Pro-apoptotic activity of mBNIP-21 depends on its BNIP-2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection

Abstract

SummaryOur previous study reported that mouse BNIP-21 (mBNIP-21) induces apoptosis through a mitochondria-dependent pathway. To map the functional domains of mBNIP-21, we performed mutational analyses and demonstrated that the BNIP-2 and Cdc42GAP homology (BCH) domain is required for apoptosis induction by mBNIP-21 targeting the mitochondria and inducing cytochrome c release. This pro-apoptotic activity was enhanced by coxsackievirus infection. However, deletion of the Bcl-2 homology 3 (BH3)-like domain, a well-known cell 'death domain' in proapoptotic Bcl-2 family proteins, did not affect the activity of mBNIP-21. These data were further supported by transfection of a mouse Bax (mBax) mutant, whose BH3 was replaced by the mBNIP-21 BH3-like domain. This replacement significantly reduced the pro-apoptotic activity of mBax. We also found that the predicted calcium binding domain has no contribution to the mBNIP-21-induced apoptosis. Further mapping of the motifs of BCH domain demonstrated that deletion of the hydrophobic motif proximal to the C-terminal of the BCH significantly reduced its proapoptotic activity. These findings suggest that mBNIP-21, as a member of the BNIP subgroup of the Bcl-2-related proteins, functions without need of BH3 but its BCH domain is critical for its activity in inducing cell elongation, membrane protrusions and apoptotic cell death.

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes

“…HeLa cells proliferating on glass coverslips in a 6-well plate at~70% confluence were co-transfected with pIRES-2A and plasmid (FLAG-DAP5-HA) expressing WT DAP5 containing an N-terminal FLAG tag and a C-terminal HA tag or G434E DAP5 (containing an N-terminal FLAG tag) and then immunostained the N-and C-terminal regions of DAP5 with FLAG and HA antibody, respectively, at 48 h after co-transfection or subjected to CVB3 infection at the indicated time points and subsequently immunostained as described previously. 57 Briefly, cells were fixed with 4% paraformaldehyde, permeabilized in methanol/acetone (50:50) at − 20°C for 20 min and stained with an anti-FLAG or anti-HA primary antibody (Santa Cruz Biotechnology Inc.). Slides were washed and stained with a goat anti-rabbit IgG (H +L) labeled with Alexa Fluor 488 or 594 (Invitrogen).…”

P58IPKinhibits coxsackievirus-induced apoptosis via the PI3K/Akt pathway requiring activation of ATF6a and subsequent upregulation of mitofusin 2

“…Immunocytochemistry and confocal microscopy p58 IPK cells and vector cells proliferating on glass coverslips at ∼ 70% confluence were stained by following the method described previously (Sall et al, 2010). Briefly, the cells were fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100 for 10 min and stained with an anti-mitofusin 2 primary antibody for 1 h and then the slides were washed and treated with goat anti-rabbit IgG (H + L) labelled with red fluorescent Alexa Fluor 594 (Invitrogen, A11012).…”

Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

“…Transfection of truncated BNIP-2 (i.e., the GrB generated cleavage fragment (tBNIP-2)) confirms cleavage at the IEAD 28 motif of full-length BNIP-2. However, whereas previous studies [11,23,24] reporting on the overexpression of full-length and/or truncated hBNIP-2 led to moderate poly (ADP) ribose polymerase 1 (PARP-1) cleavage and/or caspase activation, indicative for apoptosis, when transfecting (truncated) human and mouse BNIP-2 variants, we were unable to observe caspase activity or any of the typical apoptosis related hallmarks using fluorimetric caspase assays and flow cytometry (data not shown).…”

“…Various signaling pathways, including activation of the intrinsic apoptotic pathway [23] or direct caspase activation [24], have been proposed to explain the apoptotic potential of BNIP-2.…”