Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.

TLDR

Bcl‑2 is an apoptosis‑regulatory protein that localizes to mitochondrial and nuclear membranes, and its ability to inhibit apoptosis remains unclear, though early apoptotic events involve loss of mitochondrial inner membrane potential, implicating mitochondrial factors. The study identifies an apoptosis‑inducing factor (AIF) released upon mitochondrial membrane potential collapse, whose activity is blocked by the protease inhibitor Z‑VAD.fmk, and examines how Bcl‑2 affects AIF formation, release, and action. Bcl‑2 hyperexpression blocks mitochondrial permeability transition and prevents AIF release without altering its formation or nuclear activity, indicating that Bcl‑2 inhibits apoptosis by retaining the apoptogenic protease within mitochondria.

Abstract

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.