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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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Physical (A and B) and functional (C) interaction between Vpr and liposomes containing ANT. (A) Dose–response curve of FITC-labeled Vpr52-96 binding on ANT liposomes and plain liposomes. (B) Binding of FITC–Vpr52-96 (2 μM) to plain liposomes, ANT proteoliposomes, in the presence or absence of BA (50 μM). (C) Permeabilization of ANT proteoliposomes by Vpr (X ± SD, n = 3). Liposomes were loaded with 4-MUP and exposed for 60 min to Atr (200 μM) or the indicated Vpr-derived peptides (1 μM), in the presence or absence of BA (50 μM), ADP (800 μM), and/or the indicated peptides (same as in B, 0.5 μM, preincubated with Vpr52-96 for 5 min). Then, alkaline phosphatase was added to convert liposome-released 4-MUP into the fluorochrome 4-MU, and the percentage of 4-MUP release induced by Vpr-derived peptides was calculated as described in Materials and Methods.
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Figure 2: Physical (A and B) and functional (C) interaction between Vpr and liposomes containing ANT. (A) Dose–response curve of FITC-labeled Vpr52-96 binding on ANT liposomes and plain liposomes. (B) Binding of FITC–Vpr52-96 (2 μM) to plain liposomes, ANT proteoliposomes, in the presence or absence of BA (50 μM). (C) Permeabilization of ANT proteoliposomes by Vpr (X ± SD, n = 3). Liposomes were loaded with 4-MUP and exposed for 60 min to Atr (200 μM) or the indicated Vpr-derived peptides (1 μM), in the presence or absence of BA (50 μM), ADP (800 μM), and/or the indicated peptides (same as in B, 0.5 μM, preincubated with Vpr52-96 for 5 min). Then, alkaline phosphatase was added to convert liposome-released 4-MUP into the fluorochrome 4-MU, and the percentage of 4-MUP release induced by Vpr-derived peptides was calculated as described in Materials and Methods.

Mentions: Surface plasmon resonance measurements indicate that purified detergent-solubilized ANT protein binds to the immobilized Vpr COOH-terminal moiety biotin–Vpr52-96 (but to a far lesser extent to mutated biotin–Vpr52-96[R73,80A]) with an affinity in the nanomolar range (Fig. 1A and Fig. B). This interaction was modulated by two ANT ligands which differentially affect ANT conformation 35, namely the PTPC activator (opener) Atr, which favored Vpr binding, and the PTPC inhibitor (closer) bongkrekic acid (BA), which reduced Vpr binding (Fig. 1 C). Bcl-2–like proteins bind to a motif of ANT (amino acids 105–155; reference 8) whose implication in apoptosis control has been confirmed by deletion mapping 36. This motif partially overlaps with the second ANT loop (amino acids 92–116), a regulatory domain exposed to the intermembrane space 3738. A peptide corresponding to the overlap between the Bcl-2 binding motif and this loop (ANT104-116) inhibited the ANT–Vpr interaction (Fig. 1 C), presumably via direct association with Vpr52-96 (Fig. 1 D). Neither a topologically related peptide motif derived from the human phosphate carrier nor mutated and scrambled versions of ANT104-116 (control peptides in Fig. 1 C) had such inhibitory effects. Vpr also interacted with ANT incorporated into liposomal membranes. Indeed, Vpr52-96 binding to membranes was greatly facilitated in liposomes in which ANT has been reconstituted compared with protein-free liposomes (Fig. 2 A). The ANT-facilitated incorporation of Vpr into membranes was inhibited by BA (Fig. 2 B). In conclusion, Vpr binds to ANT, at least in part via an interaction with a domain of ANT (ANT104-116) that is exposed to the intermembrane side of this protein, coinciding with the apoptogenic portion of ANT.


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)

Physical (A and B) and functional (C) interaction between Vpr and liposomes containing ANT. (A) Dose–response curve of FITC-labeled Vpr52-96 binding on ANT liposomes and plain liposomes. (B) Binding of FITC–Vpr52-96 (2 μM) to plain liposomes, ANT proteoliposomes, in the presence or absence of BA (50 μM). (C) Permeabilization of ANT proteoliposomes by Vpr (X ± SD, n = 3). Liposomes were loaded with 4-MUP and exposed for 60 min to Atr (200 μM) or the indicated Vpr-derived peptides (1 μM), in the presence or absence of BA (50 μM), ADP (800 μM), and/or the indicated peptides (same as in B, 0.5 μM, preincubated with Vpr52-96 for 5 min). Then, alkaline phosphatase was added to convert liposome-released 4-MUP into the fluorochrome 4-MU, and the percentage of 4-MUP release induced by Vpr-derived peptides was calculated as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Physical (A and B) and functional (C) interaction between Vpr and liposomes containing ANT. (A) Dose–response curve of FITC-labeled Vpr52-96 binding on ANT liposomes and plain liposomes. (B) Binding of FITC–Vpr52-96 (2 μM) to plain liposomes, ANT proteoliposomes, in the presence or absence of BA (50 μM). (C) Permeabilization of ANT proteoliposomes by Vpr (X ± SD, n = 3). Liposomes were loaded with 4-MUP and exposed for 60 min to Atr (200 μM) or the indicated Vpr-derived peptides (1 μM), in the presence or absence of BA (50 μM), ADP (800 μM), and/or the indicated peptides (same as in B, 0.5 μM, preincubated with Vpr52-96 for 5 min). Then, alkaline phosphatase was added to convert liposome-released 4-MUP into the fluorochrome 4-MU, and the percentage of 4-MUP release induced by Vpr-derived peptides was calculated as described in Materials and Methods.
Mentions: Surface plasmon resonance measurements indicate that purified detergent-solubilized ANT protein binds to the immobilized Vpr COOH-terminal moiety biotin–Vpr52-96 (but to a far lesser extent to mutated biotin–Vpr52-96[R73,80A]) with an affinity in the nanomolar range (Fig. 1A and Fig. B). This interaction was modulated by two ANT ligands which differentially affect ANT conformation 35, namely the PTPC activator (opener) Atr, which favored Vpr binding, and the PTPC inhibitor (closer) bongkrekic acid (BA), which reduced Vpr binding (Fig. 1 C). Bcl-2–like proteins bind to a motif of ANT (amino acids 105–155; reference 8) whose implication in apoptosis control has been confirmed by deletion mapping 36. This motif partially overlaps with the second ANT loop (amino acids 92–116), a regulatory domain exposed to the intermembrane space 3738. A peptide corresponding to the overlap between the Bcl-2 binding motif and this loop (ANT104-116) inhibited the ANT–Vpr interaction (Fig. 1 C), presumably via direct association with Vpr52-96 (Fig. 1 D). Neither a topologically related peptide motif derived from the human phosphate carrier nor mutated and scrambled versions of ANT104-116 (control peptides in Fig. 1 C) had such inhibitory effects. Vpr also interacted with ANT incorporated into liposomal membranes. Indeed, Vpr52-96 binding to membranes was greatly facilitated in liposomes in which ANT has been reconstituted compared with protein-free liposomes (Fig. 2 A). The ANT-facilitated incorporation of Vpr into membranes was inhibited by BA (Fig. 2 B). In conclusion, Vpr binds to ANT, at least in part via an interaction with a domain of ANT (ANT104-116) that is exposed to the intermembrane side of this protein, coinciding with the apoptogenic portion of ANT.

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