Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Altered abundance of 26S proteasome subunits and associated factors in rpn10-2 1 .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Arabidopsis RPN13 interacts with RPN2a and RPN2b. ( A ) RPN13 is readily pulled down by GST-fused RPN2a or RPN2b. ( B ) Coexpression of AD-fused RPN13 with BD-fused RPN2a or RPN2b activates the HIS3 reporter, as shown by histidine auxotrophic growth. ( C ) Coexpression of AD-fused wild-type RPN13, site-specific-variant RPN13-101A, or RPN13-22A with BD-fused RPN2a or RPN2b activates the HIS3 reporter, as shown by histidine auxotrophic growth. ( B , C ) Positive (+) and negative (−) controls are p53-SV40 (SV40 T-antigen) and LAMIN (lamin C)-SV40 protein pairs, representing known interacting and non-interacting partners. ( D ) Wild-type RPN13 (Wt) and RPN13 variants A47 and Q70, but not R67 and R88, are readily pulled down by GST-fused RPN2a or RPN2b. ( A , D ) Amounts of prey and bait used in pull-down assays are 5 μg for all RPN13 variants and 35 μg, 350 μg, and 320 μg for GST, GST-RPN2a, and GST-RPN2b, respectively. One-hundredth of the input prey (50 ng) and one-tenth of the pull-down products were analyzed by immunoblotting against α-T7. One-tenth of the pull-down products (Baits) was examined by staining with Brilliant Blue R to confirm approximately equivalent immobilization. The pull-down product against GST alone was analyzed as a negative control.

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Variant Assay, Western Blot, Staining, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: Arabidopsis RPN13 interacts specifically with UCH2 but not with UCH1. ( A ) GST-fused UCH2 but not UCH1 readily pulled down RPN13. ( B ) GST-fused RPN13 but not RPN13-A1A2 readily pulled down UCH2. ( C ) GST-fused RPN13 could not pull down UCH1. ( A – C ) Sample amounts used in pull-down assays were 5 μg for all preys (RPN13, UCH1, and UCH2) and ~30 μg, 105 μg, 126 μg, 130 μg, and 130 μg for GST, GST-UCH1, GST-UCH2, GST-RPN13, and GST-RPN13-A1A2, respectively. One-hundredth of the input prey (50 ng) and one-tenth of the pull-down products were analyzed by immunoblotting against α-T7. One-tenth of the pull-down products (Baits) was examined by staining with Brilliant Blue R to confirm approximately equivalent immobilization. The pull-down products against GST alone were analyzed as negative controls. ( D ) Coexpression of AD-fused RPN13 with BD-fused UCH2 but not UCH1 activated the HIS3 reporter, as shown by histidine auxotrophic growth. The positive (+) and negative (−) controls used were the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: The C-terminal 246–254 region in the Arabidopsis RPN13 DEUBAD domain is critical for UCH2 interactions. ( A ) Schematic diagram shows BD-fused UCH2 (bait) and AD-fused RPN13 variants (preys), including wild-type and site-specific, C-terminal deletion, and alanine scanning mutants for Y2H assays (see the main text for details). The 330-amino-acid UCH2 coding region is boxed in green with its unique 12-amino-acid C-terminal extension designated by a red box. Coordinates for full-length proteins, mutation sites, and alanine scanning region are indicated. ( B ) Four conserved residues, E208, L209, K275, and D279, in Arabidopsis RPN13 are not involved in UCH2 binding. Similar to AD-fused wild-type RPN13, two AD-fused dual-alanine substituted (EL-AA and KD-AA) and one AD-fused quadruple-alanine substituted (ELKD-A4) RPN13 variants could still activate the HIS3 reporter when coexpressed with BD-fused UCH2. ( C , D ) When coexpressed with BD-fused UCH2, only the AD-fused C-terminal deleted RPN13 variants CΔ1 and CΔ2, but not CΔ3–6, could activate the HIS3 reporter. ( E ) When coexpressed with BD-fused UCH2, similar to AD-fused wild-type RPN13, each of the five subregion-mutated RPN13 variants (A1–A5) could activate the HIS3 reporter. ( F ) When coexpressed with BD-fused UCH2, similar to AD-fused wild-type RPN13, each of the three serial C-terminally combined subregion-mutated AD-fused RPN13 variants (A4–5, A3–5, and A2–5) could activate the HIS3 reporter. ( G ) When coexpressed with BD-fused UCH2, unlike AD-fused wild-type RPN13, all four N-terminally combined subregion-mutated AD-fused RPN13 variants (A1–2, A1–3, A1–4, and A1–5) could not activate the HIS3 reporter. ( B – G ) The positive (+) and negative (−) controls used are the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Mutagenesis, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

doi: 10.3390/ijms252111650

Figure Lengend Snippet: The 12-amino-acid C-terminal extension of Arabidopsis UCH2 is critical for RPN13 interactions. ( A ) Schematic diagram shows AD-fused RPN13 (prey) and BD-fused UCH1 and UCH2 variants (baits), including wild-type (BD-UCH2 and BD-UCH1), a C-terminal-deleted UCH2 (BD-UCH2-CΔ1), alanine scanning UCH2 mutants, and three C-terminally swapped UCH1 mutants (UCH1 2C , UCH1 2C-A2 , and UCH1 2C-A3 ) for Y2H assays (see the main text for details). The UCH2 coding region and its unique 12-amino-acid C-terminal extension are illustrated in the same way as in A. The 334-amino-acid UCH1 coding region is boxed in cyan blue. Coordinates for full-length proteins, deletion/swapping sites, and alanine scanning region are indicated. ( B ) When coexpressed with AD-fused RPN13, unlike BD-fused UCH1 (UCH1) and the C-terminal-deleted BD-UCH2 fusion (UCH2-CΔ1), the BD-fused wild-type UCH2 (UCH2), a C-terminal-swapped BD-UCH1 fusion (UCH1 2C ), and a BD-fused UCH2 variant harboring all-alanine substitutions in one of three subregions of the UCH2 C-terminus (UCH2-A1) could activate the HIS3 reporter. ( C ) When coexpressed with AD-fused RPN13, similar to BD-fused UCH2, two BD-fused UCH2 variants harboring all-alanine substitutions in each of two subregions of the UCH2 C-terminus (UCH2-A2 and UCH2-A3) could activate the HIS3 reporter. Also, similar to the C-terminal-swapped BD-UCH1 fusion (UCH1 2C ), when coexpressed with the AD-fused RPN13, two C-terminal-swapped BD-UCH1 variants harboring all-alanine substitutions in each of two subregions of the swapped UCH2 C-terminus (UCH1 2C-A2 and UCH1 2C-A3 ) could activate the HIS3 reporter. ( D ) When coexpressed with AD-fused RPN13, similar to BD-fused UCH2 (UCH2), each of the four BD-fused UCH2 variants harboring all-alanine substitutions in two or all three sub-regions of the UCH2 C-terminus (A1–2, A2–3, A1/A3, and A1–3) could activate the HIS3 reporter. ( B – D ) The positive (+) and negative (−) controls used are the same as those described in .

Article Snippet: To examine the association of RPN13 with proteasome complexes, the latter were separated using 4% native PAGE and further separated via second-dimensional SDS-PAGE following immunoblotting using rabbit polyclonal antisera against recombinant Arabidopsis RPN13 (custom-made by Genesis Biotech, Taipei, Taiwan) or moss 20S proteasomes (a kind gift from Dr. Pirre-Alain Girod).

Techniques: Variant 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: Expression was induced with 1 mM IPTG at the exponential phase (Rpn10) or using AutoInducible Medium (Rpn1, Rpn13) (CondaLab).

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: Expression was induced with 1 mM IPTG at the exponential phase (Rpn10) or using AutoInducible Medium (Rpn1, Rpn13) (CondaLab).

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: Expression was induced with 1 mM IPTG at the exponential phase (Rpn10) or using AutoInducible Medium (Rpn1, Rpn13) (CondaLab).

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) 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: Expression was induced with 1 mM IPTG at the exponential phase (Rpn10) or using AutoInducible Medium (Rpn1, Rpn13) (CondaLab).

Techniques: Western Blot

Journal: The EMBO Journal

Article Title: Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

doi: 10.1002/embj.201386906

Figure Lengend Snippet: A–E HEK293F cells were transfected with siRNA against proteasome-associated ubiquitin ligases Ube3c/Hul5 (A), Ube3a/E6AP (B), Rnf181 (C), Huwe1 (D), and Ubr4 (E). After BTZ treatment (1 μM, 4 h), cells were lysed, and ubiquitination of Rpn13 (A–E) was measured by WB. Knockdown of Ube3c blocked Rpn13 ubiquitination.

Article Snippet: The following antibodies were used: Ubr4 (Abcam ab86738, 1:500), Ube3c/Hul5 (GeneTex Inc. GTX119102, 1:1,000), RNF181 (GeneTex Inc. GTX117747, 1:1,000), Ube3a/E6AP (Santa Cruz Biotechnology sc-25509, 1:1,000), Huwe1 (Abcam ab78397, 1:1,000), Usp14 (Epitomics S3270, 1:1,000), Uch37 (Epitomics 3904-1, 1:10,000), Rpt1 (Enzo Lifesciences BML-PW8825, 1:10,000), Rpt5 (Enzo Lifesciences BML-PW8770, 1:10,000), Rpn1 (Abcam ab21749, 1:10,000), S5a (Enzo Lifesciences BML-PW9250-0100, 1:1,000), GAPDH (Sigma, G8795, 1:10,000), Ub (Enzo Lifesciences BML-PW8810, 1:5,000), Rpn13 (Enzo Lifesciences, BML-PW9910, 1:3,000), 20S (α1,2,3,5,6,7) (Enzo Lifesciences, BML-PW8195, 1:10,000), P4D1 (ubiquitin) (Santa Cruz Biotechnology, 1:1,000), FLAG (Sigma-Aldrich, F1804, 1:2,000), tubulin (Rockland, 600-401-880, 1:3,000), Gadd34 (Proteintech, 10449-1-AP, 1:1,500), and ATF3 (Santa Cruz, sc-188, 1:1,000).

Techniques: Transfection

Journal: The EMBO Journal

Article Title: Autoubiquitination of the 26S Proteasome on Rpn13 Regulates Breakdown of Ubiquitin Conjugates

doi: 10.1002/embj.201386906

Figure Lengend Snippet: Normally, Ub chains on the substrate bind initially to Rpn13 and S5a/Rpn10, and once the polypeptide becomes committed to degradation, it is translocated through the ATPase ring into the 20S (Finley, 2009; Peth et al, 2013). The Ub ligase, Ube3c, presumably functions to extend Ub chains to facilitate substrate degradation and ensure its processivity (Crosas et al, 2006). However, when proteasomes are stalled due to difficult-to-degrade substrates, or during proteotoxic stresses (heat-shock or arsenite exposure), or in vitro when 20S function is inhibited with bortezomib, 19S function slowed with ATPγS, or substrate deubiquitination by Rpn11 is blocked with a Zn2+ chelator, Ube3c ubiquitinates Rpn13 and prevents further substrate binding in vivo.

Article Snippet: The following antibodies were used: Ubr4 (Abcam ab86738, 1:500), Ube3c/Hul5 (GeneTex Inc. GTX119102, 1:1,000), RNF181 (GeneTex Inc. GTX117747, 1:1,000), Ube3a/E6AP (Santa Cruz Biotechnology sc-25509, 1:1,000), Huwe1 (Abcam ab78397, 1:1,000), Usp14 (Epitomics S3270, 1:1,000), Uch37 (Epitomics 3904-1, 1:10,000), Rpt1 (Enzo Lifesciences BML-PW8825, 1:10,000), Rpt5 (Enzo Lifesciences BML-PW8770, 1:10,000), Rpn1 (Abcam ab21749, 1:10,000), S5a (Enzo Lifesciences BML-PW9250-0100, 1:1,000), GAPDH (Sigma, G8795, 1:10,000), Ub (Enzo Lifesciences BML-PW8810, 1:5,000), Rpn13 (Enzo Lifesciences, BML-PW9910, 1:3,000), 20S (α1,2,3,5,6,7) (Enzo Lifesciences, BML-PW8195, 1:10,000), P4D1 (ubiquitin) (Santa Cruz Biotechnology, 1:1,000), FLAG (Sigma-Aldrich, F1804, 1:2,000), tubulin (Rockland, 600-401-880, 1:3,000), Gadd34 (Proteintech, 10449-1-AP, 1:1,500), and ATF3 (Santa Cruz, sc-188, 1:1,000).

Techniques: In Vitro, Binding Assay, In Vivo