Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

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

Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

BACKGROUND: The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. PRINCIPAL FINDINGS: The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis. CONCLUSIONS: Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated

Analysis of the effects of caspase-8, Bid and tBid on the Laurdan fluorescence of CL⁺ and CL⁻ liposomes.

<p>Generalised polarisation (GP, arbitrary units, a.u.) determined from Laurdan fluorescence measurements. GP values are reported for the various preparations, as described in the materials and methods.</p

αH6 and BH3 domains are both required for tBid cytochrome <i>c</i> release activity.

<p>(A) and (B) Jurkat cells were electroporated with plasmids encoding tBid, tBidΔH6, tBidG94E, tBidKKAA and tBidG94EKKAA and the kinetics of cytochrome <i>c</i> release in the cytosolic fractions were detected by ELISA.</p

tBid, a bifunctional molecule.

<p>(A) Current model of the BH3-dependant function of tBid. tBid interacts through its BH3 domain and directly activates Bax, which undergoes conformational changes that induce the exposure of its N-terminal domains. This results in the stable insertion and subsequent oligomerization of Bax in the mitochondrial outer membrane leading to the release of cytochrome <i>c</i> and apoptosis. This model highlights the importance of protein-protein interactions between tBid and Bax. (B) Refined model of the pro-apoptotic function of tBid: importance of tBid/CL interactions. First, tBids binds to CL present at the contact sites via its helix αH6 and destabilizes the mitochondrial membrane. This may affect the activity of the electron transport chain complexes and lead to an acidification of the cytosol, mitochondrial ROS production and mitochondrial lipid peroxidation. This environment may prime the activation of Bax and/or Bak. Then, tBid interacts through its BH3 domain with Bax and/or Bak to promote their oligomerization and subsequently induce cytochrome c release and apoptosis.</p

Binding of Bid and caspase-8 to CL-containing large unilamellar liposomes (LUVs).

<p>(<b>a</b>) Schematic diagram of caspase-8 autoprocessing during Fas-mediated apoptosis. Upon dimerisation, procaspase-8 (p55) is initially cleaved between its two active subunits, p18 and p10, to generate the p43/p10 heterodimer; p43 is then cleaved between the death effector domain (DED) and the p18 subunit, to produce the fully active p18/p10 form. (<b>b</b>) Western blot analysis of caspase-8 binding to the “contact site mimetic” liposomes or similar liposomes without CL, in which the CL was replaced with PE (22%) (<b>c</b>) Caspase-8 binding, as detected by caspACE FITC-VAD-fmk binding to the active site, to liposomes of various compositions (monolipid liposomes made from PA, PC, PE, PI, PG or cholesterol, and mixed liposomes composed of DOPC+CL, DOPC+PE, DOPC+CL+PE at various molar ratios, contact site mimetic liposomes; for details see materials and methods). (<b>d</b>) Flow cytometric analysis of CL⁺ and DOPC-only liposomes in the presence or absence of Bid_Alexa488. The black spectrum correspond to control vesicles whereas the red spectrum correspond to the vesicles plus Bid_Alexa488. The blue spectrum results from an alkaline wash of the CL⁺ liposomes. The alkaline wash involved centrifugation of liposomes and resuspending them in 0.1 M Na₂CO₃, pH 11.5. The liposomes were then analysed directly by flow cytometry. Fm: fluorescence mean value, in arbitrary units (a.u.).</p

Confocal microscopy study of the binding of Bid and caspase-8 to giant unilamellar vesicles containing cardiolipin.

<p>Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100∶0) vesicles are presented in panels <b>a</b> to <b>c</b> and DOPC/CL (90∶10) vesicles in panels <b>d</b> to <b>f</b>. Top: in <b>a</b> and <b>d</b>, protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05% of the hydrophobic dye DiO, as shown in (<b>b, c</b>) and in red, as shown in (<b>e, f</b>); bottom: overlay of green and red images (<b>c, f</b>). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and Bid_Alexa488, resulting in a non-fluorescent tBid.</p

tBid required its helix αH6, but not its BH3 domain, to induce superoxide anion production and mitochondrial lipid peroxidation.

<p>(A) and (B) Wild-type hepatocytes were transfected with a control vector (control) or plasmids encoding tBid, tBidΔH6, tBidΔBH3 and tBidKKAA and NAD(P)H and SNARF-1AM fluorescence (pH indicator) were measured by FACS. Data are given as % of the control ± SD. pH units were determined using a calibration curve generated using nigericin-permeabilized cells kept in buffer of different pH values. (C) AtT20 cells or DKO Mefs were transfected with empty vector (control) or plasmids encoding tBid, tBidΔH6, tBidΔBH3, tBid tBidKKAA, tBidΔH6–H7, tBidΔBH3ΔH6H7. Cells were then stained with hydroethidine (HE, Invitrogen/Molecular probes) to measure superoxide anion production. The percentages of transfected cells are indicated by the dotted line. (D) Purified mice liver mitochondria were energized using succinate (+ rotenone) and treated using 10 nM tBid, H6-5G-BH4, αH6 and αH6m. Mitochondrial superoxide anion production was measured using HE (a.u.  =  arbitrary units) whereas hydroperoxide was measured using amplex red. (E) Wild-type hepatocytes were treated with TNFα/cycloheximide, anti-Fas antibody or transfected with a plasmids encoding tBid and tBidΔH6. Mitochondrial lipid peroxidation was measured by FACS using MDA. We used the antioxidants trolox (2 mM), MnTBAP (1 mM) and MitoQ10 (1 µM).</p