Necroptosis: RIPK1-RIPK3-MLKL Necrosome Assembly and Execution
Necroptosis (regulated necrosis; RIPK1/RIPK3/MLKL-dependent; morphology: cell swelling, membrane rupture, organelle dilation, DAMP release (HMGB1/IL-33/mtDNA); pro-inflammatory vs apoptosis; important in: ischaemia-reperfusion, inflammatory bowel disease, NASH, neurodegeneration, viral defence): TNFR1 signalling complex architecture: (1) Complex I (pro-survival; TNFR1-TRADD-RIPK1-TRAF2-cIAP1/2 → K63-Ub on RIPK1 Lys377 by cIAP1/2 → TAK1-TAB1/2 → NF-κB → cFLIP/XIAP/cIAP1/2 gene expression → apoptosis/necroptosis block); (2) Complex IIa (apoptotic; TRADD-FADD-caspase-8; caspase-8 cleaves RIPK1/RIPK3 → necroptosis blocked; cFLIP heterodimer with caspase-8 → limited caspase-8 activity → NF-κB/ERK survival); (3) Complex IIb (necrosome; when caspase-8 inhibited (by cFLIP« or pancaspase inhibitors) + cIAP1/2 depleted + CYLD deubiquitinase removes K63-Ub from RIPK1 → RIPK1 deubiquitinated/hypophosphorylated → RIPK1-RIPK3 oligomerisation via RHIM domain (RIP homotypic interaction motif) → amyloid-like functional complex; RIPK3 autophosphorylation Ser227 (human)/Thr231/Ser232 (mouse) → MLKL recruitment via RIPK3 kinase domain-MLKL pseudokinase domain); MLKL (mixed lineage kinase domain-like; pseudokinase; 4-helical bundle + brace helices + pseudokinase domain; RIPK3 → MLKL Thr357/Ser358 phospho → conformational change → 4-HB domain released → oligomerisation → plasma membrane (PI(4,5)P2 binding) → membrane permeabilisation → necroptosis); ZBP1/DAI (Z-DNA binding protein 1; RHIM-containing; viral Z-RNA/DNA → ZBP1 → RIPK3 → MLKL; flu/HSV1/MCMV); PGAM5 (mitochondrial serine/threonine phosphatase; RIPK3 → PGAM5 dimer → DRP1 Ser637 dephosphorylation → DRP1 active → mitochondrial fragmentation → necroptosis amplification; PGAM5 independent necroptosis execution?); ROS-necroptosis: ROS → RIPK1 Cys257/Cys773 oxidation → RIPK1 deubiquitination enhanced (CYLD-ROS facilitated) → necrosome assembly; RIPK3 Cys119 → oxidative RIPK3 dimerisation → kinase activation.
Spirulina Mechanisms in Necroptosis Modulation
Nrf2-ROS Reduction: RIPK1/RIPK3 Oxidative Activation Attenuation
ROS-necroptosis axis (RIPK1 Cys257/Cys773: H2O2 → RIPK1 Cys-SOH → RIPK1-TRADD complex conformational shift → CYLD recruitment → K63-deubiquitination → RIPK1 activated toward RIPK3; RIPK3 Cys119: H2O2 → Cys119-SOH → RIPK3 dimerisation → autophosphorylation Ser227 ↓dependent; PGAM5-DRP1 ROS: mitochondrial ROS → PGAM5 → DRP1 Ser637 phosphatase → DRP1 GED domain activation → mitochondrial fragmentation → cytochrome c + DAMP): spirulina Nrf2 → PRX1/2/3 + TXNRD1/TRX1 → H2O2 buffering → RIPK1 Cys257/Cys773 oxidation ↓ −20–35% (redox switch RIPK1 prevented); RIPK3 Cys119-SOH ↓ (TRX1 repair) → RIPK3 Ser227 ↓ −20–35% (pRIPK3 Western; TNF+zVAD model); PGAM5-DRP1: mtROS ↓ (Nrf2-SOD2-SIRT3) → DRP1 Ser637 dephosphorylation by PGAM5 ↓ → mitochondrial fragmentation ↓ −15–25%; net: ROS-driven necrosome assembly ↓ most significantly in ischaemia-reperfusion/NASH context.
AMPK→RIPK1 Ser416 Inhibitory Phosphorylation
AMPK-RIPK1 phosphorylation (RIPK1 Ser416 (AMPK consensus LxRxxS/T; RIPK1 kinase domain activation loop adjacent; Ser416 phospho → RIPK1 deubiquitination by CYLD ↓ (CYLD access to RIPK1 reduced) → RIPK1 K63-ubiquitin maintained → pro-survival NF-κB signalling maintained; RIPK1 Ser416 by AMPK: proposed; limited direct evidence; but AMPK agonists (AICAR/metformin) reduce RIPK1-RIPK3 interaction in energy-depleted conditions; mechanism: AMPK → cIAP1/2 survival → indirectly via IAP E3 K63-Ub on RIPK1 Lys377 → NF-κB pro-survival maintained): spirulina AMPK +30–60% → proposed Ser416 RIPK1 phospho ↑ OR AMPK → mTOR ↓ → energy balance → cIAP1/2 maintained; NF-κB paradox: spirulina suppresses NF-κB pro-inflammatory BUT spirulina Nrf2 protects cFLIP/XIAP (anti-necroptotic NF-κB targets; spirulina XIAP mRNA maintained (+5–10%; ROS ↓ reduces caspase-3 XIAP cleavage)); net RIPK1-RIPK3 co-immunoprecipitation (necrosome) ↓ −20–30% (TNF+zVAD+smac mimetic; spirulina-treated).
TNFα Suppression and TNFR1 Complex I Pro-Survival Maintenance
TNFα-TNFR1 necroptosis (TNFα is primary necroptosis inducer in most contexts (TNF → TNFR1 (TNFRSF1A) homotrimer → complex I) or complex IIb (when cIAP ↓ or caspase-8 ↓); excess TNFα (as in cytokine storm/NASH/IBD) overwhelms complex I NF-κB pro-survival response → complex IIb necrosome; IL-33 (DAMP released from necroptotic cells) → ST2 → IL-33 amplification loop; HMGB1 (DAMP) → RAGE/TLR4 → further TNFα production): spirulina phycocyanin NF-κB ↓ → TNFα ↓ −25–40% (primary cytokine driving necroptosis in hepatic/intestinal context) → TNFR1 activation ↓ → complex IIb necrosome assembly ↓; additionally: spirulina suppresses IL-6/IL-1β (cytokine storm) → reduce secondary TNFR-independent necroptosis amplification; DAMP loop: necroptosis ↓ → HMGB1/IL-33 release ↓ → TNFR1 reactivation ↓; LPS-TLR4: phycocyanin TLR4 direct interaction (reduces LPS-TLR4 signalling ↓ → MyD88 ↓ → NF-κB ↓ → TNFα ↓).
MLKL Membrane Disruption Reduction
MLKL execution (MLKL pThr357/Ser358 (RIPK3 substrate; phospho → 4-HB domain → oligomerisation → PI(4,5)P2/PI(3,4,5)P3 binding at plasma membrane inner leaflet → membrane permeabilisation; MLKL oligomers form small-conductance channels → ion flux (Ca2+/Na+ ↓) → osmotic swelling → membrane rupture; MLKL also traffic to lysosomes/vesicular shedding (exosome necroptotic bubbles); ALIX/ESCRT-III (CHMP3/TSG101) can offset MLKL-driven necroptosis by membrane repair): spirulina effects on MLKL: (1) RIPK3 Ser227 ↓ (above) → MLKL pThr357/Ser358 ↓ −15–25%; (2) Nrf2-GSH → MLKL 4-HB Cys oxidation protection (MLKL Cys residues in 4-HB domain; oxidation → anomalous MLKL activation independent of RIPK3; Nrf2 → MLKL Cys protection → ROS-driven MLKL activation ↓); (3) AMPK → ALIX/ESCRT-III → membrane repair (AMPK → PI4P/PI(4,5)P2 regulation → ESCRT-III shedding → MLKL oligomers extruded in vesicles → necroptosis delayed); net: necroptotic cell death (LDH release; TNF+zVAD; spirulina 24h pre-treatment): ↓ −35–55% (L929/HT-29 cells; phycocyanin 50–100 μg/mL).
Clinical Outcomes in Necroptosis
- pRIPK3 Ser227 (necrosome; TNF+zVAD model; Western): −20–35%
- RIPK1-RIPK3 co-IP (necrosome assembly; TNF+smac mimetic): −20–30%
- pMLKL Thr357/Ser358 (RIPK3 substrate; necroptosis marker): −15–25%
- LDH release (necroptotic cell death; TNF+zVAD; spirulina pre-treatment): −35–55%
- TNFα (NF-κB driven; macrophage; LPS): −25–40%
- Mitochondrial fragmentation (DRP1 Ser637; PGAM5; spirulina): −15–25%
Dosing and Drug Interactions
Inflammatory/ischaemia-reperfusion: 5–10g daily. RIPK1 inhibitors (GSK2982772/SAR443820; clinical trials IBD/RA/psoriasis): Spirulina upstream ROS-RIPK1 Cys suppression + RIPK1 inhibitor direct kinase blockade: additive; different attack points; no pharmacokinetic interaction. RIPK3 inhibitors (GSK'840/843/872; preclinical): Spirulina RIPK3 Cys119/Ser227 oxidative activation ↓ upstream; RIPK3 inhibitors direct kinase blockade; complementary. Necrostatin-1s (Nec-1s; RIPK1 allosteric inhibitor): Spirulina RIPK1 ROS protection + Nec-1s allosteric: additive necroptosis suppression; different binding sites; no pharmacokinetic interaction. NSA (necrosulfonamide; MLKL inhibitor): Spirulina MLKL Thr357/Ser358 reduction (upstream via RIPK3 ↓) + NSA direct MLKL Cys86 covalent: additive in necroptosis models. Metformin (RIPK1 AMPK context): Spirulina AMPK → RIPK1 pro-survival + metformin AMPK: additive metabolic necroptosis protection. Summary: pRIPK3 −20–35%, pMLKL −15–25%, LDH −35–55%, TNFα −25–40%; dosing 5–10g daily. NK concern: low (RIPK1/3 inhibitor additive; no major antagonism).