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Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Jacotot E, Ferri KF, El Hamel C, Brenner C, Druillennec S, Hoebeke J, Rustin P, Métivier D, Lenoir C, Geuskens M, Vieira HL, Loeffler M, Belzacq AS, Briand JP, Zamzami N, Edelman L, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2001)

Bottom Line: Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies.Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes.In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F-94805 Villejuif, France.

ABSTRACT
Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

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Oxidative properties of purified mitochondria exposed to Vpr. Oxygen consumption curves after addition of the indicated agents. Trace a: control mitochondria (no pretreatment). Trace b: mitochondria pretreated for 10 min with 1 μM Vpr52-96. Numbers along the traces are nanomoles of O2 consumed per minute per milligram of protein.
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Figure 4: Oxidative properties of purified mitochondria exposed to Vpr. Oxygen consumption curves after addition of the indicated agents. Trace a: control mitochondria (no pretreatment). Trace b: mitochondria pretreated for 10 min with 1 μM Vpr52-96. Numbers along the traces are nanomoles of O2 consumed per minute per milligram of protein.

Mentions: Compared with untreated organelles (Fig. 4 A, trace a), purified mitochondria preincubated with Vpr52-96 (Fig. 4 A, trace b) exhibited a gross deficiency in respiratory control (RC). Vpr increased succinate oxidation preceding ADP addition and abolished both the inhibitory effect of oligomycin (a specific ATPase inhibitor) and the stimulatory effect of uncoupling by the protonophore CCCP. Thus, Vpr52-96 (but not Vpr1-51) reduced the RC (ratio of oxygen consumption with oligomycin versus CCCP) to a value of 1.1, compared with 5.3 in control mitochondria (Table ). The entire Vpr protein (Vpr1-96), and a short peptide corresponding to the minimum “mitochondriotoxic” motif of Vpr (Vpr71-82) 2223, also reduced the RC values (Table ). Noticeably, the Vpr-induced loss of RC was not associated with a significant decrease of the oxidation rate (Fig. 4 A), suggesting that no major loss of membrane-bound cytochrome c occurred on short-term incubation with Vpr52-96. Accordingly, adding cytochrome c to Vpr52-96–treated mitochondria oxidizing succinate did not stimulate the rate of oxygen uptake (Fig. 4 A, trace b). The observation of Vpr-mediated uncoupling of the respiratory chain prompted us to test its capacity to induce IM permeabilization. The IM being essentially impermeable to NADH 41, no significant oxygen uptake could be measured when NADH was added to control mitochondria (Fig. 5 A, trace a). However, addition of Vpr52-96 prompted a significant, NADH-stimulated oxygen consumption (Fig. 5 A, trace b). This indicates that Vpr permeabilized the IM both to protons (leading to uncoupling; Fig. 4 A, trace b) and NADH (Fig. 5 A, trace b). Moreover, addition of ADP (the physiological substrate of ANT) strongly inhibited the Vpr-dependent, NADH-stimulated oxygen consumption (Fig. 5 A, trace c), suggesting that the ANT–Vpr interaction is essential for inner MMP by Vpr.


Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Jacotot E, Ferri KF, El Hamel C, Brenner C, Druillennec S, Hoebeke J, Rustin P, Métivier D, Lenoir C, Geuskens M, Vieira HL, Loeffler M, Belzacq AS, Briand JP, Zamzami N, Edelman L, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2001)

Oxidative properties of purified mitochondria exposed to Vpr. Oxygen consumption curves after addition of the indicated agents. Trace a: control mitochondria (no pretreatment). Trace b: mitochondria pretreated for 10 min with 1 μM Vpr52-96. Numbers along the traces are nanomoles of O2 consumed per minute per milligram of protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195906&req=5

Figure 4: Oxidative properties of purified mitochondria exposed to Vpr. Oxygen consumption curves after addition of the indicated agents. Trace a: control mitochondria (no pretreatment). Trace b: mitochondria pretreated for 10 min with 1 μM Vpr52-96. Numbers along the traces are nanomoles of O2 consumed per minute per milligram of protein.
Mentions: Compared with untreated organelles (Fig. 4 A, trace a), purified mitochondria preincubated with Vpr52-96 (Fig. 4 A, trace b) exhibited a gross deficiency in respiratory control (RC). Vpr increased succinate oxidation preceding ADP addition and abolished both the inhibitory effect of oligomycin (a specific ATPase inhibitor) and the stimulatory effect of uncoupling by the protonophore CCCP. Thus, Vpr52-96 (but not Vpr1-51) reduced the RC (ratio of oxygen consumption with oligomycin versus CCCP) to a value of 1.1, compared with 5.3 in control mitochondria (Table ). The entire Vpr protein (Vpr1-96), and a short peptide corresponding to the minimum “mitochondriotoxic” motif of Vpr (Vpr71-82) 2223, also reduced the RC values (Table ). Noticeably, the Vpr-induced loss of RC was not associated with a significant decrease of the oxidation rate (Fig. 4 A), suggesting that no major loss of membrane-bound cytochrome c occurred on short-term incubation with Vpr52-96. Accordingly, adding cytochrome c to Vpr52-96–treated mitochondria oxidizing succinate did not stimulate the rate of oxygen uptake (Fig. 4 A, trace b). The observation of Vpr-mediated uncoupling of the respiratory chain prompted us to test its capacity to induce IM permeabilization. The IM being essentially impermeable to NADH 41, no significant oxygen uptake could be measured when NADH was added to control mitochondria (Fig. 5 A, trace a). However, addition of Vpr52-96 prompted a significant, NADH-stimulated oxygen consumption (Fig. 5 A, trace b). This indicates that Vpr permeabilized the IM both to protons (leading to uncoupling; Fig. 4 A, trace b) and NADH (Fig. 5 A, trace b). Moreover, addition of ADP (the physiological substrate of ANT) strongly inhibited the Vpr-dependent, NADH-stimulated oxygen consumption (Fig. 5 A, trace c), suggesting that the ANT–Vpr interaction is essential for inner MMP by Vpr.

Bottom Line: Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies.Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes.In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F-94805 Villejuif, France.

ABSTRACT
Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.

Show MeSH
Related in: MedlinePlus