Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: PI31 inhibition of latent and z-YA-activated 20Sc and 20Si activities was compared in multiple assays of proteasome function. Panel A . Effect of PI31 on latent and z-YA-activated hydrolysis of Suc-LLVY-AMC (upper) and casein (lower) . Mean values for rates of substrate hydrolysis for triplicate assays were determined as described under Experimental Procedures. Values for activity in the absence of z-YA and PI31 were set to 100% (Control) and other values were calculated as a percentage of Control, + standard deviation. Differences among results of different assay conditions were analyzed by 3-way ANOVA. Pair-wise differences were analyzed post-hoc by Tukey’s HSD. PI31 significantly inhibited z-YA activated 20Sc- and 20Si-catalyzed hydrolysis of both substrates (20Sc, p < 0001; 20Si, p = 0.0193). PI31 inhibition of 20Si was significantly attenuated compared to that of 20Sc for both substrates (p < 0.0001, denoted by *). Panel B . z-YA-activated 20Sc and 20Si hydrolysis of α-synuclein was determined in the presence (•) or absence (O) of PI31 (20S, 150 nM; PI31, 500 nM). At indicated times, samples were subjected to SDS-PAGE and stained with Coomassie Blue R-250. α-synuclein content was quantified using Image Studio software (LiCOR). Values at time 0 were set to 100% and other values within each group are represented as a percentage. Similar results were obtain in five independent experiments. Panel C . Me 4 BodipyFL-Ahx 3 Leu 3 -VS labeling of 20Sc and 20Si in the presence (•) or absence (O) of 5 mM z-YA and/or PI31. Labeling was conducted as described in Experimental Procedures and quantified using Image Studio software (LiCOR). Mean values for replicates of labeling of each subunit in the absence of z-YA and PI31 were set to 100%. Other values within that group are represented as percentages of that value. Similar results were obtain in two other independent experiments. Panel D . Purified 20Sc and 20Si were assayed for hydrolysis of Suc-LLVY-AMC and casein in the presence or absence of 5 mM z-YA at indicated concentrations of PI31. Mean values for rates of hydrolysis of triplicate assays in the absence of PI31 were set to 100% and other values were determined as a percentage of that value. Differences in activity between 20Sc and 20Si were analyzed by repeated measures of 2-way ANOVA. Pair-wise, concentration-matched differences in activity between 20Sc and 20Si were analyzed by Šídák’s multiple comparisons test, and significant differences are indicated at p ≤ 0.05 by (*) for respective PI31 concentrations. Similar results were obtained in four independent experiments.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Inhibition, Activity Assay, Standard Deviation, SDS Page, Staining, Software, Labeling, Purification, Concentration Assay

Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: Panel A, (left) . Purified latent (O) and z-YA-activated (•) 20Sc and 20Si (200 nM) were incubated with PI31 (1 μM) at 37° C for indicated times. Reactions were terminated with SDS samples buffer. Samples were subjected to SDS-PAGE and western blotting with an antibody against the C-terminus of PI31. PI31 bands were quantitated with Image Studio software (LiCOR). Values within each group are expressed as a percentage of the initial value. Similar results were obtained in five independent experiments. Panel A (right) . z-YA-activated 20Sc and 20Si were incubated as described above in the presence (•) or absence (O) of 0.5 mM MG132 prior to incubation with PI31, as in Panel A, right. Pane B (upper) . As in Panel A, but the gel was stained with Coomassie Blue R-250 (CB). Panel C . As in Panel A, but gel was western blotted with anti-His antibody. Panel B (lower). Quantification of PI31 bands from upper panel. Similar results were obtained in three independent experiments. Panel C (upper). PI31 was incubated with z-YA-activated 20Sc or 20Si as in Panel A. After 90 mins reactions were terminated with SDS samples buffer and subjected to western blotting using an anti-His antibody. Panel C (lower) . Quantification of intact (T0) PI31 content from upper panel. Similar results were obtained in four independent experiments.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Purification, Incubation, SDS Page, Western Blot, Software, Staining

Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: Panel A . Schematic representation of domain structure of wild type ( –271) PI31. The positions of regions of PI31shown previously to interact with the β5 (yellow), β2 (green), and β1 (blue) catalytic residues of the proteasome are indicated. Structures of PI31 mutants with C-terminal truncations are shown for comparison. Panel B. Indicated mutants of PI31 were expressed as N-terminal His-tagged recombinant proteins in E. coli and purified as described under Experimental Procedures. Each mutant PI31 was incubated at 37°C for indicated times with z-YA-activated 20Sc or 20Si. Samples were subjected to western blotting with anti-His antibody. Similar results were obtained in two independent experiments. Panel C . Wild-type PI31 and indicated PI31 mutants (1.5 μM) were assayed for inhibition of proteasome activity of casein hydrolysis by 20Sc and 20Si (150 nM), as described under Experimental Procedures. Mean values for triplicates assays of proteasome activity of each proteasome were set at 100% and other values were expressed as a corresponding percentage. Differences in relative proteasome activity for 20Sc and 20Si incubated with different truncations of PI31 were analyzed by 2-way ANOVA. 20Sc was significantly inhibited by PI31 1-271 (Tukey’s HSD; adj. p < 0.0001) and PI31 1-244 (Tukey’s HSD; adj. p < 0.0001). 20Si was significantly inhibited by PI31 1-271 (Tukey’s HSD; adj. p < 0.0001). PI31 1-271 inhibition of 20Si was significantly less than that of 20Sc (Tukey’s HSD; adj. p < 0.0001). Similar results were obtained in two independent experiments.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Comparison, Recombinant, Purification, Mutagenesis, Incubation, Western Blot, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: Intact mass spectrometry was performed on recombinant His-PI31 before and after incubation with z-YA-activated 20Si. Panel A. Reverse phase chromatogram of purified, full-length HisPI31 injected for LC-MS. Panel B. Mass spectra of full-length HisPI31 from Panel A. Panel C. Reverse phase chromatogram of purified, HisPI31 20Si degradation product injected for LC-MS. Panel D. Mass spectra of HisPI31 20Si degradation product from Panel C. Panel E . Representation of domain structure of PI31, and the 20Si hydrolysis site derived from comparisons of Panels B and D. The dominant mass peaks before and after incubation with 20Si ( Panel B vs. D ) differ by 8,110.35 Da, a value consistent with the C-terminal 78 residues of PI31.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Mass Spectrometry, Recombinant, Incubation, Purification, Injection, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: Panel A. Latent and z-YA-activated 20Sc and 20Si (150 nM) were assayed for proteasome activity in the presence or absence of full length PI31 (His-PI31 1-271 ) and a mutant consisting of only the PI31 C-terminal domain (His-PI31 152-271 ) (2.3 μM). Data are reported as mean values ( + standard deviation) of triplicate assays for rates of casein hydrolysis. Similar results were obtained in three independent experiments. Panel B. Proteasome assays were conducted as in Panel A at indicated concentrations of His-PI31 152-271 . Proteasome activity in the absence of PI31 was set at 100% and other values are expressed as a percentage of that value. Similar results were obtained in three independent experiments. Panel C. 20Sc and 20Si (150 nM) were incubated with His-PI31 152-271 (1.5 μM) for 30 mins at 37° C. Reactions were terminated with SDS sample buffer and subjected to SDS-PAGE for staining with Coomassie Blue R-250 (CB) or western blotting with antibodies against His (for the N-terminus) or for the C-terminal portion of the peptide. Similar results were obtained in three independent experiments.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Activity Assay, Mutagenesis, Standard Deviation, Incubation, SDS Page, Staining, Western Blot

Journal: bioRxiv

Article Title: Differential interactions of the proteasome inhibitor PI31 with constitutive and immuno-20S proteasomes

doi: 10.1101/2023.12.11.570853

Figure Lengend Snippet: 20Sc or 20Si were preincubated with the indicated PI31 proteins and subjected to glycerol density gradient centrifugation as described under Experimental Procedures. Panel A. Distribution of His-PI31 was determined by western blotting of using anti-His tag antibody. Control gradients were performed for proteasome in the absence of PI31 using an antibody against the α4 subunit common to both 20Sc and 20Si and against PI31 in the absence of 20S. Panel B . . PI31 bands in Panel A were quantified in ImageStudio (LiCOR) for each fraction and expressed as a percentage of the sum total intensity for all fractions. In lanes where multiple bands appear, the area containing all bands was used for quantification Similar results were obtained in three independent experiments.

Article Snippet: Proteins transferred to nitrocellulose membranes were blocked and blotted using primary antibodies against: 6xHis (mouse monoclonal, Invitrogen MA1-135); 20S α4 PSMA7 (mouse monoclonal anti-human, Enzo PW8120); PI31 C-terminus residues 241-271 (rabbit polyclonal anti-human, Enzo BML-PW9710); 20S β2i PSMB10 (rabbit polyclonal anti-human, Cell Signaling 78385S); Rabbit polycolonal antibodies against human 20S β1C PSMB6, β1i PSMB9, β5c PSMB5, and β5i PSMB8 subunits were prepared in our laboratory using C-terminal peptides of the respective subunits, as described and characterized previously ( ).

Techniques: Gradient Centrifugation, Western Blot