ABSTRACT
The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.
Subject(s)
Electron Transport Complex IV/antagonists & inhibitors , Mitochondria/drug effects , tat Gene Products, Human Immunodeficiency Virus/pharmacology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Brain/drug effects , Brain/enzymology , Cytochromes c/metabolism , Electron Transport Complex IV/metabolism , Humans , Ion Transport , Liver/drug effects , Liver/enzymology , Membrane Potential, Mitochondrial , Mice , Mice, Inbred BALB C , Mitochondria/enzymology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Myocardium/enzymology , Oxidative Phosphorylation , Permeability , Proto-Oncogene Proteins c-bcl-2/metabolism , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/physiologyABSTRACT
The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.
Subject(s)
Endothelial Cells/physiology , Gene Products, vpr/pharmacokinetics , Integrin alphaVbeta3/metabolism , Mitochondria/metabolism , Peptides/pharmacokinetics , Amino Acid Sequence , Animals , Apoptosis , Cell Survival , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Gene Products, vpr/pharmacology , Humans , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Mitochondrial Membranes/metabolism , Molecular Sequence Data , Peptides/pharmacology , PermeabilityABSTRACT
During development as well as in pathological situations, neurons that fail to find appropriate targets or neurotrophic factors undergo cell death. Using primary cortical neurons subjected to acute serum-deprivation (SD), we have examined caspases activation, mitochondrial dysfunction and cell death parameters. Among a panel of metabolic, signaling and caspases inhibitors only those able to interfere with caspase-2 like activity protect primary neurons against SD-induced cell death. In situ detection and subcellular fractionation demonstrate a very early activation of cytosolic caspase-2, which controls Bax cleavage, relocalization and mitochondrial membrane permeabilization (MMP). Both z-VDVAD-fmk and a siRNA specific for caspase-2 abolish Bax changes, mitochondrial membranes permeabilization, as well as cytochrome c release-dependent activation of caspase-9/caspase-3, nuclear alterations, phosphatidylserine exposure, neurites dismantling and neuronal death. Hence, caspase-2 is an early checkpoint for apoptosis initiation in primary neurons subjected to serum deprivation.
Subject(s)
Apoptosis , Caspase 2/metabolism , Neurons/cytology , Neurons/enzymology , Serum , Animals , Apoptosis/drug effects , Caspase 2/deficiency , Cell Survival/drug effects , Cells, Cultured , Cytochromes c/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Mice , Mitochondria/drug effects , Mitochondria/enzymology , Mitochondria/metabolism , Models, Biological , Neurons/drug effects , Peptides/pharmacology , Protein Transport/drug effects , RNA Interference , bcl-2-Associated X Protein/metabolismABSTRACT
We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developed specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.
