26s proteasome  (R&D Systems)

Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: The impairment can be mitigated by proteasome activators. (A) 20S Proteasome chymotrypsin-like peptidase activity is inhibited by oligomeric Aβ42, but not by Aβ42 monomers or fibrils. N = 4. Asterisks denote statistically significant differences (p<0.05). Right: atomic force microscopy (AFM) images of Aβ particles (tapping mode in air). The occasional larger particles in the “monomer” preparation are likely spontaneously forming oligomers. ( B ) Morphometric analysis of the 20S proteasome particles imaged by AFM (tapping mode in liquid) reveals shifts in the particles’ dimensions upon incubation with oligomeric Aβ42. 827 control 20S particles (incubated with a vehicle) and 1181 particles incubated with 2 µM oligomeric Aβ42 were analysed. Solid lines are fittings for the frequencies of control (black) and oligo-treated (red) particles. Since almost all particles are in to-view position, the “length” parameter generated during the particle analysis corresponds to the diameter of the 20S α face. The diameters are raw numbers without correction for tip broadening. When the correction of 2 pixels for SNL probe is applied, the diameter for peak 1 (raw: 14 - 15 nm) falls into 10 – 11 nm range, in excellent agreement with the crystal structure of the human 20S proteasome . See Results for putative assignment of proteasome forms to the numbered peaks. (C) Incubation with oligomeric Aβ42 shifts the conformational equilibrium of 20S core particles imaged by AFM (tapping mode in liquid) toward less open-gate and closed-gate forms, but more intermediate forms. (D) Oligomeric Aβ42 does not significantly affect degradation of oxidized hemoglobin. Degradation of hemoglobin is enhanced by a range of oligomeric Aβ42 concentrations. N=4 samples. ( E, F ) Treatment of the 20S proteasome with activators TAT1-DEN or TAT1-TOD partially protects from inhibition inflicted by the oligomeric Aβ42. ( G ) Incubation with the proteasome activator TAT1-DEN induces a dramatic shift toward open-gate forms, even in the presence of 2 µM of oligomeric Aβ42. The numbers in columns indicate percent of conformers. The number of particles analyzed: 733 (vehicle control), 843 (with oligo Aβ42), 270 (with 1 µM TAT1-DEN) and 171 (with oligo Aβ42and TAT1-DEN). Average ± SD, n= 5 to 9 fields.

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Activity Assay, Microscopy, Incubation, Control, Generated, Particle Size Analysis, Inhibition

Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: (A) Native page immunoblot depicting purified 20S and 26S proteasome under incubation with oligomeric Aβ42. Immunoblot performed against proteasome β5 subunit and accompanying total protein silver stain. Arrows depict 26S and 20S proteasome assemblages. (B) Native page immunoblot depicting purified 26S proteasome under incubation with varying concentrations of oligomeric Aβ42. Top image shows a representative set, histogram represents N=3 per condition. (C) Model for impact of Aβ on proteasome processes. *p < 0 . 05, Student’s t test was used unless otherwise stated. N represents the number of animals or samples per group .

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Clear Native PAGE, Western Blot, Purification, Incubation, Silver Staining

Journal: bioRxiv

Article Title: Sterility-Independent Enhancement of Proteasome Function via Floxuridine-Triggered Detoxification in C. elegans

doi: 10.1101/2023.11.11.566706

Figure Lengend Snippet: (A) A scheme illustrating the method for monitoring in vivo UPS activity, employing UbV-GFP as the indicative reporter. (B) In vivo UPS activity assay showing the effect of FUdR on UbV-GFP turnover in the presence of the proteasome inhibitor bortezomib (Btz) and during the RNAi depletion of pas-1 and pbs-6 proteasome subunits. Scale bar corresponds to 400 μm. (C) FUdR’s effect on proteasome activity, as measured by trypsin-like, chymotrypsin-like, and caspase-like activity in wild-type worms with or without FUdR treatment. Bortezomib (Btz) served as the negative control. Proteasome activity is represented as slopes obtained from kinetic measurements. The experiments were conducted thrice as separate biological replicates, and significance levels (** - P ≤ 0.01) were determined using an unpaired t-test with Welch’s correction. (D) Western blot showing levels of PAS-2, PAS-3, PAS-5, and PAS-7 proteasome subunits levels in wild-type worms following the RNAi depletion of RPN-9, PBS-6, PBS-2 and PAS-1 proteasome components in the presence or absence of FUdR, as depicted by using anti-proteasome 20S alpha 1+2+3+5+6+7 antibody. The No-Stain Protein Labeling Reagent was used to confirm equal protein loading.

Article Snippet: Proteasome activity was also measured using 5 nM of recombinant human 26S proteasome (Cat: E-365; R&D Systems).

Techniques: In Vivo, Activity Assay, Negative Control, Western Blot, Staining, Labeling

Journal: bioRxiv

Article Title: Sterility-Independent Enhancement of Proteasome Function via Floxuridine-Triggered Detoxification in C. elegans

doi: 10.1101/2023.11.11.566706

Figure Lengend Snippet: (A) In vivo UPS activity assay showing the effect of different concentrations of FUdR on UbV-GFP turnover. (B) In vivo UPS activity assay showing the effect of different pyrimidine analogs, 5-fluorouracil (FU), 5-fluorocytosine (FC), and 5-fluorodeoxycytidine (FCdR), on UbV-GFP turnover. In panels A and B, the scale bar corresponds to 400 μm. (C) Effect of different concentrations of FUdR on the activity of purified human 26S proteasome in HeLa cells, as measured by trypsin-like, chymotrypsin-like, and caspase-like activity in wild-type worms with or without FUdR treatment. Bortezomib (Btz) served as the negative control. Proteasome activity is represented as slopes obtained from kinetic measurements. The experiments were conducted thrice as separate biological replicates. (D) FUdR effect on chymotrypsin-like proteasome activity in HeLa cells. Cells were treated with final concentrations of 0.4 and 2 µM of FUdR and 10 nM bortezomib (Btz) as control for 6 hr. The assay was conducted by incubating the cells with 100 µl Proteasome Assay Loading Solution for 2 h as described in the methods. Results from three technical replicates were corrected for background by subtracting the fluorescence of the medium without cells and further normalized to dimethyl sulfoxide control. The graph shows the average values obtained from either two or four biological replicates for experiments that involve FUdR or Btz, respectively. (E) Western blot showing global translation activity in wild-type and glp-1(e2144) worms, in the presence or absence of FUdR, as depicted by using the anti-puromycin antibody. The No-Stain Protein Labeling Reagent was used to confirm equal protein loading.

Article Snippet: Proteasome activity was also measured using 5 nM of recombinant human 26S proteasome (Cat: E-365; R&D Systems).

Techniques: In Vivo, Activity Assay, Purification, Negative Control, Control, Fluorescence, Western Blot, Staining, Labeling

Journal: bioRxiv

Article Title: Sterility-Independent Enhancement of Proteasome Function via Floxuridine-Triggered Detoxification in C. elegans

doi: 10.1101/2023.11.11.566706

Figure Lengend Snippet: (A) Western blot showing the impact of FUdR on UbV-GFP reporter turnover upon depletion of various 26S subunits in control worms, as depicted by using anti-GFP antibody. The No-Stain Protein Labeling Reagent was used to confirm equal protein loading. (B) Western blot showing degradation of UbV-GFP in control, atg-1 RNAi (applied at either L1 or young adult stages), and lgg-1 RNAi (applied at the young adult stage) worms co-treated with bortezomib (Btz) in the presence or absence of FUdR, as depicted by using anti-GFP antibody. The No-Stain Protein Labeling Reagent was used to confirm equal protein loading. (C) Western blot showing degradation of UbV-GFP upon RNAi depletion of fem-1 in control worms, with or without bortezomib (Btz) or FUdR, as depicted by using anti-GFP antibody. The No-Stain Protein Labeling Reagent was used to confirm equal protein loading. ( D) FUdR’s effect on proteasome activity, as measured by trypsin-like, chymotrypsin-like, and caspase-like activity in skn-1 silenced worms with or without FUdR treatment. Proteasome activity is represented as slopes obtained from kinetic measurements. The experiments were conducted thrice as separate biological replicates, and significance levels (ns - not significant) were determined using an unpaired t-test with Welch’s correction.

Article Snippet: Proteasome activity was also measured using 5 nM of recombinant human 26S proteasome (Cat: E-365; R&D Systems).

Techniques: Western Blot, Control, Staining, Labeling, Activity Assay

Journal: bioRxiv

Article Title: pVHL regulates protein stability of TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

doi: 10.1101/2021.10.14.464355

Figure Lengend Snippet: (A) In vitro cell-free GST pulldown assay revealed direct interaction between RPN10 and pVHL. Proteasome was incubated with GST or GST-pVHL and pulled down by glutathione agarose. RPN10 is a critical proteasomal subunit protein and was assessed by western blotting. Input proteins were examined by Coomassie blue staining. Red arrow indicates GST-pVHL. (B) Co-IP assay revealed that Flag-pVHL interacted with endogenous RPN10 in HEK293T cells. (C) In vitro cell-free GST pulldown assay detected binding of full-length and mutant pVHL to proteasome. Red arrows indicate GST-fusion protein. (D) Schematic illustration of mechanism by which TCF/LEF protein stability is regulated by pVHL.

Article Snippet: To test the direct interaction between pVHL and proteasome, the bait GST or GST fusion protein was incubated with purified human 26S proteasome (E365; Boston Biochem, Cambridge, MA, USA) at 4 °C for 2 h in binding buffer (1× PBS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100).

Techniques: In Vitro, GST Pulldown Assay, Incubation, Western Blot, Staining, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis