Lemieux 2017 bioRxiv 151480
|Lemieux H, Subarsky P, Doblander C, Wurm M, Troppmair J, Gnaiger E (2017) Impairment of mitochondrial respiratory function as an early biomarker of apoptosis induced by growth factor removal. bioRxiv doi: https://doi.org/10.1101/151480 .|
Abstract: Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40% inhibition. A slight increase in respiration following addition of cytochrome c, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.
• Keywords: Mitochondrial respiration, OXPHOS, cytochrome c oxidase, apoptosis, CRAF, interleukin 3 • Bioblast editor: Gnaiger E • O2k-Network Lab: CA_Edmonton_Lemieux H, AT_Innsbruck_Oroboros, AT_Innsbruck_Gnaiger E
Labels: MiParea: Respiration Pathology: Cancer Stress:Cell death Organism: Mouse Tissue;cell: Blood cells Preparation: Intact cells, Permeabilized cells Enzyme: Complex IV;cytochrome c oxidase, Marker enzyme Regulation: Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, Threshold;excess capacity, Uncoupler, Q-junction effect Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: N, S, CIV, NS, ROX HRR: Oxygraph-2k
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