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

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

Intracellular localization of Bid-EYFP, tBid-EYFP and its deletion mutants.

<p>CV-1 cells were transfected with plasmids encoding Bid-EYFP, tBid-EYFP or tBid-EYFP mutants in presence of 10 µM of Bok-D. 24 h later, cells were stained with 20 nM of the mitochondrial potential probe TMRE and the localization of the tBid-EYFP mutants was determined by calculation of the TMRE/EYFP ratios using microspectrofluorometry. A ratio of 1.0 indicates that EYFP exclusively colocalizes with TMRE at the mitochondria while a lower ratio indicates that EYFP is also localized to the cytosol. The <b>*</b> highlights the contructs that localize to the mitochondria but are unable to induce cytochrome <i>c</i> release or to alter mitochondrial bioenergetics. The constructs that localize to the cytoplasm are in <i>italic</i>.</p

αH6 and BH3 domains are both required for tBid-induced apoptosis.

<p>(A) Jurkat cells were transfected with either empty vector control or with plasmids encoding tBid, tBidΔBH3 and tBidΔH6. 12 h latter mitochondrial membrane potential ΔΨm (TMRE) and cell viability (PI) were assessed by FACS analysis. Each panels is representative of 3 independent experiments. (B) Wild-type, Bax^+/+, Bax^−/−, Bak^−/− or double-knockout (DKO) Mefs cells were transfected with either empty vector control or with plasmids encoding tBid and tBidΔH6. After 8 h, green cells were analyzed by FACS for mitochondrial potential using TMRE. The red arrow indicates the ΔΨm in DKO Mefs. (C) Mefs cells were transfected with plasmids encoding tBid and tBidΔBH3, and the kinetics of mitochondrial depolarization and cell death were measured over 60 h by FACS analysis.</p

The helix αH6 targets tBid to the mitochondria.

<p>(A) and (B) Computer-based analysis of the biophysical properties of Bid. (A) Kite & Doolittle profile of Bid (PROSTCALE, Swiss Institute of Bioinformatics). (B) Comparison of the isoelectric points (pI), hydrophobicities and charges of tBid, αH6 and αH6m. (C) Schematic representation of tBid-EYFP mutants. (D) and (E) CV-1 cells were transfected with plasmids endoding tBid-EYFP mutants in presence of 10 µM of Bok-D to inhibit caspases activation and subsequent cell death. 24 h later, cells were stained with 20 nM of the mitochondrial potential probe TMRE and the localization of the tBid-EYFP mutants was determined using confocal microscopy (D) and microspectrofluorometry analysis (E).</p

Biophysical properties of Bid and its alpha-helices: number of amino acids (AA), isolelectric point (pI), hydrophobicity and charge.

<p>Other constructs like the hairpin forming domain αH6–H7 domain are described. The calculation of hydrophobicity was performed using the PROTSCALE software from the Swiss Institute of Bioinformatics according to the method of Kyte and Doolittle. αH6 and αH6m are presented in <b><i>italic</i></b>.</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

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

The helix αH6 specifically inserts into CL-monolayers through electrostatic interactions and reorganizes them into microdomains.

<p>Dipalmitoylphosphatidylcholine (DPPC), bovine heart CL (BHCL) and tetramyristoyl CL (TMCL) were spread at an initial surface pressure of 20 mN/m. αH6 and αH6m (1 µM) were injected into the subphase of these lipids monolayers. (A) The surface pressure changes (B) and epifluorescence images (C) were recorded as described previously (Gonzalvez et al 2005, CDD).</p