Journal: bioRxiv

Article Title: Resolving the Activation Mechanism of the Human 20S Proteasome

doi: 10.64898/2026.04.13.718244

Figure Lengend Snippet: a) Structural overview of the human 26S Proteasome (PDBID: 6MSK) . The 26S complex contains the 19S proteasome activator (orange) and the 20S proteasome. The 20S contains regulatory α-subunits (blue) and catalytic β-subunits (red). Subunits are labeled with their human gene (PSMC*) and yeast homolog (Rpt*) names. b) Top view of the 20S with activation pockets (black circles) and the central gate channel (green circle) highlighted in the closed (left) and open (right) states. All α-subunits are labeled with human gene (PSMA*) and yeast homolog (α*) names. 19S subunits are shown over their corresponding binding pockets (orange boxes) with the Rpt5 subunit highlighted (cyan box). c) Rpt5 C-terminal sequences across different eukaryotic organisms. Tails containing HbYX motifs are marked in red, while tails containing YФ are marked in green and co-evolving P5L in blue. The structure of PA optimized peptide NLSYYT is shown to the right (PDBID: 6XMJ) . d) Proteasome chymotrypsin-like activity assay. Maximum activity was normalized to NLSYYT. NLSYYT peptide (salmon, EC 50 = 9.18 ± 2.25 μM) and Rpt5 (pink, EC 50 = 34.02 ± 15.51 μM) were the only peptides to stimulate proteasome activity independently. Data are plotted individually (n=3). e) Table of estimated K I values based on a fluorescence polarization competition assay. Reported values are averaged (n=3). f) Chymotrypsin-like activity assay where the 5 th C-terminal amino acid in each peptide (P5, red box) was replaced by all listed amino acids that are small hydrophobic (grey), basic (blue), acidic (red), polar (purple), aromatic (beige), or other (white). All peptides were normalized to NLSYYT (100 ± 12.77%) with Phe (61.01 ± 6.75%), Trp (55.32 ± 2.86%), and Tyr (75.78 ± 6.97%) showing reduced activity and all others showing significant loss of activity (<50%). Data are plotted individually (n=3). g) Proteasome chymotrypsin-like activity assay with peptides derived from NLSYYT with P5 substitutions (red letters). Maximum activity was normalized to NLSYYT. Average curve fits are shown with data plotted individually (n=3). NLSYYT (salmon, EC 50 = 18.36 ± 8.78 μM), NFSYYT (pink, EC 50 = 52.54 ± 32.09 μM), NWSYYT (lavender, EC 50 = 24.91 ± 3.44 μM), and NYSYYT (purple, EC 50 = 103.8 ± 12.87 μM) are the only four peptides to show significant activity. h) Table of estimated K I values from a fluorescence polarization competition assay with peptides derived from NLSYYT with P5 substitutions (red letters). Reported values are averaged (n=3).

Article Snippet: Human 20S proteasome used for in vitro experiments was purchased from R&D Systems.

Techniques: Labeling, Activation Assay, Binding Assay, Activity Assay, Fluorescence, Competitive Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: Resolving the Activation Mechanism of the Human 20S Proteasome

doi: 10.64898/2026.04.13.718244

Figure Lengend Snippet: a) Top view of h20S bound to PA26 YYT (PDB: 6XMJ) with inset showing (i) Helix 1, (ii) Gate Loop, (iii) PA Tail, and (iv) Gate Tail (top). Cartoon structure of helix 1 of α5 across all structures to demonstrate observable shifts from h20S closed gate state to fully open gate state with all other PA26 bound structures (bottom). The most defining Cα shifts during gate opening can be measured by the retraction of the gate loop, and the vertical rise of helix 1. b) Scatter plot of Cα-Cα shifts (Å) for each amino acid in helix 1 across all structures relative to the closed-gate structure. c) Bar plot of Cα-Cα shifts (Å) for the conserved gate loop proline in each α-subunit. d) Cryo-EM density maps of 20S α-subunit gate tails across unbound h20S (light blue, PDB: 6RGQ) and h20S structures bound to PA26 P5V (blue, PDB: 12CR), PA26 P5Y (purple, PDB: 12CQ), PA26 P5W (lavender, PDB: 12CP), PA26 P5F (pink, PDB: 12CN), or PA26 YYT (salmon, PDB: 6XMJ) . Each PA tail density is shown in a grid indicating the structure and corresponding α-subunit tail. Both observed states are shown for α3 in the PA26 P5W -bound structure. e) Cartoon structure overlays of the h20S gate tails across each of the α-subunits. State shifts of the Gate Tails and Gate Loops are highlighted with arrows.

Article Snippet: Human 20S proteasome used for in vitro experiments was purchased from R&D Systems.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Resolving the Activation Mechanism of the Human 20S Proteasome

doi: 10.64898/2026.04.13.718244

Figure Lengend Snippet: a) Structure of the NLSYYT sequence bound within the α5-α6 pocket (left, red circle). P1-P4 side-chains of all bound PA26 constructs are shown as an overlay (right). Important H-bonds (yellow dashes) and salt bridges (purple dashes) are highlighted in the structure. b) Cryo-EM density maps of R20 in the 20S α5 subunit and P5 when bound to PA26 YYT , PA26 P5F , PA26 P5W , PA26 P5Y , and PA26 P5V . c) P5 side-chains of each bound PA26 tail are shown as sticks alongside helix 1 of α5. All side-chain positions are shown in the majority state across particles. An arbitrary line is drawn between constructs where most gate tails are open on the left side of the line and closed on the right. d) Side-chains of R20 on the α5 gate loop are shown as sticks. An arbitrary line is drawn between R20 positions where most gate tails are closed vs. open.

Article Snippet: Human 20S proteasome used for in vitro experiments was purchased from R&D Systems.

Techniques: Sequencing, Construct, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Resolving the Activation Mechanism of the Human 20S Proteasome

doi: 10.64898/2026.04.13.718244

Figure Lengend Snippet: a) Schematic showing the design of cell-based experiments using the transpose sleeping beauty method . b) Live cell proteasome chymotrypsin-like activity assay. Activity was normalized to DMSO-treated cells for all cell lines, and activity was measured for cells treated with 1 μg/mL doxycycline. Data reported individually as biological replicates (n=3). Significance values were calculated by two-way ANOVA and compared to PA26 YYT where ns = not significant, p<0.1 = *, p<0.01 = **, p<0.001 = ***, and p<0.0001 = ****. c) Proteasome chymotrypsin-like activity assay on HEK293T lysates. Data reported individually as biological replicates (n=3). Significance values were calculated by one-way ANOVA and compared to PA26 YYT where ns = not significant, p<0.1 = *, p<0.01 = **, p<0.001 = ***, and p<0.0001 = ****. d) Native PAGE western blot of 25 μg of total protein from HEK293T lysates blotted with α-PSMA5 to visualize free, single, or double PA26-capped h20S (top). An identical native-PAGE soaked in buffer containing a fluorescent activity reporter visualizes the location of active PA26+20S on the gel (middle). Loading was validated using an SDS-PAGE loading control western blot for α-actin (bottom). e) Volcano plot summarizing TMT-MS proteomic analyses of HEK293T lines expressing PA26 YYT (left). Differentially expressed hits are shown as stabilized (blue dots) or destabilized (red dots). Hits are constrained by FDR < 0.05 (horizontal dashed line) and >30% FC change (vertical dashed lines). Reported hits are consistent across biological replicates (n=3). A bar plot shows the total number of differentially expressed proteins detected across HEK293T lines expressing PA26 YYT , PA26 P5W , PA26 P5V , and PA26 YAT (right). f) Enrichment of stabilized protein hits in the PA26 YYT -expressing cell line organized by GO annotation of function. Hits were selected using the same FDR and FC constraints as (e). Number of proteins are represented by circle size, while p-value is shown by color gradient (salmon to white). g) Enrichment of destabilized protein hits in the PA26 YYT -expressing cell line organized by GO annotation of function. Hits were selected using the same FDR and FC constraints as (e). Number of proteins are represented by circle size, while p-value is shown by color gradient (salmon to white). h) Heat maps of relative peptide abundance for proteins associated with the 20S core particle subunits, 19S regulatory PA, and UIPS PAs across the cell lines. Abundance is shown as Log 2 FC on a scale of −0.5 (blue) to 0.5 (red).

Article Snippet: Human 20S proteasome used for in vitro experiments was purchased from R&D Systems.

Techniques: Activity Assay, Clear Native PAGE, Western Blot, SDS Page, Control, Expressing

Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d , e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in ( b – e ) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: The human 20S proteasome protein (E-360) was purchased from R&D Systems.

Techniques: Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, shRNA, Purification, Incubation, Positive Control