Superoxide Activates Uncoupling Proteins by Generating Carbon-centered Radicals and Initiating Lipid Peroxidation

Abstract

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondriatargeted derivative of the spin trap ␣-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation ofUCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

“…To exclude AcUCP activation by endogenous FFAs, all measurements were performed in the presence of BSA. The maximal uncoupling effect of HNE was observed with 100 μM HNE at 0.6 mg of amoeba mitochondrial protein per ml of incubation medium, which corresponds to the 35-50 μM HNE concentration at approximately 0.35 mg of mitochondrial protein per ml of assay medium usually used in studies with mammalian mitochondria (Murphy et al 2003;Echtay et al 2003;Parker et al 2008a;Aguirre and Cadenas 2010). In addition, a 100 μM HNE concentration can be considered to be physiologically relevant (Uchida 2003).…”

“…Allosteric interactions (Murphy et al 2003) or covalent modifications (Azzu and Brand 2010) have been proposed. The mitochondria were incubated in the absence or presence of 100 μM HNE for 15 min prior to energisation as described in Fig.…”

“…The empty arrows represent the activation or high level (up) and inhibition or low level (down) 2005). Therefore, the current model proposed for animal and plant mitochondria (Murphy et al 2003;Echtay et al 2003;Considine et al 2003) for the activation of UCPs by ROS through the initiation of lipid peroxidation (the HNE pathway), leading to the feedback down-regulation of mitochondrial ROS production, can be applied to the UCPs of unicellular eukaryotes. Thus, a common regulation of UCPs through lipid peroxidation products that arise from ROS could exist in all eukaryotes that possess UCPs.…”

“…However, animal UCP2 and UCP3 and plant UCPs are generally thought to be activated by lipid peroxidation products (such as HNE) to induce a proton leak, thus providing a negative feedback regulation of mitochondrial ROS production. Specifically, ROS production is a prerequisite of lipid peroxidation and HNE production, which in turn induce mild uncoupling mediated by UCPs resulting in ROS attenuation (Murphy et al 2003;Echtay et al 2003). Thus far, it is unknown whether UCP activity can be induced by ROS or lipid peroxidation products in the mitochondria of unicellular eukaryotes.…”

“…The polyunsaturated fatty acids of phospholipids in cell membranes are especially susceptible to oxidation. The nonenzymatic and self-propagating process of lipid peroxidation produces various compounds, including carbon-centred fatty acyl radicals, peroxyl radicals, fatty acid hydroperoxides and finally multiple breakdown products (Murphy et al 2003). 4-Hydroxy-2-nonenal (HNE), a reactive unsaturated aldehyde, is the most abundant end product of lipid peroxidation resulting from the production of mitochondrial superoxide.…”