MLKL post-translational modifications: road signs to infection, inflammation and unknown destinations

Abstract

Necroptosis is a caspase-independent modality of cell death that requires the activation of the executioner MLKL. In the last ten years the field gained a substantial amount of evidence regarding its involvement in host response to pathogens, TNF-induced inflammatory diseases as well as pathogen recognition receptors (PRR)-induced inflammation. However, there are still a lot of questions that remain unanswered. While it is clear that there are specific events needed to drive MLKL activation, substantial differences between human and mouse MLKL not only highlight different evolutionary pressure, but also provide potential insights on alternative modalities of activation. While in TNF-induced necroptosis it is clear the involvement of the RIPK3 mediated phosphorylation, it still remains to be understood how certain inflammatory in vivo phenotypes are not equally rescued by either RIPK3 or MLKL loss. Moreover, the plethora of different reported phosphorylation events on MLKL, even in cells that do not express RIPK3, suggest indeed that there is more to MLKL than RIPK3-mediated activation, not only in the execution of necroptosis but perhaps in other inflammatory conditions that include IFN response. The recent discovery of MLKL ubiquitination has highlighted a new checkpoint in the regulation of MLKL activation and the somewhat conflicting evidence reported certainly require some untangling. In this review we will highlight the recent findings on MLKL activation and involvement to pathogen response with a specific focus on MLKL post-translational modifications, in particular ubiquitination. This review will highlight the outstanding main questions that have risen from the last ten years of research, trying at the same time to propose potential avenues of research.

Fig. 1. MLKL structure and functional reported post-translational modifications.

MLKL domain composition and post-translational modifications. MLKL consists of a four helical bundle domain (4HB, aa 1–125), a brace region and a pseudokinase domain (aa 190–471 in human and 191–472 in mouse). MLKL undergoes RIPK3-mediated phosphorylation at threonine 357/serine 358 in human and serine 345 in mouse, shown by purple circles. In addition, MLKL can be ubiquitinated at different lysine residues, as shown by the grey circles. Sites in bold have been published to have a specific function.

Fig. 2. TNFIFN-induced necroptosis.

Cartoon depicting TNFR1-induced signalling pathway and IFN mediated upregulation of MLKL and ZBP1, that can culminate with MLKL activation and necroptosis. Binding of TNF to TNFR1 triggers the formation of a membrane-bound complex referred to as complex-I. This complex is composed of adaptor proteins (e.g., TRADD and TRAF2), E3 ligases (e.g., cIAP1/2 and LUBAC) that synthesize poly-ubiquitin chains of different topology (i.e., K63, K48, K11 and M1) and kinases, such as RIPK1 and IKK1/2, and it leads to NF-kB and MAPKs activation and expression of pro-survival as well as pro-inflammatory genes. Alternatively, upon interferon receptors activation and/or IFN signaling activation, Mlkl and Zbp1 are transcriptionally upregulated. Under certain circumstances, described in the main text, a secondary cytoplasmic complexes forms, referred to as complex-II. This complex promotes Caspase-8 activation and apoptosis. However, upon Caspase-8 inhibition by the means of synthetic or viral encoded caspase inhibitors, RIPK1 activates RIPK3 that in turn phosphorylates MLKL. Following interferon signaling, ZBP1 is upregulated and activated following binding to double-stranded RNA. Upon activation, ZBP1 binds to RIPK3 via RHIM/RHIM interaction, triggering RIPK3 phosphorylation and the consequent MLKL activation. Phosphorylated MLKL undergoes a conformation change to expose its 4 helical bundle (4HB) domain that promotes MLKL association with the plasma membrane. Here MLKL oligomerizes and disrupt plasma membrane integrity, causing necroptosis.

Fig. 3. Toll like receptors (TLRs)-induced necroptosis.

Schematic representation of how TLR3 and TLR4 can induce MLKL activation and necroptosis. TLR4 activation by LPS triggers the formation of two different signalling complexes. One, name the Myddosome, composed of the adaptor protein TIRAP, Myd88, IRAKs and TRAF6, activates NF-κB and MAPKs for the expression of pro-inflammatory genes. The other, referred to as Triffosome, is composed of the adaptor protein TRAM and TRIF. TRIF, via its RHIM domain, can recruit RIPK1, depending on the cell type, and RIPK3. RIPK3 in turn activates MLKL via phosphorylation. Activated MLKL will in turn execute necroptosis. Activation of TLR3, that localizes at endosomal membranes, by dsRNA determines the recruitment of TRIF that in turn recruits RIPK1, depending on the cell types, and RIPK3. RIPK3 phosphorylates MLKL causing its activation, translocation to the membrane, oligomerization and, ultimately, necroptosis.

Fig. 4. MLKL-mediated necroptosis in viral responses.

Schematic representation of the involvement of MLKL-mediated necroptosis in host anti-viral responses. While Vaccinia virus, mouse herpes simplex virus (mHSV) and Influenza virus (IAV) induce the activation of necroptosis, mouse cytomegalovirus (MCMV), human herpes simplex virus (human HSV) and Poxviruses such as BAV and COTV have evolved strategies to block MLKL-induced necroptosis.

Fig. 5. Proposed roles for ubiquitin modifications on MLKL.

A TNFR1 stimulation, in the presence of caspase inhibition, promotes MLKL ubiquitination at K219, by a so far unknown E3 ligase. This ubiquitination events contributes to MLKL ability to oligomerize and induce necroptosis. B TNFR1 activation and caspase inhibition trigger MLKL multi-mono-ubiquitination, that represents a signal for MLKL degradation via the proteasome. The E3 ligase involved in this process is still unknown. C TNFR1 activation, concomitantly with caspase inhibition, stimulates ubiquitination of MLKL at K50. Ubiquitin-modified MLKL enhances then lysosome-mediated destruction of intracellular bacteria.