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

<p>(A) GST-Bfl-1(1–151) was digested with recombinant µ-calpain <i>in vitro</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Wood1" target="_blank">[26]</a> (bottom sequence). 3D structure of Bfl-1 (2VM6) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Suzuki1" target="_blank">[50]</a>. 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.</p

The Bfl-1-α9 peptide induces mitochondrial permeabilization through a membrane-destabilizing mechanism.

<p>Bfl-1-α9 peptide was incubated for the indicated times with mitochondria at lower concentrations (0.5 µM and 2.5 µM, top panels) and previously used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a> (10 µM and 25 µM, bottom panels). The release of cytochrome c and the expression of MitoHsp70 were monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a>, combined with the detection of the external membrane associated protein hexokinase 1 (HK1) and the matrix-contained protein MnSOD.</p

Subcellular localization of the GFP-tagged, Bfl-1/Bax-derived (poly)peptides.

<p>MEF (left panels) and MEF DKO (right panels) cells were co-transfected with mitoDsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. Confocal images showing GFP (green) and MitoDsRed (red) fluorescence. The DNA staining dye Topro-3 (blue) was used to visualize the nuclei. In merged images, the yellow color shows the co-localization of GFP and MitoDsRed at mitochondria. Scale bar, 10 µm.</p

Bfl-1-derived peptides have different abilities to permeabilize the MOM of mitochondria isolated from cultured cells.

<p>Peptides were incubated at different concentrations (10 µM and 25 µM) with isolated mitochondria for the indicated times (5, 15, 30 and 60 min) and the release of cytochrome c was monitored by immunoblot (IB). MitoHsp70 (mHsp70) was used as control indicative of equal-loading and proper isolation of the pellet fraction containing mitochondria (Mito) in comparison to the supernatant fraction (SN). Cytochrome c release assays were performed using iBMKW2 (wild type) and iBMKD3 (double KO Bax/Bak) for all tested peptides. For Bfl-1-α5, wild type MEF and MEF DKO (Bax/Bak −/− double KO) cells were used in parallel.</p