The role of cardiolipin in the regulation of mitochondria-dependent apoptosis

Mitochondria are known as the powerhouse of the cell due to their central role in energy generation and as the site of key metabolic pathways. Over the past 15 years, it has become unequivocally clear that most pro-apoptotic stimuli require a mitochondria-dependent step, involving the permeabilisation of the mitochondrial outer membrane to apoptogenic factors, such as cytochrome c and Smac/DIABLO. The release of these factors into the cytosol is tightly regulated by proteins of the Bcl-2 family and results in the activation of the caspase cascade, leading to cell death. This event is considered as a point of no return in the apoptotic pathway and is often inhibited in cancer. Cardiolipin (CL) is a mitochondria-specific phospholipid that contains four acyl-chains. CL has been implicated in many of the mitochondria-dependent steps that lead to the release of apoptogenic factors including interaction with the Bcl-2 family protein tBid, Bax-dependant mitochondrial outer membrane permeabilization and cytochrome c release. Despite this growing body of evidence, the mechanism by which CL and its fatty acyl chain composition regulate mitochondrial apoptotic pathways remains unresolved, mostly due to the lack of cellular model. Tafazzin is a mitochondrial enzyme, which is mutated in Barth syndrome (BTHS) and is involved in the maturation process of CL. In BTHS, loss of tafazzin activity results in a decrease in mature CL, making it a good model to investigate the role of CL in apoptosis. Using BTHS patients-derived lymphoblastoid cells and HeLa cells in which tafazzin was stably knocked-down using RNA interference, this study provides the first evidence that mature CL are required for an efficient extrinsic apoptotic pathway in type II cells. Further investigation of the impaired apoptotic pathway revealed that the major block is in the activation of caspase-8. In this work, mature CL was identified as a crucial component of a mitochondrial platform required for caspase-8 translocation, oligomerization and activation following Fas signalling in type II cells. These results support a model in which once the first cleavage of procaspase-8 occurs at the DISC, the p43/p10 heteromer product translocates and inserts into the mitochondrial membrane in a CL-dependant manner. In the mitochondria, caspase-8 further oligomerizes and auto-cleaves to adopt its fully active form p18/p10. Additionally, it is shown here that mature CL is required for the physiological association of full-length Bid, the major caspase-8 cleavage substrate, with mitochondria. Thus, Bid is directly available for active caspase-8 on the mitochondrial surface where it cleaves into tBid, which in turn inserts into the mitochondrial outer membrane and induces cytochrome c release. Therefore, by tethering full-length Bid on mitochondria and by providing an activation site for caspase-8 following Fas signalling, CL brings together both the enzyme and its substrate and provides a platform from which the mitochondrial phase of apoptosis is launched. In summary, the data presented in this thesis provide the first evidence that mature CL participates in a new mitochondrial associated platform, called the “mitosome”, required for the activation of caspase-8 in type II cells

Cardiolipin provides an essential activating platform for caspase-8 on mitochondria

Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with apoptosis. However, the lack of appropriate cellular models to date restricted analysis of its role in cell death. The maturation of cardiolipin requires the transacylase tafazzin, which is mutated in the human disorder Barth syndrome. Using Barth syndrome patient-derived cells and HeLa cells in which tafazzin was knocked down, we show that cardiolipin is required for apoptosis in the type II mitochondria-dependent response to Fas stimulation. Cardiolipin provides an anchor and activating platform for caspase-8 translocation to, and embedding in, the mitochondrial membrane, where it oligomerizes and is further activated, steps that are necessary for an efficient type II apoptotic response

Cardiolipin interactions with proteins

Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions

Capillary electrophoresis-mass spectrometry at trial by Metabo-Ring: Effective electrophoretic mobility for reproducible and robust compound annotation

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (mu(eff)) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The mu(eff) was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the mu(eff) for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.Analytical BioScience