Fig. 2: The 20S proteasome processes substrates differently than the 26S proteasome.
From: The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag
a Unmodified Cyclin B1-NT or TetraUb-Cyclin B1-NT were incubated at 37 °C with either purified 20S or 26S proteasomes at 1:150 (proteasome:substrate) molar ratio. b Unmodified Cyclin B1-NT after reaction with purified 20S or 26S proteasomes for indicated time periods was resolved by 16% Tris-Tricine denaturing gel for Coomassie staining. IB with anti-PSMA1 antibody used as proteasome loading control. c The average percentage of residual unmodified Cyclin B1-NT substrate at each time point after degradation by either 20S or 26S proteasomes (from b). Error bar represents the value for three independent experiments with ±SD. d TetraUb-Cyclin B1-NT was incubated with either purified 20S or 26S proteasomes for indicated time periods. The reaction mixture was resolved by SDS-PAGE for IB with anti-HA (substrate) or anti-PSMA1 (loading control) antibodies. e The average percentage of residual TetraUb-Cyclin B1-NT substrate at each time point after degradation by either 20S or 26S proteasomes. Error bar represents the value for three independent experiments with ±SD. f Average half-life of differentially ubiquitinated Cyclin B1-NT species in presence of either 20S or 26S proteasomes. Error bar represents the value for three independent experiments with ±SEM. g Experimental design to identify products of unmodified Cyclin B1-NT, MonoUb-Cyclin B1-NT, or TetraUb-Cyclin B1-NT incubated with either purified 20S or 26S proteasomes. Peptide products at time points shorter than substrate half-life for each reaction were isolated and analyzed by LC-MS/MS. h Heatmap of Cyclin B1-NT-derived peptides obtained from each substrate by 20S or 26S proteasomes (triplicate or duplicate experiments). Each peptide product is positioned based on its N-terminal residue along the primary sequence of Cyclin B1-NT. Color intensity reflects PSM counts for each peptide normalized to the maximum observed counts. The bar plot above (in gray) represents relative sizes of the corresponding maxima. The dendrogram to the left was obtained by performing MDS analysis on the PSM counts of the top 100 peptides and clustering the samples based on the corresponding MDS distances. i Size distribution of Cyclin B1-NT peptide products in each sample from panel (g). The horizontal box lines represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile −1.5× the interquartile range and the third quartile +1.5× the interquartile range. n = 3 independent experiments. j Scatter plot represents the relative cleavage preference for each proteasome species at P1 positions on unmodified Cyclin B1-NT. Cleavage preference of each enzyme was calculated from the MS/MS count of each peptide product contributing to a given P1 site. The relative ratio plotted as log2 (described in the “Methods” section). The Y-axis represents the amino acid residue number of the HA-Cyclin B1-NT sequence. Red/blue color dots indicate a greater-than-twofold preference for a given P1 cleavage site (red, 26S; blue, 20S; gray, no significant preference). Source data are provided as a Source data file.
