tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak Cytochrome c is normally involved in the electron transfer from the bc1 complex (complex III) to cytochrome oxidase (complex IV).
Once released into the cytosol, cytochrome c activates the caspase cascade by inducing the oligomerization of a cytochrome c dATP/Apaf 1/pro caspase 9 complex named apoptosome. Moreover, it was recently reported that changes of mitochondrial physiology appeared during many apoptotic pathways10 and could be induced by many pro and antiapoptotic factors. Although the ability of Bcl Xl and Bcl 2 to affect mitochondrial energy metabolism has already been described,11, 12 the mechanisms whereby mitochondrial physiology changes control apoptosis remains to be defined. Bid is a widespread proapoptotic factor that belongs to a subset of the Bcl 2 family, and possesses sequence homology only within the kids pandora bracelets and charms conserved BH3 domain.13 Bid has attracted increasing interest since it was identified as a substrate of caspase 8, following the activation of death receptors such as Fas. During apoptosis, cleavage near its N terminus by caspase 814, 15 produces p15 tBid, the active form of Bid, which can rapidly translocate to mitochondria and trigger cytochrome c release.16 Truncated Bid (tBid) is 10 fold more affine for Bcl XL than full length Bid and is also 100 times more effective in releasing cytochrome c from mitochondria.14 It was also reported that tBid interacts with mitochondrial contact sites by a specific interaction with cardiolipins17, 18 and is able to induce remodelling of the mitochondrial membranes.19, 20 The recent observation that tBid stimulates unravelling of the mitochondrial cristae and enhances the mobilization of cytochrome c19 indicates that the early events affecting mitochondrial structure and function are linked to the insertion of tBid into the mitochondrial membrane.19 Despite the increasing knowledge on the mechanisms of tBid interaction with mitochondria, the consequences of this interaction on mitochondrial bioenergetic properties have not been elucidated. The present study analyzes the consequences of tBid interaction with mitochondrial contact sites, and the mechanism by which tBid alters the bioenergetic properties of mitochondria. Using mitochondria isolated from wild type cells, we demonstrated for the first time that tBid acts independently of Bak and Bax to slightly increase state 4 respiration as a result of uncoupling. Moreover, tBid inhibited ADP stimulated respiration. Both these effects do not require the BH3 domain of tBid in the first place. We also report that Bcl 2 and Bcl XL protect mitochondria from these changes via direct interaction between their hydrophobic pocket and the BH3 domain of tBid. We therefore showed that tBid inhibits ADP stimulated respiration by indirect inhibition of the activity of the adenine nucleotide translocator (ANT), mediated by cardiolipin reorganization into the mitochondrial inner membrane. We also proved in this paper that the absence of cardiolipin (and consequently cardiolipin derivatives) in a cardiolipin synthase deficient yeast consistant with our observations, abolished subsequent effects of tBid. These results are based on biophysical approaches, showing that cardiolipins are required for tBid interaction with synthetic lipid monolayers. We finally showed that tBid triggered cytochrome c release from the mitochondrial intermembrane space in part by transient mitochondrial permeability transition pore (PTP) opening and also by a mitochondrial membrane permeabilization insensitive to cyclosporine A (CSA). This mitochondrial membrane permeabilization is probably due to membrane rigidification linked to tBid/cardiolipin relationship primarily at the contact sites with a subsequent action onto cardiolipin reorganization throughout the whole inner mitochondrial membrane. These findings demonstrate that tBid plays a primary role in the mitochondria mediated apoptosis pathway by disturbing mitochondrial bioenergetics. This may be a key event in predisposing mitochondria to the synergistic effect of tBid with Bax and Bak. Top of pageResultsMitochondrial bioenergetics: basic propertiesThroughout this work, we used Percoll purified liver mitochondria from C57/Bl6 wild type (WT) mice, transgenic mice that overexpress Bcl 230 or Bcl XL,31 and BAX / pandora finished bracelets and BAK / mice. First, we investigated the basic bioenergetic properties of these mitochondria by monitoring succinate oxidizing mitochondria. In particular, the oxidation rate (Voxidation), respiratory control (RC), ADP/O ratio, mitochondrial membrane potential (m), and phosphorylation rate (Vphos.) were analyzed as well as the basic conditions for PTP opening (in terms of large amplitude swelling' (Figure 1). Each parameter was obtained from the simultaneous measurement of oxygen consumption, TPP+ concentration and pH. A typical trace control (control mitochondria) is represented in Figure 2a. Oxidative phosphorylation in mouse liver mitochondria. The system shows all steps of the oxidative phosphorylation via three sets of reactions that were tested in (a), and are connected by the common thermodynamic intermediate (p).41, 42 (b) The table summarizes the main characteristics of the mitochondria (control, Bcl 2, Bcl XL, Bax+/+ and Bax / ) used for the studies. Mitochondria (0.333 mg/ml) were incubated with respiratory buffer alone (a), or when indicated with various amounts of recombinant Bid (tBid) that has been cleaved with recombinant active caspase 8 (b) in a final volume of 3 ml. In (c), mitochondria from transgenic mice overexpressing Bcl 2 were used. (a) Recording of the oxidation rate (black line), mitochondrial membrane potential (green line) and phosphorylation rate (blue line) in control mitochondria. The numbers along the trace give the oxidation rate in nmol O2/min mg protein (black), the potential in mV (green), and the phosphorylation rate in nmole ATP/min/mg of mitochondrial protein (blue). The arrows along the trace correspond to ADP 110 M additions unless otherwise noted. The uncoupler mClCCP (10 M) is added at the end of the trace. (b) tBid (10 nM) was added directly after a first state 3/state 4 transition. Two subsequent ADP additions (110 M) were performed, followed by an addition of 10 M mClCCP. (c) Same as (a) with mitochondria overexpressing Bcl 2. (d and e) Histograms presenting the Bcl 2 (d) and Bcl XL (e) inhibition of state 3 respiration counteracted by preincubation with the BH 3 domain peptide of tBid. Data presented were extracted from respiratory measurements with simultaneous recording of the mitochondrial membrane potential as shown in (a In both cases, tBid BH3 peptides are used at concentrations (1 M) that did not cause apoptosis. (d) Oxidation rate pandora charm warranty in the presence of succinate of control BcP 2 mitochondria pretreated for 10 min with 10 nM tBid. ADP 110 M was added after tBid addition into the closed glass vessel. (e) Same experiment in the presence of Bcl XL or Bcl 2 mitochondria. In (f), histograms of the oxidation rate in the presence of succinate of control mitochondria treated with 10 nM tBidG94E mutant IIIm, and with Bcl 2 or Bcl XL mitochondria are shown. (g) Same experiments in the presence of Bax or Bak deficient mitochondria. For all the histograms from (d) to (g), the oxidation rate is normalized as the percentage of the maximal oxidation. The Vmax in the control mitochondria is of 744 nmol/min/mg protein for c57/Bl6 mitochondria and of 844 and 855 nmol O2/min/mg O2 for the mitochondria purified from Bcl XL and Bcl 2 transgenic mice, respectively, whereas it is of 704 and 695 for Bax / and Bak / mitochondria respectively Full figure and legend (243K) Once added into the respiratory medium, the mitochondria began to oxidize succinate and build up a membrane potential by the proton pumping activity of the respiratory complexes, at a high value associated with state 4 respiration. The addition of limited amounts of ADP (200 M) induced phosphorylation coupled respiration (state 3 respiration) and proton influx via the ATP synthase F0 channel, leading to depolarization and a higher oxidative rate of around 71 nmol O2/min/mg protein (Figure 1b). In the case of control mitochondria, m in state 4 (m4) was around 174 mV. The respiratory control ratio (RC), which corresponds to the ratio of the respiratory rate of the phosphorylation state (Vox3) to unphosphorylation state (Vox4), reflects the coupling of the mitochondria. P1,P5 di(adenosine 5')pentaphosphate, an inhibitor of kinase adenylate), the RC is normally over 4.5. The phosphorylation yield, ADP/O, is around 1.5 for succinate oxidizing mitochondria. Despite showing similar RC and ADP/O, Bcl 2 or Bcl XL displaying mitochondria exhibit a higher oxidation rate associated with an increase in the phosphorylation rate (Figure 1b). The ability of Bcl 2 or Bcl XL containing mitochondria to phosphorylate ADP at a faster rate may be linked to their enhanced capacity to exchange ADP/ATP.37 Bcl XL acts similarly during a metabolic arrest induced by growth factor withdrawal.27 Under these conditions, Bcl XL may allow growth factor deprived cells to maintain sufficient ADP/ATP exchange to sustain coupled respiration. Moreover, Bcl 2 and Bcl XL mitochondria are less sensitive to Ca2+ induced PTP opening as revealed by the increasing dose of Ca2+ required to induce CsA sensitive swelling (Figure 1c). Our results are consistent with a previous report in which Bcl 2 potentiates maximum calcium uptake capacity in neuron derived mitochondria.43 The calcium concentrations required to induce swelling of liver mitochondria are similar to those in neural cell mitochondria. The enhanced ability of mitochondria from Bcl 2 and Bcl XL overexpressed cells to sequester large quantities of Ca2+ without any profound respiratory impairment provides a plausible explanation for the mechanism by which Bcl 2 and Bcl XL inhibit certain forms of cell death. On the other hand, the absence of Bax or Bak has no effect on mitochondrial bioenergetics. Indeed, Bax / or Bak+/+ mitochondria exhibited bionergetic properties similar to control WT mitochondria (Figure 1b). tBid disturbs mitochondrial bioenergetics: a process independent of Bax and Bak but inhibited by Bcl 2 and Bcl XLWe determined the action of recombinant tBid on mitochondrial bioenergetic parameters by adding tBid in wild type succinate oxidizing mitochondria after a first transition of phosphorylation (Figure 2b). Adding 10 nM of tBid immediately induces a 14 mV decrease in m, associated with a slight increase in the respiratory rate by approximately 3 nmol O2/min/mg of mitochondrial protein (mild uncoupling). A lower concentration of tBid (2/min/mg of mitochondrial protein, associated with a decrease in the phosphorylation rate by around 63 nmole ATP/min/mg of mitochondrial protein after the first ADP addition. As shown in Figure 2b, tBid acted in a time dependent manner. The inhibition in state 3 respiration and phosphorylation increased with time and marked 30% inhibition after 10 min incubation with tBid (Figure 2b). Inhibition of both ADP stimulated respiration and silver pandora charm bracelet phosphorylation rate also increased after a second ADP addition and reached 60% inhibition after 30 min (not shown). Three hypotheses could explain these inhibitions: (i) tBid might inhibit the respiratory chain (generator of proton gradient) but this hypothesis is inconsistent with the finding that tBid has no effect on the mClCCP uncoupled respiration rate (Figure 2b), (ii) tBid may affect the machinery of phosphorylation (consumer of proton gradient) or (iii) tBid may cause increase in proton leaks by one way or another leading to a decrease in proton gradient. As shown in Figure 2c tBid is unable to disrupt any bioenergetic parameters (m, respiration and phosphorylation rate) in Bcl 2 and Bcl XL expressing mitochondria. Indeed, Bcl 2 and Bcl XL completely protect mitochondria from tBid even after 10 min incubation. On the other hand, injecting a noncytotoxic dose of tBid BH3 peptide (1 M) together with tBid completely restored the effect on tBid in Bcl 2 and Bcl XL mitochondria. The same results were obtained using BH3 peptide derived from Bax or Bak (data not shown). Our results suggest that Bcl 2 and Bcl XL protect mitochondria from bioenergetic alterations through the interaction with the BH3 domain of tBid. We therefore investigated the effects of BH3 mutation on tBid induced alterations of mitochondrial physiology.
To this end, we used the tBid mutant mIII (tBidG94E), which is unable to interact with either proapoptotic (Bax and Bak) or antiapoptotic (Bcl XL and Bcl 2) proteins. Adding 10 nM of mutant tBid mIII inhibited ADP stimulated respiration to the same level in control mitochondria as tBid (Figure 2f), suggesting that the interaction of tBid with other apoptotic factors like Bax or Bak is not required for inhibition of ADP stimulated respiration. Moreover, the effect of tBid mIII was maintained in Bcl 2 and Bcl XL mitochondria and in mitochondria purified from bax / and bak / mice (Figure 2g).
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