Article Figures & Data
Figures
- Figure 1.
Bcl-2 mediates the binding between SOD1 and VDAC1. A–D, Bcl-2, EGFP-tagged SOD1 (WT or G93A), and flag-tagged VDAC1 were transfected into HEK293T (SOD1 and VDAC1 ± Bcl-2; VDAC1 and Bcl-2 ±SOD1; Bcl-2 and VDAC1 ± SOD1). Cells were harvested 24 h later and cell lysates were immunoprecipitated with the indicated antibodies (A–C) and probed for individual proteins. D, WB of total lysates for protein expression. E, Bcl-2 and EGFP-tagged mutSOD1–G93A were cotransfected into HEK293T at the indicated ratio, together with a constant amount of flag-tagged VDAC1. Cells were harvested 24 h later and cell lysates were immunoprecipitated with flag antibody and probed for individual proteins. F, HEK293T cells were knocked down with either control or VDAC1 siRNAs. Cells were then transfected with Bcl-2 and EGFP-tagged SOD1 (WT or G93A), lysed, immunoprecipitated, and probed with indicated antibodies.
- Figure 2.
BH3 domain exposed Bcl-2 binds to VDAC1 with increased affinity through the course of disease. A, Crude mitochondria isolated from mutSOD1–G93A mice at indicated disease stages (P, presymptomatic; O, onset; S, symptomatic; E, end stage) were lysed and immunoprecipitated with anti-Bcl-2 antibody and then probed with indicated antibodies. Quantifications of co-IPs were normalized to pulldown of Bcl-2. B, Control IPs with either anti-Bcl-2 or IgGs. C, Western blot (WB) to assess total amount of VDAC1, VDAC2, COXIV, and SOD1 throughout disease progression. GAPDH was also measured as a control at each disease stage. Quantifications of WBs were normalized to each protein level at presymptomatic stage (n = 3, *p ≤ 0.05). D, Bcl-2 was immunoprecipitated using indicated conformational specific antibodies and amount of VDAC1 co-IP was assessed over disease progression.
- Figure 3.
MutSOD1/Bcl-2 complex induces decreased mitochondrial ADP permeability and loss of cellular viability. A–C, ADP consumption curves of mitochondria isolated from HEK293T cells cotransfected with pcDNA3, SOD1-WT ± Bcl-2 (A) or SOD1-WT, mutSOD1–G93A ± Bcl-2 (WT or AAA) (with pcDNA3 and mutSOD1–G93A ± Bcl-2 results highlighted in the inset) (B) or SOD1-WT + Bcl-2 and mutSOD1–G93A + Bcl-2, and measured in the presence of 5 μm G3139 (C). D, Bcl-2 (WT or AAA) was cotransfected with SOD1 (WT or G93A, fused with EGFP) into HEK293T cells. Lysates were immunoprecipitated with anti-GFP antibody and probed with either anti-GFP or anti-Bcl-2 antibody. E, Control or VDAC1 stable knockdown (VDAC1 KD) HEK293T cells were transfected with SOD1 (WT or G93A) and pcDNA, Bcl-2 (WT or AAA). Cell viability was then measured (*p ≤ 0.05). Insert, WB of VDAC1, VDAC 2, and cytochrome c (as a mitochondrial loading control) in both cell lines.
- Figure 4.
Exon 2 of mutSOD1 is responsible for binding with Bcl-2. A, Illustration of the SOD1 exons. The G93A mutation is marked with *. B, C, HEK293T cells were cotransfected with FL or exon deletion SOD1–G93A constructs and Bcl-2. Twenty-hours post-transfection, cells were either stained for Bcl-2 and MitoTracker (B, with Z-stack on the right) or immunoprecipitated with anti-Bcl-2 and probed for indicated antibodies (C). D, EGFP-tagged SOD1 (WT or G93A) with different exon domains expressed were cotransfected into HEK293T cells with Bcl-2. The cells were harvested 24 h later and lysates immunoprecipitated with GFP antibody and probed with indicated antibodies.
- Figure 5.
Peptide corresponding to exon 2-SOD1 inhibits mutSOD1/Bcl-2 binding and resulting mutSOD1 mitochondrial clustering in HEK293T cells. A, Left, EGFP-tagged mutSOD1–G93A and Bcl-2 were cotransfected into HEK293T cells. The lysates were aliquoted and mixed with 1 μm peptide corresponding to each exon of SOD1. These mixtures were immunoprecipitated with GFP antibody and probed with antibodies indicated. Right, Quantifications of Western blots were normalized to the WT-SOD1 condition. B, EGFP-tagged mutSOD1–G93A and flag-tagged VDAC1 were cotransfected into HEK293T cells. The lysates were aliquoted and mixed with or without 1 μm peptide 2. These mixtures were immunoprecipitated with GFP antibody and probed with the antibodies indicated. C, HEK293T cells were cotransfected with mutSOD1–G93A and Bcl-2. Twenty hours post-transfection, cells were treated with peptide 2 (50, 100, or 200 ng), then stained with 300 nm MitoTracker. Mitochondrial localization and clustering of mutSOD1 were analyzed using confocal microscopy.
- Figure 6.
Peptide 2 reverses mutSOD1-caused hyperpolarization of MMP and loss of viability in NSC-34 cells. NSC-34 cells were transduced with adenovirus harboring SOD1 genes and treated with the indicated amount of peptides following protocols described (see Materials and Methods). A, Lysates were immunoprecipitated with anti-Bcl-2 antibodies and analyzed by WB. B, C, Cells were then stained with 25 nm TMRE in the presence or absence of 1 μm FCCP and analyzed via flow cytometry (B) or confocal microscopy (C). The ratio of TMRE and TMRE-FCCP staining was normalized to SOD1-WT treatment (*p < 0.05, n = 3, compared with all other groups). D, Cell viabilities were assessed with CellTiter-Glo and normalized to values of EGFP control (*p < 0.05, n = 3).
- Figure 7.
Peptide 2 rescues mutSOD1/Bcl-2-induced inhibition of mitochondrial ADP permeability. A, Western blot (WB) analysis of the isolated mitochondrial lysate (Mito Lysate) and spinal cord lysate (Total Lysate). B, Comparison of the morphology of mitochondria isolated from symptomatic stage and its age-matched control under transmission electron microscopy. Scale bar, 500 nm. C, D, ADP consumption curve of isolated spinal cord mitochondria through disease courses (black dots): (C) presymptomatic, (D) symptomatic, and their age-matched controls (empty dots). E, ADP consumption curve of isolated spinal cord mitochondria from symptomatic mutSOD1–G93A mice with or without treatment of 1 μm peptide 2.
