THE CENTRAL EXECUTIONER OF APOPTOSIS - MULTIPLE CONNECTIONS BETWEEN PROTEASE ACTIVATION AND MITOCHONDRIA IN FAS APO-1/CD95-INDUCED AND CERAMIDE-INDUCED APOPTOSIS/

According to current understanding, cytoplasmic events including activ ation of protease cascades and mitochondrial permeability transition ( PT) participate in the control of nuclear apoptosis. However, the rela tionship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1 beta converting enzyme (ICE; caspase 1) o r ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (Delta Psi(m)). In contrast, cytosolic CPP32/Y ama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the Delta Psi(m) is fully disrupted. Transfection with the cowpox protease inhi bitor crmA or culture in the presence of the synthetic ICE-specific in hibitor Ac-YVAD.cmk both prevent the Delta Psi(m) collapse and subsequ ent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells acc umulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified I CE suffices to cause isolated mitochondria to undergo PT-like large am plitude swelling and to disrupt their Delta Psi(m). In addition, ICE-t reated mitochondria release an apoptosis-inducing factor (AIF) that in duces apoptotic changes (chromatin condensation and oligonucleosomal D NA fragmentation) in isolated nuclei in vitro. AIF is a protease (or p rotease activator) that can be inhibited by the broad spectrum apoptos is inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondria l alterations (large amplitude swelling + Delta Psi(m) collapse + rele ase of AIF) induced by prooxidants or cytosols from ceramide-treated c ells, it has no effect on the ICE-induced mitochondrial PT and AIF rel ease. These data connect a protease activation pathway with the mitoch ondrial phase of apoptosis regulation. In addition, they provide a pla usible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-in duced apoptosis.