Control of Mitochondrial Membrane Permeabilization by Adenine Nucleotide Translocator Interacting with HIV-1 Viral Protein R and Bcl-2

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.

Gelsolin segment 5 inhibits HIV‐induced T‐cell apoptosis via Vpr‐binding to VDAC

“…The effects of both mitochondrial and cytotoxic Vpr are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC) [19]. Vpr favors the permeabilization of artificial membranes containing purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax.…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…Indeed, pharmacological inhibitors of these proteins (cyclosporin A for CypD and bongkrekic acid for ANT) are able to prevent cell death, at least in some models of apoptosis (in vitro and in vivo) [5,36]. Interestingly, the viral protein R (Vpr) from human immunodeficiency virus type I (HIV-1) exerts cytotoxic effects by promoting MMP via direct interaction with ANT (as assessed ex vivo, in purified mitochondria and artificial membranes containing ANT) [37][38][39].…”

Human immunodeficiency virus type 1 (HIV-1) Vpr-regulated cell death: insights into mechanism

“…Specifically, the carboxyl-terminus of Vpr interacts with the adenine nucleotide translocator (ANT) in the nM range and forms large conductance channels and, consequently, decouples the respiratory chain and induces inner mitochondria depolarization. 38 The effect can be attenuated with the overexpression of Bcl-2, which prevents the interaction of Vpr with the complex. However, the role of the mitochondria gateway factors Bak/Bax in Vpr-mediated disruption of MMP remains undetermined, 39 although the fact that Vpr can directly interact with ANT suggests that Vpr may be an aberrant apoptotic molecule that can bypass this requirement.…”