Cardiolipin (CL), the signature phospholipid of mitochondria, is required for optimal mitochondrial function and numerous cellular processes. The de novo synthesis of CL is followed by a unique remodeling pathway, which is characterized by cycles of phospholipase-mediated deacylation of CL to monolysocardiolipin (mCL), and reacylation of mCL by tafazzin (Taz) to form predominantly unsaturated CL species. Mutations in Taz cause the life-threatening genetic disorder, Barth syndrome (BTHS), highlighting the significance of CL remodeling. While it is clear that Taz deficiency leads to accumulation of mCL, the mechanism linking defective CL remodeling to the pathology in BTHS is not known. An increase in reactive oxygen species (ROS), a feature of Taz-deficient cells, has been implicated in BTHS pathogenesis. However, detailed redox lipidomic analyses of oxidized species of mCL and CL (mCLox and CLox) in the context of CL remodeling have not been performed. Our preliminary and published studies show that CL and mCL form complexes with the intermembrane space hemoprotein, cytochrome c (cyt c), converting it to a potent peroxidase that generates mCLox and CLox. The proposed study will test the innovative hypothesis whereby aberrant CL remodeling along with peroxidation by mCL/cyt c complexes leads to a vicious metabolic cycle of CL/mCL oxidation in BTHS. Our approach combines our expertise and experience in genetics, molecular biology, and cell biology (Greenberg lab) with innovative approaches in mass spectrometry technologies, oxidative lipidomics, and biochemistry of lipids (Kagan lab).
Aim 1 will test the working hypothesis that peroxidation by mCL/cyt c is exacerbated by Taz deficiency.
Aim 2 will utilize solid-state NMR and computational modeling to rigorously characterize mCL/cyt c complexes and identify properties resulting in gain of peroxidase activity. We expect these studies to provide the first detailed characterization of the role of CL remodeling in generating and eliminating mCLox/CLox.
The proposed studies will generate a new model of pathogenesis for the severe genetic disorder Barth syndrome, delineating the role of CL remodeling in the generation and remediation of oxidative damage. This model will be experimentally tested and provide a new mechanistic paradigm with which to understand the link between defective remodeling due to tafazzin deficiency and resulting mitochondrial damage in Barth syndrome. Schema 1. Vicious metabolic cycle of CL oxidation. Nascent molecular species of CL normally undergo maturation to predominantly homo-acylated unsaturated CL via sequential reactions of hydrolysis (by CLD1 in yeast and iPLA2? and other phospholipases in mammalian cells) and reacylation (by tafazzin). In Barth syndrome, a deficiency of tafazzin-catalyzed reacylation leads to accumulation of monolysoCL (mCL), including oxidizable polyunsaturated species or non-oxidizable saturated and mono-unsaturated species. All mCL species can form peroxidase complexes with cyt c, which can oxidize bound oxidizable mCLs or CLs. mCLox and CLox are preferred substrates for CLD1 or iPLA2?, and their hydrolysis yields mCL and dilysoCL (dCL), with the concomitant release of free oxygenated fatty acids (FAox).
