mouse anti bag1  (Santa Cruz Biotechnology)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Proteostasis network response to environmental chronic stress: linking survival to protein aggregation in a human neuroblastoma cellular model

doi: 10.1007/s00018-025-05884-6

Figure Lengend Snippet: Analysis of the BAG1/BAG3 triage system. a Western blot analysis of the expression of BAG1 in the RIPA-soluble protein fraction after 24 h ( n = 3) and b 72 h ( n = 4). c Analysis of the expression of soluble BAG3 after 24 h ( n = 4) and d 72 h ( n = 4). e. Analysis of BAG3 in the insoluble fraction after 72 h of treatment ( n = 3). Data were normalized against Vinculin as loading control. For the insoluble fraction, the same amount of proteins quantified in the respective soluble fractions were considered. Data are expressed as fold increase ± S.E.M and compared to normalized control, shown as dashed line. Statistical significance was determined after one-way ANOVA with Dunnet’s multiple comparison test of each treatment against control. *** p < 0.001; ** p < 0.01; * p < 0.05

Article Snippet: Primary antibodies utilized were: PARP-1, Cell Signaling Technology, 9542; BiP/Grp78, Abcam, ab32618; p-eIF2⍺, Cell Signaling Technology, 9721; eIF2⍺, Cell Signaling Technology, 9722; Ubiquitin, antibodies.com, A85455; BAG3, Invitrogen, MA5-32706; BAG1, Proteintech, 19064–1-AP; LC3, RBC Lifescience, PD014; p62/SQSTM1, Cell Signaling Technology, 8025; pTDP-43, Proteintech, 80007–1-RR −1-AP; TDP-43, Proteintech, 12892–1-AP; β-Actin, Sigma-Aldrich, A5316; Vinculin, Sigma-Aldrich, V9131.

Techniques: Western Blot, Expressing, Control, 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 ) 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