μ-calpain cleaves Bfl-1 at two major sites in its N-terminus and releases a large C-terminal fragment with cytotoxic activity.

(A) GST-Bfl-1(1–151) was digested with recombinant µ-calpain in vitro and the fragments were separated by SDS-PAGE (left panel). Bands corresponding to cleaved products (arrows) were analyzed by mass spectrometry (MS). A higher concentration of recombinant GST-Bfl-1(1–151) was treated with µ-calpain, products were separated by SDS-PAGE and blotted to PVDF (right panel). A sub-band (asterisk) was excised and subjected to Edman degradation, which indicated that Bfl-1 had been N-terminally cleaved between residues F71 and N72. (B) Schematic representation of the wild type Bfl-1 protein showing the location of the identified μ-calpain cleavage sites (asterisks, upper sequence), of mutant Bfl-1 protein with a 6 aminoacids deletion surrounding the two cleaved residues (Bfl-1DD, middle sequence), and of mutant Bfl-1 in which the region overlapping the first cleavage site was swapped with a structurally homologous region in Bcl2L10 (Bfl-1SD, bottom sequence) (C) Confirmation of the two calpain cleavage sites identified in Bfl-1 using noncleavable mutants. 293T cell lysates expressing GFP-tagged Bfl-1 constructs were exogenously treated with μ-calpain. Lysates containing equal amount of GFP-tagged Bfl-1 proteins were separated by SDS-PAGE and analyzed by western blot with an anti-GFP antibody to detect the full length protein and N-terminal truncated fragments and with a polyclonal anti-Bfl-1 antibody to detect C-terminal truncated fragments. Upper and lower panels represent two independent experiments with different time of exposure. (D) BJAB cells were cultured with or without treatment with TNF/CHX in the presence or absence of the calpain inhibitor ALLN. Lysates were separated by SDS-PAGE and the presence of a cleaved fragment was assayed by western blot using an anti-Bfl-1 antibody. (E) Secondary structure of Bfl-1 in which the nine helices of the protein are represented by boxes along with the different BH domains (left panel).The two cleavage sites mapped in (A) are indicated. A comparison with the previously published µ-calpain cleavage site in Bax is also shown [26] (bottom sequence). 3D structure of Bfl-1 (2VM6) [31] and position of the different cleaved sites (red circles) are indicated. The different Bcl-2 Homology domains are colored in yellow (putative BH4), red (BH3), green (BH1) and blue (BH2). (F) FACS assays of Annexin V staining in HT1080 cells. Chimeric GFP constructs encoding GFP alone, or fusions of GFP with full-length Bfl-1 or Bax or with the various membrane-active α-helices corresponding to the C-terminal part of Bfl-1 or Bax, i.e. α5, α6 (PFD, pore forming domain) and α9 (FE, final exon) are represented. The α-helical topology of Bax and Bfl-1 corresponds to the structures solved in aqueous environment [31], [50]. Transfected cells were stained for phosphatidylserine exposure using Cy3-conjugated Annexin V and the percentage of apoptotic GFP-expressing cells was determined by FACS 24 hours post transfection (right panel). Death of GFP-expressing and staurosporine (STS)-treated cells were also monitored as controls. Graphs shown are representative of three independent experiments.