Biomedical subjects

L Ravagnan

Publications and source records attributed to L Ravagnan.

At least 19 recordsLinked to original sources

PubMed2000

The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

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.

Apoptosis↗

PubMed1999

Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore.

The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Proteasome activation occurs at an early, premitochondrial step of thymocyte apoptosis.

Proteasomes and mitochondrial membrane changes are involved in thymocyte apoptosis. The hierarchical relationship between protease activation and mitochondrial alterations has been elusive. Here we show that inhibition of proteasomes by two specific agents, lactacystin or MG132, prevents all manifestations of thymocyte apoptosis induced by the glucocorticoid receptor agonist dexamethasone or by the topoisomerase II inhibitor etoposide. Lactacystin and MG132 prevent the early disruption of the mitochondrial transmembrane potential (delta psi(m)), which precedes caspase activation, exposure of phosphatidylserine, and nuclear DNA fragmentation. In contrast, stabilization of the delta psi(m) using the permeability transition pore inhibitor bongkrekic acid or inhibition of caspases by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone does not prevent the activation of proteasomes, as determined with the fluorogenic substrate N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-7-amido-4-methylcoumarin . Thus, proteasome activation occurs upstream from mitochondrial changes and caspase activation. Whereas the proteasome-specific agents lactacystin and MG132 truly maintain thymocyte viability, a number of protease inhibitors that inhibit nuclear DNA fragmentation (acetyl-Asp-Glu-Val-Asp-fluoromethylketone; N-Boc-Asp(OMe)-fluoromethylketone; N-tosyl-L-Phe-chloromethylketone) do not prevent the cytolysis induced by DEX or etoposide. These latter agents fail to interfere with the preapoptotic delta psi(m) disruption. Altogether, our data indicate that different proteases may be involved in the pre- or postmitochondrial phase of apoptosis. Only those protease inhibitors that interrupt the apoptotic process at the premitochondrial stage can actually preserve cell viability.

Animals↗

PubMed1998

Caspases disrupt mitochondrial membrane barrier function.

Mitochondrial intermembrane proteins including cytochrome c are known to activate caspases. Accordingly, a disruption of the mitochondrial membrane barrier function with release of cytochrome into the cytosol has been shown to precede caspase activation in a number of different models of apoptosis. Here, we addressed the question of whether caspases themselves can affect mitochondrial membrane function. Recombinant caspases were added to purified mitochondria and were found to affect the permeability of both mitochondrial membranes. Thus, caspases cause a dissipation of the mitochondrial inner transmembrane potential. In addition, caspases cause intermembrane proteins including cytochrome c and AIF (apoptosis-inducing factor) to be released through the outer mitochondrial membrane. These observations suggest that caspases and mitochondria can engage in a circular self-amplification loop. An increase in mitochondrial membrane permeability would cause the release of caspase activators, and caspases, once activated, would in turn increase the mitochondrial membrane permeability. Such a self-amplifying system could accelerate the apoptotic process and/or coordinate the apoptotic response between different mitochondria within the same cell.

Amino Acid Chloromethyl Ketones↗

PubMed1969