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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.

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Mitochondrial Vpr effects in intact cells. COS cells were treated for 3 h with 1 μM Vpr1-51 (negative control) or Vpr52-96, fixed, permeabilized, and immunostained with antibodies specific for AIF or cytochrome (Cyt.) c (both normally in the mitochondrial intermembrane space revealed PE, red fluorescence) and the mitochondrial matrix protein Hsp60 or the inner mitochondrial membrane protein COX (both revealed by FITC, green fluorescence). In addition, cells were stained with the ΔΨm-sensitive dye CMXRos (red fluorescence) and the DNA intercalating agent Hoechst 33342 (blue fluorescence). The histograms indicate the percentage of cells manifesting mitochondrionuclear AIF translocation, mitochondriocytosolic cytochrome c translocation, or a low ΔΨm after treatment with different Vpr peptides (1 μM) in the presence or absence of Z-VAD.fmk (50 μM).
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Figure 3: Mitochondrial Vpr effects in intact cells. COS cells were treated for 3 h with 1 μM Vpr1-51 (negative control) or Vpr52-96, fixed, permeabilized, and immunostained with antibodies specific for AIF or cytochrome (Cyt.) c (both normally in the mitochondrial intermembrane space revealed PE, red fluorescence) and the mitochondrial matrix protein Hsp60 or the inner mitochondrial membrane protein COX (both revealed by FITC, green fluorescence). In addition, cells were stained with the ΔΨm-sensitive dye CMXRos (red fluorescence) and the DNA intercalating agent Hoechst 33342 (blue fluorescence). The histograms indicate the percentage of cells manifesting mitochondrionuclear AIF translocation, mitochondriocytosolic cytochrome c translocation, or a low ΔΨm after treatment with different Vpr peptides (1 μM) in the presence or absence of Z-VAD.fmk (50 μM).

Mentions: In Jurkat cells, Vpr caused a loss of ΔΨm, which was followed by an increase in the production of superoxide anion ( Fig. 1 B) and nuclear apoptosis ( Fig. 1 C). This early effect on the ΔΨm (1–2 h after addition of Vpr or Vpr52-96) was transiently inhibited by CsA and BA, two inhibitors of the PTPC ( Fig. 1 B). Similar results were obtained with primary cells such as mouse thymocytes (not shown) and human primary PBLs, in which the ΔΨm reducing effect of Vpr or Vpr52-96 is counteracted by the ANT ligand BA ( Fig. 2 A). The ΔΨm loss was also inhibited by overexpression of Bcl-2 ( Fig. 1 C), an endogenous cytoprotective protein acting on the PTPC 27 29. Bcl-2 concomitantly prevented other Vpr-induced features of apoptosis, such as phosphatidylserine exposure on the plasma membrane and nuclear DNA loss ( Fig. 1 C). In contrast, the pancaspase inhibitor Z-VAD.fmk failed to prevent the ΔΨm dissipation, although it did reduce the (caspase-dependent) DNA loss resulting in hypoploidy ( Fig. 1 C). Vpr52-96 induced, in intact cells, the mitochondrionuclear translocation of AIF and the mitochondriocytosolic translocation of cytochrome c, as detected by confocal immunofluorescence microscopy ( Fig. 3). Vpr also caused nuclear chromatin condensation (measured with Hoechst 33342), as well as a dissipation of the ΔΨm, as measured with the ΔΨm-sensitive dye CMXRos ( Fig. 3). Again, Z-VAD.fmk (which did prevent end-stage nuclear chromatin condensation) had no mitochondrioprotective effects ( Fig. 2). Altogether, these findings indicate that the mitochondrial effects of Vpr are caspase independent yet suppressed by PTPC inhibitors such as CsA, BA, or Bcl-2.


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)

Mitochondrial Vpr effects in intact cells. COS cells were treated for 3 h with 1 μM Vpr1-51 (negative control) or Vpr52-96, fixed, permeabilized, and immunostained with antibodies specific for AIF or cytochrome (Cyt.) c (both normally in the mitochondrial intermembrane space revealed PE, red fluorescence) and the mitochondrial matrix protein Hsp60 or the inner mitochondrial membrane protein COX (both revealed by FITC, green fluorescence). In addition, cells were stained with the ΔΨm-sensitive dye CMXRos (red fluorescence) and the DNA intercalating agent Hoechst 33342 (blue fluorescence). The histograms indicate the percentage of cells manifesting mitochondrionuclear AIF translocation, mitochondriocytosolic cytochrome c translocation, or a low ΔΨm after treatment with different Vpr peptides (1 μM) in the presence or absence of Z-VAD.fmk (50 μM).
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Related In: Results  -  Collection

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Figure 3: Mitochondrial Vpr effects in intact cells. COS cells were treated for 3 h with 1 μM Vpr1-51 (negative control) or Vpr52-96, fixed, permeabilized, and immunostained with antibodies specific for AIF or cytochrome (Cyt.) c (both normally in the mitochondrial intermembrane space revealed PE, red fluorescence) and the mitochondrial matrix protein Hsp60 or the inner mitochondrial membrane protein COX (both revealed by FITC, green fluorescence). In addition, cells were stained with the ΔΨm-sensitive dye CMXRos (red fluorescence) and the DNA intercalating agent Hoechst 33342 (blue fluorescence). The histograms indicate the percentage of cells manifesting mitochondrionuclear AIF translocation, mitochondriocytosolic cytochrome c translocation, or a low ΔΨm after treatment with different Vpr peptides (1 μM) in the presence or absence of Z-VAD.fmk (50 μM).
Mentions: In Jurkat cells, Vpr caused a loss of ΔΨm, which was followed by an increase in the production of superoxide anion ( Fig. 1 B) and nuclear apoptosis ( Fig. 1 C). This early effect on the ΔΨm (1–2 h after addition of Vpr or Vpr52-96) was transiently inhibited by CsA and BA, two inhibitors of the PTPC ( Fig. 1 B). Similar results were obtained with primary cells such as mouse thymocytes (not shown) and human primary PBLs, in which the ΔΨm reducing effect of Vpr or Vpr52-96 is counteracted by the ANT ligand BA ( Fig. 2 A). The ΔΨm loss was also inhibited by overexpression of Bcl-2 ( Fig. 1 C), an endogenous cytoprotective protein acting on the PTPC 27 29. Bcl-2 concomitantly prevented other Vpr-induced features of apoptosis, such as phosphatidylserine exposure on the plasma membrane and nuclear DNA loss ( Fig. 1 C). In contrast, the pancaspase inhibitor Z-VAD.fmk failed to prevent the ΔΨm dissipation, although it did reduce the (caspase-dependent) DNA loss resulting in hypoploidy ( Fig. 1 C). Vpr52-96 induced, in intact cells, the mitochondrionuclear translocation of AIF and the mitochondriocytosolic translocation of cytochrome c, as detected by confocal immunofluorescence microscopy ( Fig. 3). Vpr also caused nuclear chromatin condensation (measured with Hoechst 33342), as well as a dissipation of the ΔΨm, as measured with the ΔΨm-sensitive dye CMXRos ( Fig. 3). Again, Z-VAD.fmk (which did prevent end-stage nuclear chromatin condensation) had no mitochondrioprotective effects ( Fig. 2). Altogether, these findings indicate that the mitochondrial effects of Vpr are caspase independent yet suppressed by PTPC inhibitors such as CsA, BA, or Bcl-2.

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
Related in: MedlinePlus