Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance

Abstract

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system.

Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes

“…According to a recently published study, mouse MLKL is also ubiquitinated at lysine 219, and it was suggested that this ubiquitination, as well as ubiquitination of the corresponding lysine residue in human MLKL (at position 230), serve to facilitate death induction [ 28 ]. However, in our repeated mass spectroscopy analysis we could not discern ubiquitination of lysine 230 in HT-29 cells that had been treated for 3 h with TBZ.…”

The role of Ubiquitination in Apoptosis and Necroptosis

“…Mass spectrometry identified K219 as a ubiquitination site of mouse MLKL and mutation of the site was shown to reduce K63-linked ubiquitination of MLKL, generated during TNF-α-induced necroptosis [ 119 ]. Additionally, ubiquitinated K219 was shown to predominantly promote necroptosis in mouse dermal fibroblasts (MDFs) derived from Mlkl K219R/K219R knock-in mice, which almost completely blocked TNF-α-induced necroptosis, and abrogated phenotypes associated with necroptosis-induced tissue injury were seen in Mlkl K219R/K219R mice [ 119 ]. Interestingly, Mlkl K219R/K219R MDFs cannot form nearly as many higher-order MLKL polymers in membrane fractions compared to wild-type MDFs, indicating that K219 ubiquitination promotes MLKL oligomerisation at the plasma membrane [ 119 ].…”

“…Additionally, ubiquitinated K219 was shown to predominantly promote necroptosis in mouse dermal fibroblasts (MDFs) derived from Mlkl K219R/K219R knock-in mice, which almost completely blocked TNF-α-induced necroptosis, and abrogated phenotypes associated with necroptosis-induced tissue injury were seen in Mlkl K219R/K219R mice [ 119 ]. Interestingly, Mlkl K219R/K219R MDFs cannot form nearly as many higher-order MLKL polymers in membrane fractions compared to wild-type MDFs, indicating that K219 ubiquitination promotes MLKL oligomerisation at the plasma membrane [ 119 ]. In contrast, another study showed that exogenous expression of a non-ubiquitinated MLKL-USP21 fusion protein (USP21 removes all Ub conjugates from a protein) had minimal effects on TNF-α-induced necroptosis, but did affect MLKL stability [ 120 ].…”

“… [ 101 ] OTULIN** (M1) ↑Necroptosis [ 103 ] CHIP* ↓Necroptosis [ 108 ] CYLD** ↑Necroptosis [ 112 , 113 ] RIPK3 K5 (Ms) A20** (K63) ↓Necroptosis [ 105 ] K55, K363 CHIP* ↓Necroptosis [ 108 ] K363 Pellino-1* (K48) ↓Necroptosis [ 110 ] K197, K302, K364 Parkin* (K33) ↓Necroptosis [ 107 ] K501 TRIM25*(K48) ↓Necroptosis [ 109 ] K518 USP22** ↑Necroptosis [ 106 ] MLKL K219 (Ms) ??? * (K63) ↑Necroptosis [ 119 ] ??? ???…”

Necroptosis in Pulmonary Diseases: A New Therapeutic Target

“…The phosphorylated MLKL undergoes conformational changes and disengagements from RIPK3; then, the MLKL oligomer is transferred to the cell membrane, leading to cell expansion and rupture ( Wang H. et al, 2014 ; Garnish et al, 2021 ). Recently, it has been found that MLKL ubiquitination is also correlated with MLKL activation and necroptosis, and MLKL ubiquitination at K219 imposes the cytotoxic potential of phosphorylated MLKL ( Garcia et al, 2021 ). Cell rupture is followed by the release of a large number of endogenous host-derived molecules such as adenosine triphosphate, IL-33, heat-shock proteins, and high-mobility group box 1 (HMGB1) and other DAMPs, thus causing an excessive inflammatory response and aggravating surrounding tissue damage ( Scaffidi et al, 2002 ; Iyer et al, 2009 ; Gong et al, 2020 ).…”