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Tales From the Crypt: how RIPK3 kinase unleashes the zombie protein, MLKL, to kill cells by necroptosis

Tales From the Crypt: how RIPK3 kinase unleashes the zombie protein, MLKL, to kill cells by necroptosis

; Walter and Eliza Hall 鶹Ƶ of Medical Research

James Murphy completed his undergraduate studies in his hometown of Christchurch, New Zealand, before completing his PhD studies at the Australian National University, Canberra, Australia in 2003. As a CJ Martin Fellow of the National Health and Medical Research Council of Australia (NHMRC), he completed postdoctoral training in the lab of the signalling guru, the late Tony Pawson (Toronto, Canada). He moved to the Walter and Eliza Hall 鶹Ƶ in 2007 and was appointed group leader in January 2015, and appointed Head of the Inflammation Division in 2019 where he is now Professor. He has pursued a mechanistic understanding of the roles of several pseudokinases, protein kinases, cytokines/receptors and epigenetic regulators in signal transduction, with a particular focus on MLKL, the key pseudokinase in the necroptosis cell death pathway. These studies have culminated in >170 publications to date and receipt of awards from the UK Biochemical Society and the Australia and New Zealand Society of Cell and Developmental Biology. He is highly engaged in learned society journals, including as Chair of the Publications Committee of the IUBMB, Editor-in-Chief of the reviews journal of the UK Biochemical Society, Biochemical Society Transactions; is an Associate Editor of Biochemical Journal and sits on the Editorial boards of JBC, CDD, Structure and TIBS.

In 2012, Mixed lineage kinase domain-like (MLKL), a catalytically-dead (“zombie”) cousin of conventional protein kinases, termed a pseudokinase, was implicated as the key effector in the programmed necrosis (or necroptosis) cell death pathway. This pathway has ancestral origins in innate immunity, but it is its dysregulation, such as in inflammatory diseases, that has attracted enormous interest as a therapeutic target. Our dissection of the chronology of events in this pathway using novel tools, structural biology, biochemistry, microscopy, proteomics, mouse disease models and analysis of patient biopsies, has enabled us to define a series of regulated steps in MLKL activation. I will outline our current understanding of how phosphorylation of MLKL by the upstream kinase, RIPK3, triggers its structural interconversion, oligomerization and trafficking to the plasma membrane, where MLKL accumulates into hotspots that permeabilize the plasma membrane to cause a cell's demise. Necroptotic cell death is considered pro-inflammatory, because MLKLmediated plasma membrane disruption results in the release of cellular contents into the extracellular milieu. Defining precisely where and when MLKL activation and necroptotic death occurs physiologically has proven challenging due to the nascency of the field and a dearth of robust reagents and methods. Here, I will describe our recent work defining where and when necroptotic effectors are expressed in mouse and human tissues, and their activation in Inflammatory Bowel Disease (IBD; or Crohn's and Ulcerative Colitis) patient biopsies. Under basal conditions, our data indicate barrier tissue cells are equipped to undergo necroptosis, consistent with a role for the pathway in host defence. Intriguingly, exposure to insults, such as TNF or antibiotics, perturbs expression and equips other cell types to undergo necroptotic cell death. Consistent with pathway dysregulation in disease, our study of >50 IBD patients identified activation of the terminal effectors of the necroptosis pathway, RIPK3 and MLKL, not observed in healthy controls. These data raise the prospect that pharmacologically targeting checkpoints in the necroptosis pathway may provide a new avenue to counter inflammatory diseases, such as IBD.

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