Limits...
The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2000)

Bottom Line: The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr.Again, this effect is prevented by addition of recombinant Bcl-2.Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, F-94801 Villejuif, France.

ABSTRACT
Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Show MeSH
Effects of Vpr on the PTPC or PTPC components reconstituted into liposomes. (A) Effect of Vpr on purified PTPCs. PTPCs were reconstituted into phosphatidylcholine/cholesterol liposomes  26. These PTPC liposomes were loaded with DiOC6(3), the retention of which was measured after treatment with SDS (0.1%), Atr (250 μM), or the indicated Vpr peptides ± BA (50 μM) or CsA (1μM), as detailed in Materials and Methods. (B) Effect of Vpr on liposomes containing purified individual proteins. Phosphatidylcholine/cardiolipin liposomes containing ANT (from rat heart), Bcl-2 (recombinant), and/or Bax (recombinant) at a molar ratio of 4:4:1 were treated with SDS (0.1%), Atr (100 μM), or Vpr52-96 (1 μM), followed by determination of DiOC6(3) release. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195797&req=5

Figure 9: Effects of Vpr on the PTPC or PTPC components reconstituted into liposomes. (A) Effect of Vpr on purified PTPCs. PTPCs were reconstituted into phosphatidylcholine/cholesterol liposomes 26. These PTPC liposomes were loaded with DiOC6(3), the retention of which was measured after treatment with SDS (0.1%), Atr (250 μM), or the indicated Vpr peptides ± BA (50 μM) or CsA (1μM), as detailed in Materials and Methods. (B) Effect of Vpr on liposomes containing purified individual proteins. Phosphatidylcholine/cardiolipin liposomes containing ANT (from rat heart), Bcl-2 (recombinant), and/or Bax (recombinant) at a molar ratio of 4:4:1 were treated with SDS (0.1%), Atr (100 μM), or Vpr52-96 (1 μM), followed by determination of DiOC6(3) release. Data are representative of three independent experiments.

Mentions: To confirm the hypothesis that Vpr might permeabilize mitochondrial membranes by a direct effect on the PTPC, we purified this molecular complex from brain 27 29, reconstituted it into liposomes, and measured the capacity of Vpr to permeabilize the liposomal membrane ( Fig. 9 A). Vpr or Vpr52-96 increases the permeability of liposomes containing the PTPC, and this effect is inhibited by CsA or BA ( Fig. 9 A). In addition, Vpr52-96 acts on a combination of two proteins from the PTPC, ANT plus Bax, and this effect is suppressed by recombinant Bcl-2 ( Fig. 9 B). Thus, Vpr acts on the PTPC to perturb the barrier function of mitochondrial membranes.


The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G - J. Exp. Med. (2000)

Effects of Vpr on the PTPC or PTPC components reconstituted into liposomes. (A) Effect of Vpr on purified PTPCs. PTPCs were reconstituted into phosphatidylcholine/cholesterol liposomes  26. These PTPC liposomes were loaded with DiOC6(3), the retention of which was measured after treatment with SDS (0.1%), Atr (250 μM), or the indicated Vpr peptides ± BA (50 μM) or CsA (1μM), as detailed in Materials and Methods. (B) Effect of Vpr on liposomes containing purified individual proteins. Phosphatidylcholine/cardiolipin liposomes containing ANT (from rat heart), Bcl-2 (recombinant), and/or Bax (recombinant) at a molar ratio of 4:4:1 were treated with SDS (0.1%), Atr (100 μM), or Vpr52-96 (1 μM), followed by determination of DiOC6(3) release. Data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195797&req=5

Figure 9: Effects of Vpr on the PTPC or PTPC components reconstituted into liposomes. (A) Effect of Vpr on purified PTPCs. PTPCs were reconstituted into phosphatidylcholine/cholesterol liposomes 26. These PTPC liposomes were loaded with DiOC6(3), the retention of which was measured after treatment with SDS (0.1%), Atr (250 μM), or the indicated Vpr peptides ± BA (50 μM) or CsA (1μM), as detailed in Materials and Methods. (B) Effect of Vpr on liposomes containing purified individual proteins. Phosphatidylcholine/cardiolipin liposomes containing ANT (from rat heart), Bcl-2 (recombinant), and/or Bax (recombinant) at a molar ratio of 4:4:1 were treated with SDS (0.1%), Atr (100 μM), or Vpr52-96 (1 μM), followed by determination of DiOC6(3) release. Data are representative of three independent experiments.
Mentions: To confirm the hypothesis that Vpr might permeabilize mitochondrial membranes by a direct effect on the PTPC, we purified this molecular complex from brain 27 29, reconstituted it into liposomes, and measured the capacity of Vpr to permeabilize the liposomal membrane ( Fig. 9 A). Vpr or Vpr52-96 increases the permeability of liposomes containing the PTPC, and this effect is inhibited by CsA or BA ( Fig. 9 A). In addition, Vpr52-96 acts on a combination of two proteins from the PTPC, ANT plus Bax, and this effect is suppressed by recombinant Bcl-2 ( Fig. 9 B). Thus, Vpr acts on the PTPC to perturb the barrier function of mitochondrial membranes.

Bottom Line: The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr.Again, this effect is prevented by addition of recombinant Bcl-2.Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique, F-94801 Villejuif, France.

ABSTRACT
Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Show MeSH