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).