Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.
Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.
Susin
S A
SA
Centre national de la Reccherche Scientifique-UPR420, F-94801 Villejuif, France.
Zamzami
N
N
Castedo
M
M
Hirsch
T
T
Marchetti
P
P
Macho
A
A
Daugas
E
E
Geuskens
M
M
Kroemer
G
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000187:drug effects; D010450:Endopeptidases / Q000494:pharmacology*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008930:Mitochondria, Liver / Q000187:drug effects*; D008954:Models, Biological; D010539:Permeability; D011480:Protease Inhibitors / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D011480:Protease Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D010450:Endopeptidases