Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

Abstract

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.

Initiation and perpetuation of NLRP3 inflammasome activation and assembly

“…The structurally unique diphospholipid cardiolipin is found in prokaryotes and in eukaryotic inner mitochondrial membranes and at contact sites between mitochondrial membranes (147)(148)(149). Cardiolipin is important for normal mitochondrial function, but it also regulates extrinsic apoptosis by recruiting Bid and caspase-8 to mitochondria, which is thought to require externalization of cardiolipin to the outer mitochondrial membrane (150)(151)(152)(153)(154)(155)(156)(157). Additionally, cardiolipin anchors cyt c to the inner mitochondrial membrane and during intrinsic apoptosis, cyt c oxidizes cardiolipin and is released into the cytosol (158,159).…”

NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy

“…15 Externalization of CL to the mitochondrial surface is also part of other damage-related signaling pathways, including early pro-apoptotic signaling [16][17][18] and inflammatory reactions in intracellular compartments and extracellular environments. 19 By providing a scaffold at the mitochondrial surface for recruiting and functionally altering different apoptotic proteins, including caspase 8, tBid, Bak, and Bax, [20][21][22] CL participates in launching the release of cytochrome c, [23][24][25][26][27][28] although this step may not be required for all forms of apoptosis. 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.…”

X-ray structure, thermodynamics, elastic properties and MD simulations of cardiolipin/dimyristoylphosphatidylcholine mixed membranes

“…Alternative roles for CL include the formation of cubic phases which can efficiently store lipid for the dynamic cristae (Deng and Mieczkowski, 1998) and apoptosis signaling (Kagan et al, 2009). Especially, CL seems to play a crucial role in the assembly of a passive docking platform for the binding of Bid, and the initiation of the apoptotic process (Jalmar et al, 2013). Finally, a defect in CL synthesis is the cause of Barth’s syndrome, which is a sex-linked recessive disorder, clinically characterized by the classical symptoms of cardiomyopathy, neutropenia and delayed growth with a lethal effect on young boys (Barth et al, 1983).…”