorylates MLKL at T357/S358 and these events are critical for necrosis.

MLKL was phosphorylated by RIP3 at the threonine 357 and serine 358 residues, and these phosphorylation events were critical for necrosis.

PMID:19524512

Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.

RIPK3 catalytic activity is required for necrosis.

The kinase activity of RIP3 is essential for necrosis execution.

PMID:24012422

The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism.

MLKL's non-enzymatic necroptotic role is switched on by RIPK3-mediated phosphorylation.

its essential nonenzymatic role in necroptotic signaling is induced by receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated phosphorylation

Part 4: execution phase - MLKL destabilizes the plasma membrane and the cell lyses

Execution phase of necroptosis (MLKL)Biological Processnecroptosis_execution

Phosphorylation releases the MLKL four-helix bundle from the pseudokinase latch; binding of higher-order inositol phosphates is additionally required for oligomerization and membrane recruitment; the basic patch of the 4HB domain then binds phosphatidylinositol phosphates at the plasma membrane and disrupts bilayer integrity, producing ion influx and lytic death. The three annotons are the three reviewed MLKL core functions: activation, membrane targeting, and the lethal activity itself.

execution phase of necroptosisGO:0097528

Annotons

MLKL inositol-phosphate-dependent activation

mlkl_ip6_activation

Participant: Gene Product: MLKL

Gene Product:

MLKLUniProtKB:Q8NB16

Function

inositol hexakisphosphate bindingGO:0000822

Processes

execution phase of necroptosisGO:0097528

Locations

cytosolGO:0005829

Binding of IP6 and related higher-order inositol phosphates to the N-terminal region is required, in addition to RIPK3 phosphorylation, for MLKL oligomerization and membrane recruitment - a second input to the execution phase that is not supplied by the kinase.

PMID:29883610

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Loss of higher-order inositol phosphates blocks necroptosis downstream of MLKL phosphorylation.

genetic disruption of IP kinases to abolish production of higher order inositol phosphates blocked necroptosis downstream of MLKL phosphorylation by RIPK3 and prevented the oligomerization and plasma membrane recruitment of MLKL

PMID:25288762

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death.

The pseudokinase domain restrains the four-helix bundle until activation.

the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain

MLKL phosphatidylinositol-phosphate-dependent membrane targeting

mlkl_pip_membrane_targeting

Participant: Gene Product: MLKL

Gene Product:

MLKLUniProtKB:Q8NB16

Function

phosphatidylinositol-4,5-bisphosphate bindingGO:0005546

Processes

execution phase of necroptosisGO:0097528

Locations

plasma membraneGO:0005886

A basic patch on the four-helix bundle binds plasma-membrane phosphatidylinositol phosphates, recruiting the MLKL oligomer to the bilayer it then disrupts; membrane localization of the oligomer is required for death.

PMID:24813885

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.

A basic 4HB surface patch binds PIPs and recruits MLKL to the plasma membrane.

a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane

PMID:24316671

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.

Plasma-membrane localization of oligomerized MLKL is required for necroptosis.

the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis

MLKL membrane destabilization (lethal step)

mlkl_membrane_destabilization

Participant: Gene Product: MLKL

Gene Product:

MLKLUniProtKB:Q8NB16

Function

membrane destabilizing activityGO:0140912 The reviewed MLKL core activity. Pore-forming activity (GO:0140911) is deliberately not asserted: the reviewed evidence supports bilayer destabilization and leakage rather than a defined channel.

Processes

execution phase of necroptosisGO:0097528 necroptotic processGO:0070266

Locations

plasma membraneGO:0005886

The terminal, lethal activity of the module: oligomerized, membrane-bound MLKL directly compromises bilayer integrity, causing ion influx and lysis. MLKL is the only module member whose loss blocks death downstream of every initiator.

PMID:24703947

Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.

Phosphorylated MLKL oligomerizes, moves to membranes, and directly disrupts membrane integrity.

This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.

PMID:24813885

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates.

Recombinant MLKL permeabilizes PIP-containing liposomes, and the basic patch is required.

recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX

PANTHER:PTN008689042

IBD propagation of GO:0097527 at PAINT node PTN008689042 in PTHR44329, seeded by UniProtKB:Q8NB16 and MGI:MGI:1921818.

Part 5: caspase-8/FADD/cFLIP brake - proteolytic restraint whose failure permits the chain above (optional)

Caspase-8/FADD/cFLIP brake on necroptosisRegulatory Stepcasp8_fadd_cflip_brake

In a cell with intact caspase-8 activity, FADD-recruited caspase-8 (heterodimerized with cFLIP(L)) cleaves RIPK1 and other substrates and keeps the RHIM core from forming; necroptosis is what appears when this restraint is lost, which is why caspase-8 deficiency is embryonic-lethal in a RIPK3-dependent way. The same RIPK1/FADD/caspase-8/cFLIP platform is the apoptotic alternative and is modelled as such in modules/death_receptor_apoptotic_signaling.yaml and modules/apoptotic_execution_phase.yaml; it is included here only as regulation, not as a core necroptotic step.

negative regulation of necroptotic processGO:0060546

Annotons

Caspase-8 proteolytic restraint of necroptosis

casp8_ripk1_cleavage

Participant: Gene Product: CASP8

Gene Product:

CASP8UniProtKB:Q14790

Function

cysteine-type endopeptidase activityGO:0004197

Substrates: RIPK1

Processes

negative regulation of necroptotic processGO:0060546

Locations

cytosolGO:0005829

Limited proteolysis of RIPK1 (and other signaling substrates) is the normal off-state of this module; loss of caspase-8 unmasks RIPK3-dependent death.

PMID:21368762

RIP3 mediates the embryonic lethality of caspase-8-deficient mice.

Caspase-8 suppresses RIPK3-RIPK1 complex-dependent necroptosis.

Casp8 suppresses RIP3-RIP1 (also known as RIPK3-RIPK1) kinase complex-dependent necroptosis that follows death receptor activation

PMID:21368762

RIP3 mediates the embryonic lethality of caspase-8-deficient mice.

The lethality of caspase-8 loss is RIPK3-dependent, placing the brake upstream of this module's core.

We find that RIP3 is responsible for the mid-gestational death of Casp8-deficient embryos.

PMID:24019532

Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL.

The same caspase-8 restraint governs the TLR-initiated route.

rely on caspase 8 to suppress RIP3-dependent programmed necrosis

FADD adaptor assembling the restraining platform

fadd_platform_adaptor

Participant: Gene Product: FADD

Gene Product:

FADDUniProtKB:Q13158

Function

protein-macromolecule adaptor activityGO:0030674

Processes

regulation of necroptotic processGO:0060544

Locations

cytosolGO:0005829

Brings caspase-8 (and cFLIP) into the RIPK1-containing cytosolic platform. The reviewed FADD annotations keep this necroptotic-regulatory context as non-core relative to FADD's death-receptor role, which is why it sits in the optional part here.

PMID:21737330

cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms.

The RIPK1-containing platform contains FADD, caspase-8, caspase-10 and cFLIP isoforms.

It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms.

cFLIP setting of the caspase-8 rheostat

cflar_caspase8_rheostat

Participant: Gene Product: CFLAR (c-FLIP)

Gene Product:

CFLAR (c-FLIP)UniProtKB:O15519

Function

cysteine-type endopeptidase activator activityGO:0140608

Targets: CASP8

Processes

regulation of necroptotic processGO:0060544

Locations

cytosolGO:0005829

cFLIP(L)-caspase-8 heterodimers give the limited protease activity that cleaves RIPK1 without committing to apoptosis; which cFLIP isoform is present decides whether the platform yields apoptosis or RIPK3-dependent necroptosis.

PMID:21737330

cIAPs block Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death complex differentially regulated by cFLIP isoforms.

Caspase activity set by cFLIP isoforms decides between RIPK3-dependent necroptosis and caspase-dependent apoptosis.

caspase activity is the "rheostat" that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis