WEHI Wednesday Seminar hosted by Associate Professor James Vince

Daniel Frank, PhD Student - Vince Laboratory, Inflammation division - Infection, Inflammation and Immunity Theme

RIPK3 ubiquitylation in cell death and inflammation

This is a PhD completion seminar.

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Includes Q&A session

Receptor Interacting Serine/Threonine Protein Kinase-3 (RIPK3) is essential for necroptosis, an inflammatory form of programmed cell death pathway implicated in infections, kidney ischemia reperfusion injury, and systemic inflammatory response syndrome. During necroptosis, RIPK1 drives phosphorylation and oligomerisation of RIPK3. Activated RIPK3 oligomers phosphorylate MLKL pseudokinase which subsequently perforates the plasma membrane as a prelude to cellular rupture. However, recent findings have unveiled RIPK3 as a multifaceted protein also capable of triggering caspase-8-mediated apoptosis and inducing the production and release of pro-inflammatory cytokines. Understanding the molecular regulation of RIPK3 will thereby facilitate the ongoing pre-clinical development of RIPK3 inhibitors.

Post-translational modification is a critical regulator of protein function and emerging evidence suggests that ubiquitylation, in particular, can modulate RIPK3 activities. Using mass-spectrometry, I identified a novel ubiquitylation site on the C-terminal end of RIPK3 beyond its Receptor-Interacting Protein Homotypic Interaction Motif (RHIM), a region predicted to be disordered. Complementation of RIPK3-deficient cells with a mutant which abolished RIPK3 ubiquitylation beyond-the-RHIM (BTR) demonstrated that decoration of RIPK3 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. In vivo, initial studies suggest that RIPK3 BTR ubiquitylation is required for efficient bacterial pathogen clearance. Unexpectedly, the overall ubiquitylation of BTR mutant RIPK3 was enhanced, which correlated with its increased killing efficiency compared to wild type RIPK3. Silencing this additional ubiquitylation was sufficient to dramatically reduce RIPK3 hyperactivity resulting from loss of BTR ubiquitylation. Therefore, RIPK3 BTR ubiquitylation may function to prevent RIPK3 hyper-ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerisation and consequent cell death signalling.