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Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral

E Jacotot¹, K F Ferri, C El Hamel

¹Centre National de la Recherche Scientifique, UMR 1599, Institut Gustave Roussy, F-94805 Villejuif, France.

The Journal of Experimental Medicine

|February 22, 2001

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.

Insights

HIV-1

Area of Science:

Mitochondrial biology
Virology
Molecular cell biology

Background:

Viral protein R (Vpr) from HIV-1 induces apoptosis by permeabilizing mitochondrial membranes.
This process involves the permeability transition pore complex (PTPC), including VDAC and ANT.
The precise role of ANT in Vpr-mediated mitochondrial membrane permeabilization (MMP) requires further elucidation.

Purpose of the Study:

To investigate the specific interaction between Vpr and ANT.
To determine the role of ANT in Vpr-induced MMP.
To explore the mechanism by which Bcl-2 modulates Vpr-induced MMP.

Main Methods:

Utilized a synthetic Vpr-derived peptide (Vpr52-96) for binding studies.
Employed planar lipid bilayers to assess channel formation.
Investigated effects on isolated mitochondria, including respiration and membrane potential.
Used affinity purification and surface plasmon resonance to study protein interactions.

Main Results:

Vpr52-96 binds specifically to the adenine nucleotide translocator (ANT) with nanomolar affinity.
Vpr52-96 and ANT cooperatively form conductance channels in lipid bilayers.
Vpr52-96 induces inner mitochondrial membrane permeabilization (MMP) and uncouples respiration.
Bcl-2 inhibits Vpr-induced MMP by reducing the ANT-Vpr interaction and suppressing channel formation.

Conclusions:

Vpr and Bcl-2 directly modulate mitochondrial membrane permeabilization (MMP) through interactions with ANT.
ANT plays a critical role in Vpr-mediated MMP.
Bcl-2 acts as an inhibitor by disrupting the ANT-Vpr complex formation.

Read full analysis

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