Intracellular redox changes during TNFα-mediated necroptosis
Necroptosis is a form of regulated cell death triggered by a variety of factors associated with infection and inflammation. It is distinct from the well-characterised cell death processes of apoptosis and necrosis, sharing the morphology of necrosis but driven by a carefully controlled genetic program. The cellular events leading to necroptosis include phosphorylation of the mixed lineage kinase-domain like (MLKL) pseudokinase by receptor-interacting kinase 3 (RIPK3), followed by MLKL oligomerisation, translocation to membranes and plasma membrane permeabilisation. Little is known of the regulatory mechanisms that control necroptosis signalling. One hypothesis is that the intracellular redox environment regulates necroptosis; in particular, the phosphorylation and thiol-dependent oligomerisation of MLKL. Intracellular redox changes during TNFα-mediated necroptosis were characterised in this thesis.Wild-type and MLKL-knockout mouse dermal fibroblasts were triggered to undergo necroptosis as measured by live cell imaging and flow cytometry. Western blotting revealed that MLKL phosphorylation occurred within 30 minutes of TNFα stimulation in wild-type cells. This was followed by a delay of approximately 30 minutes, until concurrent oxidation of intracellular peroxiredoxins, thioredoxins and MLKL was observed. Samples have been prepared for a mass spectrometry-based measurement of global thiol protein oxidation and sent to a collaborating laboratory for analysis. It was hypothesised that the rate of necroptosis could be increased by agents that promote thiol oxidation. Intriguingly, hydrogen peroxide had no effect on the extent of MLKL oxidation, which suggests it occurs by a tightly controlled mechanism. Three different thioredoxin reductase inhibitors showed variable responses in their ability to influence TNFα-mediated necroptosis. Sublethal doses of auranofin promoted necroptosis, whereas two specific thioredoxin reductase inhibitors were unable to increase cell death. This suggests the effect of auranofin is due to an alternate mechanism that warrants further investigation. Compounds that influence the sensitivity of cells to necroptosis may be valuable in promoting necroptosis in cancer cells resistant to therapeutic agents, or in inhibiting necroptosis in pathologies where necroptosis exacerbates tissue damage.
Advisor: Hampton , Mark
Degree Name: Bachelor of Biomedical Sciences with Honours
Degree Discipline: Department of Pathology and Biomedical Science
Publisher: University of Otago
Keywords: necroptosis; MLKL; peroxiredoxin; thiol oxidation; thioredoxin; cell death; redox
Research Type: Thesis