Journal: Science Advances

Article Title: The immunoproteasome catalytic β5i subunit regulates cardiac hypertrophy by targeting the autophagy protein ATG5 for degradation

doi: 10.1126/sciadv.aau0495

Figure Lengend Snippet: ( A ) Cluster of the proteasome gene expression profiles in saline- or Ang II–infused mouse heart at day 1 ( n = 3 samples per group). ( B ) qPCR analyses of β1, β2, β5, β1i, β2i, and β5i mRNA expression in saline- or Ang II–infused mouse heart at day 1 and control ( n = 3 per group). ( C ) Protein levels of β5i in NRCMs exposed to Ang II (100 nM) at different time points (top) and quantification (bottom, n = 3). ( D and E ) Protein levels of β5i in WT hearts after 2 or 4 weeks of Ang II infusion (1000 ng/kg per min) or TAC operation (top) and quantification (bottom, n = 3). ( F ) Neonatal rat fibroblasts were treated with Ang II for 0 to 24 hours. The β5i protein expression was examined by Western blot analysis (top) and quantification of the relative protein levels (bottom, n = 3). ( G ) Representative immunohistochemical staining of β5i and BNP proteins in the heart tissues from normal control and HF patients (left). Quantification of β5i- and BNP-positive areas (right, n = 3). ( H and I ) β5i level and chymotrypsin-like activity in blood samples of normal controls ( n = 38) and HF patients ( n = 38). Data are presented as means ± SEM, and n represents the number of samples per group. * P < 0.05 and ** P < 0.01 versus saline, sham, or normal control. RLU, relative luciferase units.

Article Snippet: The full-length clone DNA of human proteasome (prosome, macropain) subunit β type 8 (large multifunctional peptidase 7) with pCMV3-N-FLAG–expressing plasmid was purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Activity Assay, Luciferase

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: PLoS Pathogens

Article Title: Hepatitis C virus induces oxidation and degradation of apolipoprotein B to enhance lipid accumulation and promote viral production

doi: 10.1371/journal.ppat.1009889

Figure Lengend Snippet: (A) Huh-7 cells were infected with HCV (MOI = 1) for 4 days or treated with 1 μM H 2 O 2 for 24 hours. Intracellular ROS levels were analyzed using a Fluorometric Intracellular ROS Kit. (B and C) Huh-7 cells were infected with HCV (MOI = 1) for 4 days prior to treatment with 1 mM NAC or 100 μM PDTC for 12 hours. ApoB was immunoprecipitated, and carbonyl groups generated by oxidation were derivatized to DNP and detected by western blotting with an anti-DNP antibody. The protein levels of ApoB and the core were also analyzed. Actin was used as the loading control. (D) Huh-7 cells were infected with HCV at different MOIs. Cell lysates were separated on a native-PAGE gel. Proteasomes were detected by western blotting with an anti-PSMB5 antibody. (E) Huh-7 cells were infected with HCV at different MOIs. Cell lysates were separated on a native-PAGE gel. The proteolytic activities of the 26S proteasome and the 20S proteasome (in the presence of 0.02% SDS) were analyzed with the proteasome substrate suc-LLAV-AMC. The stained gel was analyzed using a UV trans-illuminator at 365 nm wavelength. (F) ApoB was immunoprecipitated from mock-infected, HCV-infected (MOI = 1), and HCV-infected NAC/PDTC-treated cells prior to treatment with MG-132. Immunoprecipitated ApoB was incubated with purified 20S proteasome for the indicated timepoints in vitro. ApoB and the 20S proteasome in the reaction mixtures were analyzed by immunoblotting. (G) ApoB was immunoprecipitated from mock-infected and HCV-infected cells (MOI = 1) at day 4 after infection. Protein oxidation was analyzed as described in Methods. (H) A GST pulldown assay was performed to evaluate the interaction of the 20S proteasome subunit PSMA1-PSMA7 with ApoB from HCV-infected (MOI = 1) and MG-132-treated Huh-7 cells. (I) A GST pulldown assay was performed to evaluate the interaction of the 20S proteasome subunits PSMA5 and PSMA6 with ApoB from mock-infected, HCV-infected (MOI = 1), or HCV-infected Huh-7 cells treated with MG-132. The data in C and F are presented as the means ± SDs from densitometry analyses of n = 2 or 3 independent experiments, and representative gels from each specific assay are shown. The statistical significance was determined by unpaired two-sided Student’s t -tests. n.s., not significant. * P < 0.05. ** P < 0.01.

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

Techniques: Infection, Immunoprecipitation, Generated, Western Blot, Control, Clear Native PAGE, Staining, Incubation, Purification, In Vitro, GST Pulldown Assay

Journal: PLoS Pathogens

Article Title: Hepatitis C virus induces oxidation and degradation of apolipoprotein B to enhance lipid accumulation and promote viral production

doi: 10.1371/journal.ppat.1009889

Figure Lengend Snippet: ApoB is retrotranslocated from the ER lumen to cytosolic LDs via a process requiring Derlin-1. HCV infection induces oxidative stress and results in ApoB oxidation. Oxidized ApoB is recognized and degraded by the 20S proteasome. The degradation of ApoB impairs lipid secretion and contributes to lipid accumulation, which might lead to enhanced HCV production.

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

Techniques: Infection

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Proteasome activity measured as in-gel ABP fluorescence and Western blot analysis of PSMB1, POMP and GAPDH levels in HEK293 cells transfected with each of three different siRNAs targeting PSMB1 or scrambled control (Scrm) and cultured for 72 hr before analysis. Total protein staining was used as a loading control. (B) Analysis of experiments like the one shown in A. Quantification of PSMB1 protein levels shows significant depletion with all three siRNAs, with siRNA-A and siRNA-B producing the strongest effects. Quantification of proteasome activity by in-gel ABP fluorescence indicates significantly reduced activity following PSMB1 knockdown. POMP protein levels are significantly increased in response to PSMB1 depletion. ns=p>0.05, *p≤0.05, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Dunnett’s test, n = 4 biological replicates, mean±SD, FC=fold change. (C) Proteasome activity measured as in-gel ABP fluorescence and Western blot analysis of PSMB7, POMP and GAPDH levels in HEK293 cells transfected with each of three different siRNAs targeting PSMB7 or scrambled control (Scrm) and cultured for 72 hr before analysis. Total protein staining was used as a loading control. (D) Analysis of experiments like the one shown in C. Quantification of PSMB7 protein levels shows significant depletion with all three siRNAs. Quantification of proteasome activity by in-gel ABP fluorescence indicates significantly reduced activity following PSMB7 knockdown. POMP protein levels are significantly increased in response to PSMB7 depletion. *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA with post-hoc Dunnett’s test, n = 4 biological replicates, mean±SD, FC=fold change. (E) Proteasome activity measured as in-gel ABP fluorescence and Western blot analysis of POMP and GAPDH levels in HEK293 cells transfected with each of three different siRNAs targeting POMP or scrambled control (Scrm) and cultured for 72 hr before analysis. Total protein staining was used as a loading control. (F) Analysis of experiments like the one shown in E. Quantification of POMP protein levels shows a significant depletion with siRNA A and C. Quantification of proteasome activity by in-gel ABP fluorescence indicates significantly reduced activity following POMP knockdown. ns=p>0.05, *p≤0.05, **p<0.01, ***p<0.001, one-way ANOVA with post-hoc Dunnett’s test, n = 4 biological replicates, mean±SD, FC=fold change. (G) Principal component analysis (PCA) of transcriptomic data from cells transfected with siRNAs targeting POMP, PSMB1, PSMB7 or scrambled control. Samples of all three knock-down conditions cluster away from scrambled, consistent with the fact that all three result in proteasome inhibition. However, while PSMB1 and PSMB7 are interspersed with one another and cannot be clustered apart, POMP samples form a distinct cluster, suggesting that POMP knock-down gives rise to a distinct transcriptional signature. (H) Analysis of the induction of CP subunits’ mRNAs (Log 2 FC, vs Scrm) following PSMB (POMP + ) and POMP (POMP - ) knock-down. Data show a significant reduction in CP transcript levels upon POMP depletion, suggesting that POMP is required to facilitate expression of CP mRNAs. ***p<0.001, unpaired two-tailed t-test, n=14, each data point representing a different CP subunit. Boxplots show the median (line), interquartile range (box), and Min-Max whiskers. (I) KEGG pathway enrichment analysis of differentially expressed genes upon POMP knockdown (POMP - ). Dot size corresponds to the number of genes per pathway; color indicates adjusted p-value (FDR). Pathways are ranked based on fold enrichment. The POMP-specific transcriptional signature is characterised by the activation of pro-inflammatory and cancer-related pathways. (J) KEGG pathway enrichment analysis of differentially expressed genes upon PSMB knockdown (POMP + ). Dot size corresponds to the number of genes per pathway; color indicates adjusted p-value (FDR). Pathways are ranked based on fold enrichment. The PSMB-specific transcriptional signature (POMP is characterised by a rewiring of cellular metabolism, ribosome biogenesis and a neurodegeneration-like transcriptional signature. (K) Volcano plots of RNA-seq differential expression analysis comparing siRNA-POMP (POMP - ) to scrambled control. Significantly differentially regulated genes are highlighted in blue. Following POMP knock-down cells downregulate the expression of ribosomal genes, are unable to mount robust compensatory expression of proteasome CP transcripts and strongly induce proinflammatory factors, proto-oncogenes and cell cycle inhibitors. The dashed horizontal and vertical lines indicate the thresholds used for differential gene expression. (L) Volcano plots of RNA-seq differential expression analysis comparing siRNA-PSMB (POMP + ) to scrambled control. Significantly differentially regulated genes are highlighted in blue. Following PSMB knock-down cells are able to maintain expression of ribosomal genes at control levels, induce compensatory expression of proteasome CP transcripts and prevent induction of proinflammatory factors, proto-oncogenes and cell cycle inhibitors. The dashed horizontal and vertical lines indicate the thresholds used for differential gene expression. (M) Volcano plot comparing differential gene expression between PSMB7 and PSMB1 knock-downs. Significantly regulated genes are shown in blue. Only few genes are differentially regulated and no clear transcriptional signature emerges from this comparison. The dashed horizontal and vertical lines indicate the thresholds used for differential gene expression.

Article Snippet: Pre-designed siRNA oligo duplex sets against POMP (SR309742), TC11/NRF1 (SR303155), HSF1 (SR320556), PSMB1 (SR303824) and PSMB7 (SKU SR303830) were purchased from OriGene.

Techniques: Activity Assay, Fluorescence, Western Blot, Transfection, Control, Cell Culture, Staining, Knockdown, Inhibition, Expressing, Two Tailed Test, Activation Assay, RNA Sequencing, Gene Expression, Comparison

Journal: bioRxiv

Article Title: The proteasome maturation factor POMP moonlights as a stress-induced transcriptional regulator

doi: 10.1101/2025.04.25.650603

Figure Lengend Snippet: (A) Density plots comparing transcriptomic features of differentially regulated genes (blue, “List”) to background gene sets (red, “Background”) in PSMB KD (POMP present, left) and POMP KD (POMP absent, right) conditions. Features include coding sequence length, transcript length, genome span, UTR lengths, and GC content. (B) Linear discriminant analysis (LDA) based on sequence and structural features shows partial separation between gene sets regulated upon PSMB KD (magenta) and POMP KD (green). (C) Feature importance plot from a random forest classifier trained to distinguish between POMP-dependent and POMP-independent gene sets. Top contributing features include CDS GC content, minimum free energy (mfe), exon/intron lengths, and sequence skew metrics. (D) Feature enrichment analysis histograms showing distributions of exon counts (top panels) and transcript isoforms per coding gene (bottom panels) in PSMB KD (left, pink) and POMP KD (right, green) compared to genome-wide expectations (light blue). Chi-squared p-values indicate deviation from expected distributions. (E) Juxtaposed transcript usage over gene expression plots for POMP KD (left) and PSMB KD (right). The x-axis reports differential gene expression and the y-axis differential transcript usage. Genes showing differential gene expression (DGE), differential transcript usage (DTU), or both (DTE) are color-coded; selected genes that show differential transcript usage regulation and are involved in DNA-repair and stress response are labeled. (F) Venn diagram depicting the overlap between genes that, by DRIM-Seq analysis, show differential transcript usage in POMP KD and PSMB KD conditions. (G) Gene ontology (GO) enrichment analysis of biological process (BP) terms for genes that show differential transcript usage uniquely in the POMP-dependent condition (PSMB KD only,1055 genes). Top terms are related to DNA damage response and cellular stress response. (H) Schematic diagrams of splicing reporters used to monitor exon retention vs. exon skipping. Upper one is the RG6 general splicing reporter, which incorporates an artificial version of the chicken cardiac troponin (cTNT) exon 5 and is not selective for specific splicing factor. Lower one is the IBB reporter, which contains exon 9 from the CFTR gene and is selective for TDP-43-mediated splicing events. The two reporters work in the way that exon retention/skipping events will lead to different ratios of GFP:RFP signal. (I) DRIMseq analysis of differential isoform usage for PARP3 in HEK293 cells transfected with siRNAs against PSMB1, PSMB7, POMP and scrambled control (Scrm) for 72 hr. n=3 (Scrm), 6 (PSMB1, PSMB7 and POMP). Boxplots show the median (line), interquartile range (box), and Min-Max whiskers. (J) Western blot analysis of PARP3 isoform usage in HEK293 cells transfected with siRNAs against PSMB1, PSMB7, POMP and scrambled control for 72 hr. Knock-down of proteasome subunits, but not POMP, leads to an increase in the levels of one of the lower molecular weight isoforms of PARP3 (indicated as iso_1 and _2). Total protein stain is reported and was used as loading control. The dashed lines mark where the gel was spliced. (K) Quantifications of the relative abundance of PARP3 FL and its two isoforms in experiments like the one shown in I. PSMB1 and PSMB7 knock-down data were merged into a common PSMB term. The data show that in response to proteasome subunit knock-down expression of PARP3 FL decreases slightly in favour of its alternative isoforms, in particular iso_2. By contrast, POMP knock-down leads to a reduction in the levels of the two isoforms compared to scrambled control. n=2 (Scrm, POMP) and 4 (PSMB) biological replicates.

Article Snippet: Pre-designed siRNA oligo duplex sets against POMP (SR309742), TC11/NRF1 (SR303155), HSF1 (SR320556), PSMB1 (SR303824) and PSMB7 (SKU SR303830) were purchased from OriGene.

Techniques: Sequencing, Genome Wide, Gene Expression, Labeling, Transfection, Control, Western Blot, Knockdown, Molecular Weight, Staining, Expressing

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: Cryo-EM Structures of the Recombinant Human 20S Proteasome and 20S-PA200 Complex (A) Cryo-EM structure of the recombinant human 20S proteasome with a fitted atomic model (see also Figure S2 ). (B and C) Two close-up views of the structure shown in (A), which has well-resolved side chains throughout. (D) Cryo-EM structure of the recombinant human 20S-PA200 complex with a fitted atomic model (see also and ). (E and F) Two close-up views of the structure shown in (D), which has well-resolved side chains throughout. (G) Overall view of the 20S-PA200 atomic model, with major domains indicated. (H) Close-up views of the 20S-PA200 cryo-EM map (gray mesh) with a fitted atomic model (cartoon representation), with each subunit color-coded as indicated at the top. In (A)–(F) The cryo-EM maps are shown as mesh and the protein models as cartoons (A and D) or sticks (B, C, E, and F).

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Cryo-EM Sample Prep, Recombinant

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: Biochemical Characterization of Recombinant Human 20S Proteasome and 20S-PA200 Complexes (A) SDS-PAGE of endogenous (e20S) and recombinant (r20S) human 20S proteasomes and recombinant human 20S-PA200 complexes (r20S-PA200) (see also A and S1B). (B) Proteolytic activities of e20S, r20S, and r20S-PA200 against the fluorogenic substrates Z-LLE-AMC (blue), Boc-LRR-AMC (magenta), and Suc-LLVY-AMC (red), specific for the proteasome β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like active sites, respectively. Similar 20S proteasome molarities were used in each assay, as shown in (A). (C) Proteolytic activities of recombinant human 20S proteasomes in the presence of increasing concentrations of PA200, color coded as in (B) (see also Figure S1 C). Error bars are represented as mean ± SD.

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Recombinant, SDS Page

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: PA200 Main Docking Sites on the Proteasome α Rings (A) Cartoon representation of the PA200 structure, showing the two major anchor regions of PA200 at the proteasome α ring, one involving the PA200 loop formed by residues 561–576 (dashed circle, left) and the other involving the PA200 C terminus (solid circle, right). (B) Close-up view of interactions between the PA200 loop, residues 561–576, and the proteasome subunits α1 (orange) and α2 (green). (C) Close-up view of interactions between the PA200 C terminus and the proteasome subunits α5 (blue) and α6 (red). In (A)–(C) the 20S-PA200 structure is oriented to best depict the interactions highlighted. In (B) and (C), the cryo-EM maps are shown as gray mesh and the atomic models as sticks. The protein-protein interaction network involving these two PA200 anchor regions and the 20S proteasome are represented in A and S4B. See also Figure S5 .

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Cryo-EM Sample Prep

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: PA200 Induced Conformational Changes in the 20S Proteasome α Rings (A) The closed α ring outer surface of the 20S proteasome. (B) Superimposition of the α ring outer-surface atomic models of the recombinant human 20S proteasome (gray cartoon) and 20S-PA200 (cartoon with subunits color coded). The H0 helices of the 20S proteasome α1–α2 and α4–α7 are indicated by solid circles color coded as in 20S-PA200, whereas the H0 helix of α3, which is disordered in 20S-PA200, is encircled by a black dashed circle. (C) The open α ring outer surface of the 20S-PA200 complex. In (A) and (C), the cryo-EM maps are shown as gray mesh, and the atomic models are represented as cartoons.

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Recombinant, Cryo-EM Sample Prep

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: Interaction between the N-Terminal Loops of the Proteasome α Subunits with PA200 (A) Cartoon representation of the N-terminal tails of the proteasome α5–α7 subunits, indicated by arrows, at the PA200 dome inner surface. (B–D) Close-up views of the N termini of the proteasome subunits α7 (B), α6 (C), and α5 (D). The cryo-EM maps are shown as gray mesh and the atomic models as sticks. In (A)–(D), the 20S-PA200 structure is oriented to best depict the highlighted interactions. The protein-protein interaction network involving the N-terminal tails of the proteasome α5–α7 subunits and PA200 are represented in C–S4E.

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Cryo-EM Sample Prep

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet: Comparison of the Proteolytic Active Sites in the Human 20S Proteasome and 20S-PA200 Complexes Shown are van der Waals surface representations, colored by charge, of the three proteasome active sites (β1, β2, and β5) of the recombinant 20S proteasome (top row) and 20S-PA200 complexes (bottom row), viewed from the proteasome inner cavity. White dashed circles indicate the S1 pocket of each active site.

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Comparison, Recombinant

Journal: Molecular Cell

Article Title: Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

doi: 10.1016/j.molcel.2019.07.014

Figure Lengend Snippet:

Article Snippet: Recombinant human 20S proteasomes, endogenous human 20S proteasomes (Enzo) and recombinant human 20S-PA200 complexes, all at 7.5 nM in 25 mM HEPES, pH 7.5 and 0.5 mM EDTA, were incubated with 50 μM substrate (stock at 5 mM in DMSO) for 30 minutes at room temperature.

Techniques: Virus, Recombinant, Transfection, Software, Single Particle

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

doi: 10.1073/pnas.0901982106

Figure Lengend Snippet: Apparent K i ′ and k assoc values for the chymotrypsin-, trypsin-, and caspase-like activity of human 20S proteasome

Article Snippet: The biochemical proteasome assays were performed as described in ref. 15 , with commercially available human erythrocyte 20S proteasomes from Biomol.

Techniques: Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

doi: 10.1073/pnas.0901982106

Figure Lengend Snippet: X-ray analysis of the complex of SylB (2) and the 20S proteasome and comparison with other syrbactins. (A) Structure of syringolin A (1) and B (2). (B) Electrostatic potential surface [contoured from +15 kT/e (intense blue) to −15 kT/e (intense red)] of SylB covalently bound to β5. (C) Stereo representation of SylB (2) bound to the chymotryptic like active site in complex with 20S proteasome (rose, subunit β5; gray, subunit β6). (D) Structural superimposition of SylA (1, yellow), SylB (2, green), and GlbA (3, light gray).

Article Snippet: The biochemical proteasome assays were performed as described in ref. 15 , with commercially available human erythrocyte 20S proteasomes from Biomol.

Techniques: Comparison