anti bag 1  (Cell Signaling Technology Inc)

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Generation of Bag1 exon 5 mutant mice. (a) Structure of human BAG1 and mouse Bag1 genes and the coding regions of Bag1L. Lines and boxes indicate the Bag1 gene and seven exons (Ex1–Ex7), respectively. Small arrows indicate primers for PCR to verify the genotypes of mice. Bag1 protein was illustrated with the UbL and BAG domains (shadowed boxes). Dark shadowed boxes indicate the BAG domain helix 2, which is encoded in exon 5. The target position of Bag1 is Cys282 (Cys272 in humans). The structures of putative ORFs of mutant Bag1S with the insertion of 49 bp in exon 5 (if generated by normal splicing) and the mutant Bag1S with the deletion of exon 5 due to in-frame exon skipping (see below) are shown in a square box. (b) Genotyping of F1 pups from the candidate Bag1 mutant mouse. A genome-edited F0 candidate was mated with a Bag1 WT C57/BL6J mouse to generate F1 pups. To distinguish the Bag1 mutant mice, the exon 5 region was amplified from 8 F1 pups by PCR and analyzed by agarose gel electrophoresis. Arrows indicate the PCR products of Bag1 WT (WT) and mutated exon 5. Pups with the mutant allele are shown using boldface numbers. (c) Genotyping of mice F2 pups from the possible F1 mice carrying the hetero allele (#1 mated with #6). The genome was analyzed by PCR. (d) Bag1 mutant genome sequence is shown in alignment with the Bag1 WT sequence. A homozygous mouse (#29) genome was amplified by PCR. The exon 5 region is indicated. The full and partial oligo DNA sequences in the #29 genome are underlined. Abbreviations used: ORF, open reading frame; UbL, ubiquitin-like; PCR, polymerase chain reaction; WT, wild type.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Mutagenesis, Generated, Amplification, Agarose Gel Electrophoresis, Sequencing, Polymerase Chain Reaction

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: The mutant exon 5 is skipped by splicing to generate alternative mRNA encoding mutant Bag1 with helix 3 deletion that is undetectable. (a) Analysis of Bag1 cDNA. RT-PCR of Bag1 mRNA using the indicated primer pair generated 1281 bp of cDNA product of Bag1 WT (WT). The exon 5 mutant exhibited an approximately 100-bp smaller cDNA size. Pups carrying only the shorter cDNA (exon 5 mutant Bag1 ) are shown in boldface numbers. (b) Genes and mRNA structures of Bag1 WT and Bag1 Δex5 mice are illustrated with the frequency of pups' appearance. The inserted oligo on exon 5 in a mutant mouse is indicated by pink coloration. Arrowheads indicate primers to verify the genotypes of mice and clone cDNA. The frequency of live birth among Bag1 Δex5 mice are shown. (c) Homo Bag1 WT and Bag1 Δex5 mouse embryos at E13.5 are shown. (d) Expression of Bag1 mRNA in male Bag1 WT/WT and Bag1 Δex5/Δex5 mice was examined using RT-PCR. Total RNAs from organs and tissues derived from ectoderm, mesoderm, or endoderm are shown. Positions of Bag1 WT (WT) and Bag1 Δex5 (Δ5) PCR fragments are indicated by arrows. (e) Bag1 proteins in male Bag1 WT (WT) and Bag1 Δex5 (Δ5) mice were examined. Positions of Bag1L, Bag1S, and GAPDH are indicated. Protein lysate of Bag1 WT MEFs (M) was used as positive controls. The 63 kDa protein that appeared in both WT and Δ5 lysates is a non-Bag1 protein that reacted with the monoclonal antibody used, as it could not be detected with the anti-Bag1 rabbit antibody (see ). Abbreviations used: MEF, mouse embryonic fibroblast; RT-PCR, reverse transcription-PCR; cDNA, complementary DNA; mRNA, messenger ribonucleic acid; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; WT, wild type; PCR, polymerase chain reaction.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Generated, Expressing, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Effect of the insertion of a neo marker knockout cassette upstream of Bag1 on head-to-head gene Chmp5 expression. (a) Chromosomal region of Bag1 (exon 1–7)-Chmp5 (exon 1–8), including the predicted regulatory future from the Ensemble regulation resources ( https://oct2022.archive.ensembl.org/Mus_musculus/Gene/Summary?db=core;g=ENSMUSG00000028419;r = 4:40945802–40951599;t = ENSMUST00000030128;gene_summary=regulatory_build=normal ). Constructs of Chmp5-fused nLuc reporter genes carrying 8.4 kbp (F1-cLuc) and 5.0 kbp (F3-cLuc) are shown. The upstream region was replaced with the reporters, with the neo cassettes illustrated (F1-neo-nLuc and F3-neo-nLuc; see text for detail). (b) nLuc activity was observed by 3 different experiments (n = 3). Normalized activity of F1-nLuc (F1), F1-neo-nLuc (F1neo), F3-nLuc (F3), and F3-neo-nLuc (F3neo) is shown. The values are shown as the means ± SEM. Significance was determined using the Student’s t-test. Abbreviation used: SEM, standard error of the mean.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Marker, Knock-Out, Expressing, Construct, Activity Assay

Journal: Cell Stress & Chaperones

Article Title: Bag1 protein loss sensitizes mouse embryonic fibroblasts to glutathione depletion

doi: 10.1016/j.cstres.2024.05.003

Figure Lengend Snippet: Establishment and characterization of Bag1 Δex5 MEFs. (a) Bag1 protein expression in Bag1 WT and Bag1 Δex5 MEFs was examined by immunoblotting. Bag1 isoforms (Bag1L and Bag1S) are indicated by arrows. GAPDH signals were used as a loading control. No stress (Cont, lanes 1 and 3), and 100 μM BSO for 30 min (BSO, lanes 2 and 4) groups are shown. (b) Relative sensitivity of Bag1 Δex5 MEF to that of Bag1 WT MEF, and the indicated concentration of DOX for 24 h. Relative growth levels were normalized against 0 µM values set as 100%, with the values shown as means ± SEM (n = 3, i.e., 3 independent experiments). (c) Synthesis and redox cycles of GSH. Glutamate cysteine ligase (GCL) is responsible for the first step of GSH synthesis. GSH is regenerated by GSSG reduction by glutathione reductase (GR). GR and glutathione peroxidase (GPx) are also indicated. BSO inhibited GCL. (d) Sensitivity to BSO was determined over 24 h. Growth levels (%) were shown as the means ± SEM from three independent experiments. * P < 0.05 and ** P < 0.01 when compared to viability without BSO (one-way analysis of variance followed by a Dunnett’s post hoc test for multiple parameter comparisons). (e) Intracellular GSH/GSSG ratios in Bag1 WT and Bag1 Δex5 MEFs were determined with and without BSO (200 μM BSO for 24 h). The values are shown as the means ± SEM from four independent experiments. The P -values are indicated using Student's t -test. (f) GR protein levels in Bag1 WT and Bag1 Δex5 MEFs were determined by immunoblotting using an anti-GR antibody. β-tubulin signals were used as a loading control. MEFs were treated with BSO (100 μM for 24 h, lanes 2 and 4). No treatment controls are shown (lanes 1 and 3). (g) Cellular H 2 O 2 levels were determined for Bag1 WT and Bag1 Δex5 MEFs. The values are shown as the means ± SEM from four independent experiments. The P -values are indicated using Student's t-test. Abbreviations used: BSO, buthionine sulfoximine; DOX, doxorubicin; GSH, glutathione; GSSG, oxidized glutathione; MEFs, mouse embryonic fibroblasts; SEM, standard error of the mean; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Immunoblotting was performed using antibodies against Bag1 (AF815, R&D systems, Minneapolis, MN, USA), Gsr (sc-133245, Santa Cruz Biotechnology, Dallas, TX, USA), and Glyceraldehyde 3-phosphate dehydrogenase (G8795, Merk, Darmstadt, Germany), respectively.

Techniques: Expressing, Western Blot, Control, Concentration Assay

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: ( a ) SEC analysis of Bag1 interaction with the proteasome subunit Rpn1. The shift in the elution profile of the sample containing both Bag1 and Rpn1 (orange) indicates the formation of a complex compared to Bag1 (red) and Rpn1 (goldenrod) alone. ( b ) SEC analysis of different combinations of Hsp70, Rpn1, Bag1 and a model substrate RCMLA. The sample containing Hsp70, Rpn1 and Bag1 (green) elutes prior to Bag1:Rpn1 complex (orange), showing a formation of a ternary complex. Upon addition of RCMLA to the ternary complex (purple), a shift in the elution peak was observed, showing that the model substrate interacts with the ternary complex of Hsp70:Bag1:Rpn1. ( c ) Different views of the cryo-EM map (4.8 Å resolution) of the Hsp70 NBD :Bag1:Rpn1 ternary complex. AlphaFold prediction of Hsp70 NBD (blue) and full-length Bag1 (Bag1 BD in red and Bag1 UBL in green) are docked into the final map. The remaining density, which is presumably attributed to part of Rpn1, is colored in wheat. Bag1 interfaces to the putative Rpn1 density are indicated with black asterisks. (d) Cryo-EM reconstruction of the Bag1-bound 26S proteasome in S BAG1 (EMDB:52097) at 3.8 Å resolution. Only the UBL domain of Bag1 (Bag1 UBL ) is observed, with the BAG domain missing in the map. Colors are as follows: CP (white), ATPase domain of Rpts (rosy brown), OB domain of Rpts (orange), Rpn1 (beige), Bag1 UBL (light green), Rpn11 (light yellow), Lid (light blue). (e) Binding of Bag1 UBL to the T2 site of Rpn1 in the proteasome. The inset shows contacts between Rpn1 and Bag1 UBL.

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: (a,b) Structural comparison of the cryo-EM reconstruction of the 26S proteasome in S BAG1 (EMDB: 52097 in goldenrod) with the S D4 state (PDB: 7W3K in teal), focusing on Rpn1 (a) and ATPase ring (b) . Bag1 UBL is shown in light green and the rest of densities are shown in light grey. The Changes in shift (Å) and angle (°) are indicated. (c-e) Comparison of individual subunits in S BAG1 and S D4 (PDB: 7W3K) states. Structural differences in Rpn1 (c) , Rpt2 (d), and Rpt4 (e) are shown. Two structures are aligned to the CP α ring. The atomic model of the 20S CP is shown in white. Rpn1 is shown in beige for S BAG1 and in teal for S D4 (c) . Rpt2 and Rpt4 in the S BAG1 are depicted in dark salmon and pale violet red, respectively, while the structures in the S D4 are shown in transparent (d,e) . (f) Superimposition of the S BAG1 (EMDB: 52097, PDB: 9HEU) and S D4 (EMDB: 32283, PDB: 7W3K) Rpn1 and ATPase ring structures. The two cryo-EM structures are aligned to the CP α ring. In S BAG1 , the ATPase ring (rosy brown) protrudes outward relative to the 20S CP, compare to the S D4 (blue green). Rpn1 (beige) shifts and rotates toward the ATPase ring. Atomic models for each map are shown. (g) Structural comparison of the ATPase ring (rosy brown) and Rpn1 (beige) in the S BAG1 (left) and S D4 (right) reveals that the ATPase ring in S BAG1 is deformed and creates a large cavity at the center. (h) Averages of the contact area between the AAA+ domains of adjacent Rpt subunits in different conformational states. Individual values for each structure are shown in dots and the median with a black dashed line. The S BAG1 has overall contact surfaces 3.5-fold smaller than the other conformational states.

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Comparison, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: (a) Cross-section of cryo-EM map of the proteasome in S BAG1 and S D4 focusing on the interface between the ATPase and CP rings. Rpn1 (tan), OB ring (orange), ATPase ring (rosy brown), and CP (white) are colored separately, as indicated. In S BAG1 , the central channel is deformed and a large cavity is observed on top of the CP gate, whereas the interior of the ATPase ring is packed in S D4 . (b) In S BAG1 , the atypical positioning of the ATPase subunits creates a large cleft (highlighted in light green) between the OB (orange) and ATPase (rosy brown) rings. The structure contrasts with the S D4 structure (EMDB: 32283) (PDB: 7W3K) in (b) . The atomic models of Rpt1, Rpt4 and Rpt5 are colored in sky blue, pink and goldenrod, respectively

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: (a) Cross-section of cryo-EM maps in S D4 state (upper panel) (EMDB: 32282; PDB: 7W3K) and Bag1-bound conformations in S BAG1 (S BAG1 PDB: 9HEU, EMDB: 52097), focusing on the interface between the OB and ATPase rings. Conformational changes in Rpt4 and Rpt3 (see schematic representation in (c) ), result in a significant opening at the interface between the OB and ATPase domains. Cryo-EM densities are shown in transparent. (b) Cryo-EM segmentation of the ATPase ring in S D4 and S BAG1 (PDB: 9HEU; EMDB: 52097). A large cavity is observed in the middle of the ATPase ring in all three Bag1-bound conformations, while the ATPase ring is tightly packed in S D4 . Atomic models of the subcomplexes, and individual Rpt subunits are colored as followings; OB ring (orange), 20S CP (white), Rpt1 (light blue), Rpt2 (salmon), Rpt6 (purple), Rpt3 (green), Rpt4 (hot pink), Rpt5 (yellow). (c) Schematic representation of the movement of the ATPase ring from a top-view. Structural models in S BAG1 and S D4 are aligned with the 20S CP and only the ATPase rings are shown with the same color code in . The S D4 is shown in transparent. (d) Structural comparison between S BAG1 and S D4 focusing on the conformational change of Rpt3 (olive) and Rpt4 (hot pink). The S D4 structure is shown in transparent. (e) Structural comparison of the Rpt2 large domains at the interface of Rpt1 between S BAG1 (PDB: 9HEU) and S D4 (PDB: 7W3K). Two structures are superimposed with Rpt1 large domain (light blue). Only Rpt1 subunit in S BAG1 is shown (large and small domains in light blue and in blue, respectively). Rpt2 large domains in S BAG1 (salmon) and S D4 (light yellow) are shown for comparison. Rpt2 position is shifted and Arg(R)-finger Arg343 is positioned far from ATP in S BAG1 , implying a lack of ATPase activity. Detailed view is shown in the dotted square. ( f ) Structural comparison of the Rpt4 large domain at the interface of Rpt3 between S BAG1 and S D4 . Two structures are superimposed with Rpt3 large domain (olive). Only Rpt3 subunit in S BAG1 is shown (large and small domains in olive and green). Rpt4 large domains in S BAG1 (hot pink) and S D4 (light pink) are shown for comparison. Rpt4 large domain in S BAG1 shifts by ∼40 Å and rotates 65.5° in comparison to SD4 . Rpt4 Arg-finger Arg291 is highlighted.

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Cryo-EM Sample Prep, Comparison, Activity Assay

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: (a) ATPase activity of the 26S proteasome (black dash line) upon Bag1 (blue) Bag1 BD (red) or Bag1 UBL (green) titration. Whereas Bag1 BD shows no effect, full-length Bag1 and Bag1 UBL decrease 26S ATPase activity. The data represent the mean ± SD for n=5 independent experiments (represented with dots). (b) Insertion of Rpt C-terminal tails into CP a ring pockets. The RP–CP interface and insertion of Rpt C-terminal tail into the α-pockets of the CP in S BAG1 are shown. The cryo-EM density of the CP is shown in white, whereas the C-terminal tails of Rpt2, Rpt3 and Rpt6 are colored in dark salmon, green and purple respectively. Empty pockets are indicated with red asterisks. The EM density of the N-terminal tail of α3 in the ‘down’ state is shown in yellow. (b,c) N-terminal tail of α3 exhibits ‘up’ and ‘down’ states. The ‘up’ conformation (light green) corresponds to the ‘open’ gate, while the ‘down’ conformation (yellow) represents the ‘closed’ gate. Side view of the α3 subunit highlights the movement of the N-terminal tail (c) . (d) The pore-2 loop of Rpt2 moves lower towards the CP gate, by approximately 2 Å distance and interacts with N-terminal tails of α4 and α5. (e) Zoom-in of the cryo-EM density of the Rpt2 pore-2 loop and the N-terminal tails of α4 and α5. (f) The pore-2 loop of Rpt5 moves lower towards the CP gate, approximately 4 Å distance and interacts with N-terminal tails of α6 and α7. (g) Zoom of the cryo-EM density of the Rpt5 pore-2 loop and the N-terminal tails of α6 and α7. For (d-g) comparison with S D4 (PDB: 7W3K) was used.

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Activity Assay, Titration, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Structures of the 26S proteasome in complex with the Hsp70 cochaperone Bag1 reveal a novel mechanism of ubiquitin-independent proteasomal degradation

doi: 10.1101/2025.01.22.633148

Figure Lengend Snippet: (a) Structural model of the Hsp70-Bag1-bound 26S proteasome created based on Hsp70 NBD :Bag1:Rpn1 complex and the 26S:Bag1 complex together with an AlphaFold prediction of the ADP-bound Hsp70 and Bag1 complex. Hsp70 SBD (dark blue) is positioned adjacent to the OB-ATPase cleft, indicating a direct transfer of unfolded proteins to the 20S CP for degradation. (b) Summary of western blot results (left panel) analyzing proteasomal degradation of α-synuclein in the absence of ATP at 0, 8, and 24 hours. Statistical analysis (right panel) reveals that Bag1 alone (red) and with Hsp70 (orange) significantly enhance synuclein degradation compared to the proteasome alone (grey), while Hsp70 alone (yellow) shows stronger effects at later times. MG-132, as expected, inhibits degradation (dark blue). Data (n=4-5) analyzed via two-way ANOVA (*p=0.0402, ****p<0.0001).

Article Snippet: Then three washing steps of 10 min in PBST were performed to remove the blocking solution, and the membranes were later incubated with primary antibodies (all diluted in PBST) against Rpn1 (PSMD2 A11, Santa Cruz Biotechnology, 1:300 dilution), Hsp70 NBD (501043, PALEX,1:1000 dilution), Bag1 (α-Histidine tag coupled to horseradish peroxidase, Santa Cruz Biotechnology, 1:4000 dilution) and α-synuclein coupled to horseradish peroxidase (Santa Cruz Biotechnology, 1:100 dilution), 1 h RT with gentle shaking.

Techniques: Western Blot