Mitochondrial Redox Opto-Lipidomics Reveals Mono-Oxygenated Cardiolipins as Pro-Apoptotic Death Signals

Abstract

While opto-genetics has proven to have tremendous value in revealing the functions of the macromolecular machinery in cells, it is not amenable to exploration of small molecules such as phospholipids (PLs). Here, we describe a redox opto-lipidomics approach based on a combination of high affinity light-sensitive ligands to specific PLs in mitochondria with LC-MS based redox lipidomics/bioinformatics analysis for the characterization of pro-apoptotic lipid signals. We identified the formation of mono-oxygenated derivatives of C18:2-containing cardiolipins (CLs) in mitochondria after the exposure of 10-nonylacridine orange bromide (NAO)-loaded cells to light. We ascertained that these signals emerge as an immediate opto-lipidomics response, but they decay long before the commencement of apoptotic cell death. We found that a protonophoric uncoupler caused depolarization of mitochondria and prevented the mitochondrial accumulation of NAO, inhibited the formation of C18:2-CL oxidation product,s and protected cells from death. Redox opto-lipidomics extends the power of opto-biologic protocols to studies of small PL molecules resilient to opto-genetic manipulations.

Known unknowns of cardiolipin signaling: The best is yet to come

“…A recently developed optolipidomics approach based on a combination of high affinity light-sensitive ligands for CL with LC-MS based lipidomics demonstrated selective accumulation of hydroxy- and hydroperoxy-C18:2-containing CL species in mouse embryonic fibroblasts and HeLa cells triggered to apoptosis [159]. In this case, selectivity of intrinsic apoptotic death has been achieved by photo-oxidation of mitochondria-accumulated and CL-selective dye, nonyl-acridine orange and confirmed by a bioinformatics n-gram analysis.…”

NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy

“…29 In contrast to the CL that triggers mitophagy through recognition via LC3, the CL externalized during apoptosis has mostly peroxidized acyl chains, due to the CL oxidase function of cytochrome c, 30 and this oxidation step seems to be important for execution of the apoptotic program. 31,32 Externalized CL also participates in the complex activation the Nlrp3 inflammasomes trough interaction with the Nlrp3 LRR3 domain, leading to pyroptotic cell death. 33,34 Finally, when released into the extracellular space, CLs on the surface of mitochondria or mitochondrial fragments, again mostly in their oxidized form, 35 can act as pro-phagocytotic 'eat me' signals via CD36 scavenger receptor and/or as suppressors of cytokine production potentially leading to immunoparalysis.…”

Elimination of the unnecessary: Intra- and extracellular signaling by anionic phospholipids

“…While it is commonly accepted that the phagocytosis of apoptotic cells triggers the resolution of inflammation [75, 76], the actual meaning of each individual signal and combinations of different molecular signals in pro-/anti-inflammatory conditions has not been deciphered. With the development of redox (phospho)lipidomics the identification of the role of individual PS and oxidized PS species as “orchestral players” of inflammation can be unraveled [77]. …”

“…For some time, it remained unclear which of them are, indeed, directly related to the execution of apoptosis. This conundrum has been recently resolved by using a protocol of redox opto-lipidomics [77]. It has been demonstrated that only few out of a large number of detectable CL oxidation products represent real apoptotic signals.…”

“…It has been demonstrated that only few out of a large number of detectable CL oxidation products represent real apoptotic signals. In fact, in cells exposed to several pro-apoptotic treatments such as actinomycin D, staurosporine, ionizing radiation or 10-nonylacridine orange, only C18:2 mono-oxygenated species of CLs were identified as predictive biomarkers of apoptosis [77]. …”