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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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Vpr-induced swelling and ΔΨm dissipation in isolated mitochondria. Rat liver mitochondria were exposed to Vpr (2 μM) or the indicated Vpr derivative (1 μM), CsA (5 μM; added 1 min before Vpr to mitochondria), BA (50 μM; added 1 min before Vpr to mitochondria), a random eicosadesoxynucleotide (DNA; 1 μM), total cell RNA (1 μM), or Ncp7 (10 μM; added to Vpr 1 min before joint addition to mitochondria). Mitochondrial swelling (measured as 90° light scattering at 545 nm) or the ΔΨm (measured as Rh123 dequenching) were monitored continuously. ΔΨm and swelling determinations yielded concordant results. Representative curves obtained with either of the two methods are shown.
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Figure 5: Vpr-induced swelling and ΔΨm dissipation in isolated mitochondria. Rat liver mitochondria were exposed to Vpr (2 μM) or the indicated Vpr derivative (1 μM), CsA (5 μM; added 1 min before Vpr to mitochondria), BA (50 μM; added 1 min before Vpr to mitochondria), a random eicosadesoxynucleotide (DNA; 1 μM), total cell RNA (1 μM), or Ncp7 (10 μM; added to Vpr 1 min before joint addition to mitochondria). Mitochondrial swelling (measured as 90° light scattering at 545 nm) or the ΔΨm (measured as Rh123 dequenching) were monitored continuously. ΔΨm and swelling determinations yielded concordant results. Representative curves obtained with either of the two methods are shown.

Mentions: The release of mitochondrial proteins induced by Vpr in vitro was blocked by the PT pore inhibitor CsA ( Fig. 4B and Fig. C). Moreover, mitochondria isolated from Bcl-2–overexpressing cells were refractory to the Vpr-induced release of AIF activity ( Fig. 4 D). The fact that some of the Vpr effects were inhibited by PTPC inhibitors (CsA, BA, or Bcl-2) suggested that Vpr can act on the mitochondrial PT pore, the opening of which can be a rate-limiting step of the apoptotic process. Accordingly, Vpr induced two hallmarks of PTPC opening when added to purified mitochondria, namely mitochondrial volume increase and ΔΨm dissipation ( Fig. 5), and both of these effects were inhibited by CsA and BA. The effect of free holo Vpr on isolated mitochondria is fully mimicked by Vpr52-96 but not by Vpr52-96 R73A, Vpr52-96 R77A, or Vpr52-96 R80A ( Fig. 5). Preincubation of Vpr with a molar excess of RNA or DNA (which bind to the Vpr71-82 motif; reference 53) abolished its effects on isolated mitochondria ( Fig. 5), correlating with the data obtained in cells (not shown). In contrast, synthetic HIV-1 nucleocapsid protein NCp7 (which binds to the extreme COOH terminus of Vpr; reference 39) does not inhibit Vpr effects on mitochondria ( Fig. 5). Thus, the structural motifs of Vpr responsible for direct, presumably PTPC-mediated mitochondrial effects in vitro ( Fig. 5) and apoptosis induction in intact cells ( Fig. 1 C) are the same. This fact is also underscored by the comparison of the ED50 of different Vpr peptides determined on intact cells ( Fig. 6 A) and purified mitochondria ( Fig. 6 B).


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)

Vpr-induced swelling and ΔΨm dissipation in isolated mitochondria. Rat liver mitochondria were exposed to Vpr (2 μM) or the indicated Vpr derivative (1 μM), CsA (5 μM; added 1 min before Vpr to mitochondria), BA (50 μM; added 1 min before Vpr to mitochondria), a random eicosadesoxynucleotide (DNA; 1 μM), total cell RNA (1 μM), or Ncp7 (10 μM; added to Vpr 1 min before joint addition to mitochondria). Mitochondrial swelling (measured as 90° light scattering at 545 nm) or the ΔΨm (measured as Rh123 dequenching) were monitored continuously. ΔΨm and swelling determinations yielded concordant results. Representative curves obtained with either of the two methods are shown.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: Vpr-induced swelling and ΔΨm dissipation in isolated mitochondria. Rat liver mitochondria were exposed to Vpr (2 μM) or the indicated Vpr derivative (1 μM), CsA (5 μM; added 1 min before Vpr to mitochondria), BA (50 μM; added 1 min before Vpr to mitochondria), a random eicosadesoxynucleotide (DNA; 1 μM), total cell RNA (1 μM), or Ncp7 (10 μM; added to Vpr 1 min before joint addition to mitochondria). Mitochondrial swelling (measured as 90° light scattering at 545 nm) or the ΔΨm (measured as Rh123 dequenching) were monitored continuously. ΔΨm and swelling determinations yielded concordant results. Representative curves obtained with either of the two methods are shown.
Mentions: The release of mitochondrial proteins induced by Vpr in vitro was blocked by the PT pore inhibitor CsA ( Fig. 4B and Fig. C). Moreover, mitochondria isolated from Bcl-2–overexpressing cells were refractory to the Vpr-induced release of AIF activity ( Fig. 4 D). The fact that some of the Vpr effects were inhibited by PTPC inhibitors (CsA, BA, or Bcl-2) suggested that Vpr can act on the mitochondrial PT pore, the opening of which can be a rate-limiting step of the apoptotic process. Accordingly, Vpr induced two hallmarks of PTPC opening when added to purified mitochondria, namely mitochondrial volume increase and ΔΨm dissipation ( Fig. 5), and both of these effects were inhibited by CsA and BA. The effect of free holo Vpr on isolated mitochondria is fully mimicked by Vpr52-96 but not by Vpr52-96 R73A, Vpr52-96 R77A, or Vpr52-96 R80A ( Fig. 5). Preincubation of Vpr with a molar excess of RNA or DNA (which bind to the Vpr71-82 motif; reference 53) abolished its effects on isolated mitochondria ( Fig. 5), correlating with the data obtained in cells (not shown). In contrast, synthetic HIV-1 nucleocapsid protein NCp7 (which binds to the extreme COOH terminus of Vpr; reference 39) does not inhibit Vpr effects on mitochondria ( Fig. 5). Thus, the structural motifs of Vpr responsible for direct, presumably PTPC-mediated mitochondrial effects in vitro ( Fig. 5) and apoptosis induction in intact cells ( Fig. 1 C) are the same. This fact is also underscored by the comparison of the ED50 of different Vpr peptides determined on intact cells ( Fig. 6 A) and purified mitochondria ( Fig. 6 B).

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