2.2.3 MLKL
In the human genome, exceed five hundred protein kinases have been found and authenticated, in which about 10% of the protein kinases seems have no enzyme activity and having been classified as pseudokinases (Manning et al. 2002). MLKL, as one of pseudokinase, is made up of a C-terminal pseudokinase domain, a two-helix brace or linker, and an N-terminal four-helix bundle (4HB) (Murphy et al. 2013). ​ It was originally recognized as a RIP3-binding protein via its C-terminal kinase-like domain. It is reported that phosphorylation of MLKL can cause the change of the pseudokinase domain conformation, and resulting in exposure of the 4HB domain (Petrie et al. 2018). The phosphorylation of MLKL activated by RIPK3 is a symbol of necroptosis. Recruitment of MLKL relies on auto-phosphorylation of RIPK3 at Ser227 for human RIPK3 or Ser232 for mouse RIPK3 (Sun et al. 2012b). The late formation of micro pores with an approximately 4 nm diameter is a key process in necroptosis (Ros et al. 2017). Evidences have shown that activated MLKL can form membrane destruction pores through the interaction between its N-terminus and phospholipids, leading to membrane leakage. This concept expands researchers’ understanding of morphological changes following necroptosis occurs in vivo (Zhang et al. 2016a).
2.3necroptosis, a developmental signaling pathway
In general, necroptotic signal pathway could be activated by several stimuli covering ambient pressure, variety of chemotherapy medicines, mechanical damage, inflammation, and infection et al (Lalaoui et al. 2015). Current studies show that lipopolysaccharide (LPS) can promote necroptosis through TLRs (Kim and Li 2013). The necroptosis-promoting activities of type I IFN sectionally depends on TRIF and derives from the continuous provoke of signal transducer and activator of transcription 1 (STAT1), STAT2, and interferon regulatory factor 9 (IRF9) (McComb et al. 2014a). IFNAR1 and interferon gamma receptor 1 (IFNGR1) can also induce necroptosis in macrophages (Robinson et al. 2012, Thapa et al. 2013). Among these stimulus factors, TNFR superfamily was regarded as the most intensively studied (Grootjans, Vanden Berghe and Vandenabeele 2017). Therefore, the activation of the necroptotic signaling pathway can be summarized by the events triggered by TNF-α/TNFR. When the organism is subjected by various external stimuli, the tissue microenvironment will release lots of inflammatory factors including TNF-α. Then, the TNF-α combined with TNFR1 induces comformational change of TNFR1 trimers, which further recruit variety of proteins, covering RIPK1, tumor necrosis factor receptor type 1-associated death domain (TRADD), cellular inhibitor of apoptosis protein 1 (cIAP1), cIAP2, TNFR-associated factor 2 (TRAF2) and TRAF5, forming complex I (Moriwaki, Balaji and Ka-Ming Chan 2020). Particularly worth mentioning is the protein of RIPK1 in complex I, which is a powerful cytokine regulatory factor determines the life and death of cell. RIPK1 can polyubiquitinated by cIAP1/2, which induce classical nuclear factor kappa-B signaling pathway and promote cell survival (Gong et al. 2019). When the continuous activation of nuclear factor NF-kappa-B (NF-κB) is blocked, the apoptotic pathway is tending to be activated. Therefore, RIPK1, caspase-8, TRADD and FAS-associated death domain protein (FADD) recruit each other to forming Complex II and activating caspase-8 (Hitomi et al. 2008). Then, the activated caspase-8 can start apoptosis-promoting caspase activation cascade and finally contribute to the occurrence of cell apoptosis (Wu, Liu and Li 2012). When caspase-8 is inhibited due to certain physiological changes or external stimuli, the cell death mode will converted from apoptosis to necroptosis. At this time, RIPK1 is activated by phosphorylated, which result from the serine residue 161(S161) autophosphorylation at its N-termini (Degterev et al. 2008). The active RIPK1 will interact with RIPK3 to cause its phosphorylation and forming a necrosome complex (Bedoui, Herold and Strasser 2020). In addition, RIPK3 can also be triggered by TLR through a process of TIR domain-containing adapter molecule 1 (TRIF) inducing interferon-β. Active RIPK3 phosphorylates its well-featured functional substrate MLKL pseudokinase. Then, MLKL oligomerizes and transfers to the plasmalemma to trigger necroptosis and destroy the integrity of the plasmalemma by forming micro pores. The resulting inrush of water and sodium and potassium outflow cause cell swelling, destruction of membrane potential, and ultimately cell death characterized by loss of cell and organelle integrity (Sun et al. 2012a). Another study showed that Zα domains of ZBP1 can sense endogenous Z-form nucleic acids to activite RIPK3-dependent necroptosis (Jiao et al. 2020). At present, the downstream target of MLKL is still unclear. Research has shown that phosphorylated RIPK3 activate MLKL to form a homotrimer by its amino-terminal coiled-coil domain and locates to the cell plasmalemma, further mediate transient receptor potential melastatin related 7 induce Ca(2+) influx (Cai et al. 2014). Another research demonstrated that activated MLKL outcomes in the producing of broken, plasmalemma ”bubbles” with uncovered phosphatidylserine that are liberated from the outside of the otherwise intact cell. The ESCRT-III machinery is required for forming these bubbles and acts to maintain survival of the cell when MLKL activation is limited or reversed (Gong et al. 2017).
(Show in Fig 3).