title: Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death.
abstract: The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 1-125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human monocyte-derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli.
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000, Ljubljana, Slovenia.
authors.forename: Katja Hrovat
authors.initials: KH
authors.lastname: Arnež
authors.affiliation: Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research, and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Michaela
authors.initials: M
authors.lastname: Kindlova
authors.affiliation: Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research, and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Nilesh J
authors.initials: NJ
authors.lastname: Bokil
authors.affiliation: Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia Department of Medical Biology, University of Melbourne, Parkville, Victoria 3050, Australia.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.affiliation: Institute for Molecular Bioscience, IMB Centre for Inflammation and Disease Research, and Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sweet
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000, Ljubljana, Slovenia gregor.guncar@fkkt.uni-lj.si.
authors.forename: Gregor
authors.initials: G
authors.lastname: Gunčar
mesh_terms: D002470:Cell Survival; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008264:Macrophages / Q000201:enzymology* / Q000473:pathology; D008958:Models, Molecular; D009336:Necrosis; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D007527:Isoenzymes; C570693:MLKL protein, human; D011494:Protein Kinases
keywords: MLKL; cell death; isoform; macrophage; mixed lineage kinase domain-like; necroptosis