µ-Calpain Conversion of Antiapoptotic Bfl-1 (BCL2A1) into a Prodeath Factor Reveals Two Distinct alpha-Helices Inducing Mitochondria-Mediated Apoptosis

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance

A noncomitogenic CD2R monoclonal antibody induces apoptosis of activated T cells by a CD95/CD95-L-dependent pathway.

Clonal expansion of activated T and B cells is controlled by homeostatic mechanisms resulting in apoptosis of a large proportion of activated cells, mostly through interaction between CD95 (Fas or Apo-1) receptor and its ligand CD95-L. CD2, which is considered as a CD3/TCR alternative pathway of T cell activation, may trigger activation-induced cell death, but the role of CD95/CD95-L interaction in CD2-mediated apoptosis remains controversial. We show here that the CD2R mAb YTH 655.5, which does not induce comitogenic signals when associated with another CD2 mAb, triggers CD95-L expression by preactivated but not resting T cells, resulting in CD95/CD95-L-mediated apoptosis. The critical role of CD95/CD95-L interaction was supported by complete inhibition in the presence of the antagonist CD95 mAb ZB4 and by blocking CD95-L synthesis and surface expression by cycloheximide, cyclosporin A, EGTA, or cytochalasin B. YTH 655.5 was shown to stimulate p56lck phosphorylation and enzymatic activity. However, p56lck activation is not sufficient to trigger apoptosis, because other CD2R and CD4 mAbs that activate p56lck do not induce apoptosis. In conclusion, CD2 can mediate nonmitogenic signals, resulting in CD95-L expression and apoptosis of CD95+ cells