BAG3 binds αB-crystallin directly through BAG3 intermediate region.

<p>(A) <i>BAG3 interacts with αB-crystallin in rat cardiomyocytes</i>. Cardiomyocytes were isolated from rat hearts, and Flag-tagged BAG3 was expressed using adenovirus. Cell extracts were immunoprecipitated with anti-Flag antibody, and the sample was applied to SDS-PAGE. Anti-αB-crystallin antibody was used to detect αB-crystallin. The same membrane was blotted with anti-Hsc70 and anti-αActinin antibody for a positive and negative control, respectively. No actinin interacting was observed (negative control). (B) <i>Schematic representation of the primary structure of wild-type BAG3 and its mutants</i>. The BAG3ΔC construct lacks the BAG domain. The BAG3 intermediate domain and amino acids from 87–101 and from 200 to 213 are deleted in BAG3ΔM1 and BAG3ΔM2, respectively. Amino acid sequences between residues 87–101 and 200–213 are shown. (C) <i>Co-precipitation of αB-crystallin and BAG3</i>. Flag-tagged BAG3 or mutants were expressed with αB-crystallin in HEK293 cells, and cell extracts were immunoprecipitated with anti-Flag antibody. αB-crystallin was detected with anti-αB-crystallin antibody. Anti-Hsc70 antibody was used to detect Hsc70 precipitation with BAG3. The cell extracts were also applied to an immunoblotting assay to verify expression. (D) <i>BAG3 interacts with αB-crystallin via amino acids 87–101 and 200–213</i>. His-tagged BAG3 or BAG3M2 was expressed with αB-crystallin in HEK293 cells, and BAG3 was precipitated with anti-His antibody. Co-precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane was blotted with anti-His antibody (bottom panel). (E) <i>Direct interaction of BAG3 with αB-crystallin</i>. Left panel: GST protein fused to BAG3 was expressed in <i>Escherichia coli</i>, and purified fusion proteins were incubated with purified αB-crystallin, followed by precipitation with GSH beads. After SDS-PAGE, αB-crystallin was detected using anti-αB-crystallin antibody. Right panel: GST fusion proteins used in the experiment were visualized with CBB staining to verify the quality and quantity. <i>(F) Competition assay with peptides</i>. Purified GST or GST-BAG3 was incubated with purified αB-crystallin in the presence or absence of peptides corresponding to amino acids 87–101 or 200–213 of BAG3 at indicated concentrations. The precipitated samples with GSH beads were loaded onto SDS-PAGE, followed by an immunoblotting assay using anti-αB-crystallin antibody.</p

BAG3 attenuates the toxicity of mutant αB-crystallin.

<p><i>BAG3 inhibits apoptosis caused by αB-crystallin mutant in C2C12 cells</i>. <i>α</i>B-crystallin wild type or R120G was expressed with or without Flag-tagged BAG3 in C2C12 myoblast cells, and cells were cultured with differentiation media to induce myotube differentiation. After fixing cells, αB-crystallin and cell nuclei were stained with anti-αB-crystallin antibody and Dapi solution, respectively. Cells with nuclear fragmentation typical of apoptosis were counted and statistically analyzed.</p

BAG3 recognizes folding status of αB-crystallin.

<p><i>(A) BAG3 binds to mutant type of αB-crystallin strongly.</i> Wild type (WT) or mutant (R120G) αB-crystallin was expressed in HEK293 cells with Flag-tagged BAG3, and the cell lysate was mixed with anti-Flag antibody. Precipitated samples were analyzed by SDS-PAGE using anti-αB-crystallin (upper panel), Flag-tagged BAG3 (middle panel), and actin (lower panel). The sample before immunoprecipitation was also loaded to confirm protein expression (right four lanes). <i>(B) Direct recognition of mutant αB-crystallin by BAG3.</i> Purified GST or GST-fused BAG3 beads were incubated with purified αB-crystallin wild type (WT) or mutant (R120G), and a pull-down assay was performed. Precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane stained with Ponceau is shown below. <i>Mutated αB-crystallin preferentially binds to BAG3.</i> Purified GST, GST-fused αB-crystallin wild type (WT) or mutant (R120G) was mixed with purified BAG3 for a pull-down assay. The detection of BAG3 was achieved with anti-BAG3 antibody after SDS-PAGE. The same membrane was stained with Ponceau (lower panel).</p

Overexpression of BAG3 suppresses aggregation of mutant αB-crystallin and increases its solubility.

<p>(A) <i>Aggregation of mutant αB-crystallin is cleared by BAG3.</i> HEK293 cells were transfected with plasmids for wild type or mutant αB-crystallin with or without Flag-tagged BAG3. Two days later, cells were fixed and αB-crystallin and Flag-tagged BAG3 were stained with anti-αB-crystallin antibody (red in color images) and Flag-antibody (green in color images), respectively. Merged images are also shown (Merge). (B) <i>BAG3 cleared aggregation caused by αB-crystallin mutant.</i> After staining of αB-crystallin, the number of cells containing αB-crystallin aggregation were counted and statistically analyzed. The Y-axis indicates the percentage of cells displaying αB-crystallin aggregation. <i>(C) BAG3 increases stability of mutant type of αB-crystallin in HEK293 cells.</i> HEK293 cells were cultured in a 24-well dish and plasmids for αB-crystallin wild type (WT) or mutant (R120G) were transfected (0.2 µg each) with or without the plasmid for Flag-tagged BAG3. The amount of BAG3 plasmid was increased gradually (0.05, 0.15, and 0.3 µg). After two days, cells were lysed in lysis buffer A (10 mM Tris, pH 8.0, 150 mM NaCl, 2% SDS, 10 mM NaF, 2 mM Na₃VO₄, 2 mM PMSF, and 1∶50 protease inhibitor cocktail), and sonicated. The sample was loaded onto SDS-PAGE, and an immunoblotting assay performed with anti-αB-crystallin (first panel), Flag (second panel), and actin antibody (third panel). GFP plasmid was transfected together to monitor transfection efficiency, and detected with anti-GFP antibody (bottom panel). <i>(D) BAG3 increases solubility of mutant type of αB-crystallin in HEK293 cells.</i> The same set of transfections described above in “C” were done, but lysis buffer B was used instead of lysis buffer A (lysis buffer B; 20 mM Tris, pH 7.4, 150 mM NaCl, 0.001% Tween-20, 10 mM NaF, 2 mM Na₃VO₄, 2 mM PMSF, and 1∶50 protease inhibitor cocktail). After homogenization, the sample was transferred to a tube, and centrifuged at 14,000×g for 15 minutes at 4°C. The supernatant was transferred to a new tube to represent the soluble fraction. The samples were subjected to an immunoblotting assay to detect αB-crystallin (first panel), Flag-tagged BAG3 (second panel), actin (third panel), and GFP (last panel). (E) <i>BAG3 clears aggregation of mutant αB-crystallin independent with Hsc70.</i> HEK293 cells were transfected with indicated plasmids, and cultured for two days. Panel #1 (upper three panel): wild type αB-crystallin+pcDNA3 empty vector (mock). Panel #2–7 represents transfectants of αB-crystallin R120G mutant with plasmid indicated at the left of each three panels. After fixation, αB-crystallin (αBCry) and Flag-tagged proteins were stained with anti-αB-crystallin antibody (red in color images) and Flag-antibody (green in color images), respectively. Merged images are also shown (Merge). The number of cells containing αB-crystallin R120G aggregation were counted and statistically analyzed. The percentage of cells displaying αB-crystallin aggregation is shown in the right panel. (F) <i>Expression of Hsc70 and BAG3 proteins in HEK293 cells used above.</i> HEK293 cells were transfected with plasmids for αB-crystallin with or without Flag-BAG3 or Flag-Hsc70. Two days later, cells were lysed for western blotting. The expression levels of Hsc70 and BAG3 proteins in HEK293 cells were verified using anti-Hsc70 antibody (Hsc70), anti-BAG3 antibody (BAG3), and anti-Flag antibody (Flag). Actin was used as a loading control (actin). Hsc70 antibody detected endogenous Hsc70 as shown at the left three lanes in the second panel and overexpressed Flag tagged Hsc70 with endogenous protein at the right three lanes. Flag antibody detected BAG3FL (2 and 5 lane), BAG3▵C (3 and 6 lane) and Hsc70 (4, 5 and 6 lane).</p