Necrosome core machinery: MLKL

Abstract

In the study of regulated cell death, the rapidly expanding field of regulated necrosis, in particular necroptosis, has been drawing much attention. The signaling of necroptosis represents a sophisticated form of a death pathway. Anti-caspase mechanisms (e.g., using inhibitors of caspases, or genetic ablation of caspase-8) switch cell fate from apoptosis to necroptosis. The initial extracellular death signals regulate RIP1 and RIP3 kinase activation. The RIP3-associated death complex assembly is necessary and sufficient to initiate necroptosis. MLKL was initially identified as an essential mediator of RIP1/RIP3 kinase-initiated necroptosis. Recent studies on the signal transduction using chemical tools and biomarkers support the idea that MLKL is able to make more functional sense for the core machinery of the necroptosis death complex, called the necrosome, to connect to the necroptosis execution. The experimental data available now have pointed that the activated MLKL forms membrane-disrupting pores causing membrane leakage, which extends the prototypical concept of morphological and biochemical events following necroptosis happening in vivo. The key role of MLKL in necroptosis signaling thus sheds light on the logic underlying this unique "membrane-explosive" cell death pathway. In this review, we provide the general concepts and strategies that underlie signal transduction of this form of cell death, and then focus specifically on the role of MLKL in necroptosis.

Keywords: MLKL; Necroptosis; Necrosome; Pore-forming protein; Regulated cell death.

Fig. 1

Necrosome assembly and signal propagation. Ligation of members of the tumor necrosis factor receptor superfamily (TNFRSF, including TNFR1, CD95, TRAILR) leads to the recruitment of receptor-interacting protein kinase 1 (RIP1). RIP1 and RIP3 form a pro-necrotic death complex via their RHIM domains. RIP3 is then activated by auto-phosphorylation at Ser227 (Ser232 for mouse RIP3). The activated RIP3 recruits and phosphorylates its substrate MLKL at Thr357/Ser358 (Ser345 for mouse MLKL), a step which defines the formation of the functional necrosome. Besides TNFRSF, activation of type I interferon receptor (IFNAR1) also triggers the formation of a RIP1–RIP3–MLKL-containing necrosome. Moreover, pattern recognition receptors (PRRs) drive necrosis in an RIP1-independent manner. Toll-like receptor-induced TRIF–RIP3–MLKL necrosome formation (for TLR3, e.g., by sense dsRNA such as poly I:C; for TLR4 in response to LPS) also depends on RHIM interaction between TRIF and RIP3. Likewise, infection with M45 mutant murine cytomegalovirus (MCMV) leads to RHIM-mediated interaction between DAI and RIP3. Of all necrosome protein complexes reported to date, MLKL binds to activated RIP3 to propagate the necrotic death signal, regardless of what RHIM protein RIP3 utilizes for its self-activation. Activated MLKL binds phosphatidylinositol phosphates (PIPs), cardiolipin (CL) and phosphatidylglycerol (PG), which navigate necrosomes to different phospholipid-rich cellular compartments. Once targeted to membranes, MLKL disrupts membrane integrity and finally causes necroptotic cell death

Fig. 2

Overview of the membrane-punching mechanisms of pore-forming proteins. a Oligomerized MLKL punches membranes. The MLKL monomer is sequestered in an inactivated state by its C-terminal kinase-like domain (KLD). Phosphorylation of MLKL releases the auto-inhibition on the amino-terminal MLKL and enables MLKL to bind with PIPs or CL. NSA blocks MLKL from oligomerization and membrane translocation. b A representative α-PFT protein, cytolysin A (ClyA, also known as HlyE), forms a pore on a target membrane. The β-tongue, consisting of two β-strands between the third and fourth helices, locks ClyA in a compacted soluble state. Once ClyA binds to the membrane, the C-terminal helix is released from the bundle, touches the membrane and further recruits other molecules. After assembled into oligomers, the aggregated C-terminal helices insert into the membrane to form pores. c Structural transitions of CDC pore-forming proteins. Beta-PFTs are assembled into oligomeric pre-pores and bind to the membrane. The tandem transmembrane helical (TMH) units that are initially buried in the core of the pre-pore intermediate will be exposed and inserted into the membrane as a β-barrel. Perforin and the complement share similar primary structures of both monomer and oligomer, whether there is a transition station remains unclear