Abstract
We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.
Highlights
Novel Bcl-2-expressing vector (Bcl-2) homology 3 (BH3) Domain Only Protein SOUL Facilitates Cell Death branes [15], and myristoylation of tBid further enhances its membrane avidity [16]
We show that SOUL promotes the permeabilization of both outer and inner mitochondrial membranes in oxidative stress and that its effect can be reversed by deleting the putative Bcl-2 homology 3 (BH3) sequence from SOUL as well as by inhibition of mitochondrial permeability transition either by cyclophilin D suppression or overexpression of antiapoptotic Bcl-2 members
A multiple alignment of the BH3 domains of Bcl-2-associated proteins show high homology to a sequence found in SOUL (Fig. 1A)
Summary
All of the chemicals for cell culture, cyclosporine A (CsA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and rhodamine-1,2,3 (Rh-123) were purchased from Sigma. The vector-containing SOUL or ⌬BH3-SOUL or the empty pcDNA3.1 vector was transfected into NIH3T3 cells with Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). Cell clones were subsequently screened by Western blot analysis with antiSOUL polyclonal antibody for an increase in SOUL or ⌬BH3-SOUL protein expression relative to that in the pcDNA-transfected cells. After subjecting the cells to the appropriate treatment indicated in the figure legends, coverslips were rinsed twice in phosphate-buffered saline and were put upside down onto a silicon isolator placed on top of a microscope slide to create a small chamber, which was filled with phosphate-buffered saline supplemented with 0.5% fetal calf serum and containing 5 g/ml JC1-dye (Molecular Probes). Flow Cytometry Analysis—Cell death was induced with 300 M hydrogen peroxide (Sigma) for 24 h in sham-transfected or SOUL-overexpressing NIH3T3 cells They were either co-overexpressing or not co-overexpressing Bcl-2 or Bcl-xL proteins or were pretreated with 30 nM anti-cyclophilin D siRNA for 24 h. Cells in each category are expressed as a percentage of the total number of stained cells counted and are presented as pie charts
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