The 20S as a stand-alone proteasome in cells can degrade the ubiquitin tag

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.

enzyme was calculated from the MS/MS count of each peptide contributing to a given P1 site and the relative ratio plotted as log2 (described in Methods). The Y-axis represents the amino acid residue number of the HA-CyclinB1-NT sequence. Red/Blue dots indicate a ≥2 fold preference (Red, 26S; Blue, 20S; grey, no significant preference). c, Venn diagrams represent the unique and common peptides generated by each proteasome from the given substrates. iodoacetamidean inhibitor for cysteine-based DUBa; OPA, 1,10(O)-phenanthrolinean inhibitor for Rpn11; Na3VO4, sodium ortho-vanadatean ATPase inhibitor; EDTAa metal ion chelator (for trace metallo-proteases or as an ATPase inhibitor by neutralizing residual MgCl2); geldanamycinan HSP90 inhibitor; PMSFa broad specificity protease inhibitor.
The reaction mixture was separated by 14% Tris-Tricine SDS-PAGE followed by either Coomassie staining or immunoblotting with anti-Ub, anti-Myc and anti-HA antibodies. Source data are provided as a Source Data file. two gates all appear symmetrically closed. c, Resolution estimation of these obtained maps according to the gold standard FSC criterion of 0.143. d, Local resolution estimation for the 20S alone map. The resolution color bar (in Å) is also shown. e, Density details for 20S only map before post-processing (3.72Å) at α-helix (L73-G92) of β2-subunit and β-sheet (A192-T199) of β6-subunit.
of 20S proteasome incubated with MonoUb-CyclinB1-NT. b, 3D classification and refinement procedures. After 2D and 3D classifications to eliminate bad particles, there are 282,834 remaining cleaned-up particles, based on which we obtained an overall map for 20S+MonoUb-CyclinB1-NT (named as S0). A further 3D classification into 5 classes generated a class (class 1) with an obviously asymmetric gate configuration (in magenta, 29.1% of the population), which is distinct from the other classes. This class was further processed to generate a map denoted as S1. For the other three classes with reasonably good structural features (2, 4, and 5), since they all appear symmetric between the two rings and show highly similar features, their particles were combined and refined into a map denoted as S2. c, Resolution estimation of these obtained maps according to the gold standard FSC criterion of 0.143. d, Local resolution estimations for the S0, S1, and S2 maps. Their resolution color bars (in Å) are also shown. e, Density details for S1 map (4.47Å) at α-helix (L73-G92) of β2-subunit and β-sheet variability components for the 20S only map. Positive (red) and negative (blue) values correspond to density to be added and subtracted from the mean density, respectively. b, Component 1 of 20S only map depicts a squashed motion of the whole complex. The dashed circles in the first two panels are identical perfect circles. It can be seen that the distances between the edge of the structure and the circle are different in the arrow-indicated direction (from blue to red map). c, Central slices of the first three 3D variability components for the 20S+monoUb-CyclinB1-NT map. Positive (red) and negative (blue) values correspond to density to be added and subtracted from the mean density, respectively. d, Component 1 of 20S+monoUb-CyclinB-NT exhibits dynamics in the gate region (indicated by an orange dashed circle) without an obvious overall shape change. Here, the red and blue maps are the two representative extreme maps. This rendering style is followed.
periods. Native cell lysates were prepared at each time points and proteasome peptidase activity (LLVY-AMC) was measured. The graph shows the average proteasome peptidase activity of three technical repeats at each time-point. d, Native cell lysates from panel c were resolved by 4% native gel for proteasome in-gel proteolytic assay to evaluate proteasome activity or for transfer and immunoblotting of intact complexes using anti-PSMA1 antibody. Source data are provided as a Source Data file.
For synthesis of HA-CyclinB1-NT, the sequence was divided into two fragments:

Synthesis of fragment 1, CyclinB1-NT (53-97):
CLGDIGNRVSEQLQARNlePMRK (64)EARPSATGRVIDRRLPRPLERVPNle The synthesis was carried out using Fmoc-SPPS on Rink amide resin ( Cleavage from the resin: The resin was washed with DMF, MeOH, DCM and dried. The peptide was cleaved using TFA:triisopropylsilane (TIS):water (95:2.5:2.5) cocktail for 2 h. The cleavage mixture was filtered and the combined filtrate was added drop-wise to a 10 fold volume of cold ether and centrifuged. The precipitated crude peptide was dissolved in acetonitrile-water (1:1) and was further diluted to ~30% with water and lyophilized. The HPLC analysis was carried out on a C18 analytical column using a gradient of 0-60% B over 30 min.
For preparative HPLC, the same gradient was used to purify the fragment 1 in ~50% yield. complete the cyclization. The resin was washed using DMF (3 × 5 mL). Cleavage and purification were carried out as described above to afford the fragment 2 in 40-50% yield.
Cleavage from resin: The resin was washed with DMF, DCM and dried over high vacuum. A cocktail of TFA:DCM:triisopropylsilane:water (90:5:2.5:2.5) was added to resin and reaction mixture was shaken for 2 h at RT. The resin was filtered, and the combined filtrate was added dropwise to a 10 fold volume of cold ether and centrifuged. The precipitated crude peptide was dissolved (~70%) in acetonitrilewater (1:1) and was further diluted to ~30% with water and lyophilized. Myc tag-EQKLISEEDL and Flag tag-DYKDDDDK

Thz deprotection of Myc-Ub1K48
The crude peptide (50 mg) was dissolved in 6M Gn·HCl buffer to a final concentration of ~3 mM. Hydrazine hydrate (80% solution, 100 equivalents) was added and kept at 25⁰C for 1 h

Synthesis of Ub2 and Ub3 a) Conversion of Ub2K48-N-methyl cysteine to 3-mercaptopropionic acid ester
Ub2K48-N-methyl cysteine (50 mg) was dissolved in 6 M Gn·HCl buffer (2.82 mL, 2 mM) and was subjected for the UV irradiation (365 nm, 2 h) to remove photolabile 2-nitrobenzyl protecting group of the C-terminal Cys, followed by the pH adjustment (~1.5-2) and incubation with 20% (vol/vol) 3-mercaptopropionic acid (MPA) at 42⁰C for 20 h. The reaction was followed by HPLC using a C18 analytical column and a gradient of 0-60% B over 30 min and LC-MS analysis. For preparative HPLC, the same gradient was used to afford the Ub2K48-MPA thioester at a yield of ∼10% (∼4.9 mg).

b) Thz deprotection of Ub3K48-N-methyl cysteine
Similar procedure used for the thz deprotection of Myc-UbK48-N-Methyl cysteine was used for thz deprotection of Ub3K48-N-methyl cysteine to yield the product in 9% yield (~4.5 mg). The reaction was followed by HPLC using a C4 analytical column and a gradient of 5-55% B over 60 min and LC-MS analysis. For preparative HPLC, the same gradient was used to afford the Myc-DiUb K48 -MPA thioester, 4 at a yield of ∼38% (∼3.6 mg).

Expression of HA-CyclinB1-NT in HEK293 cells
When HA-cyclinB1-NT was expressed through pCMV10 plasmid in HEK293 cells, we could not detect any protein expression although there was expression of the mRNA. Since, CyclinB1-NT (1-88 aa) is an unstructured polypeptide, it most likely degrades rapidly after translation. Hence, in order to provide a stability as well as a marker for its protein expression we attached a self-immolating tag "NS3-protease" with its own cleavage site in between NS3domain and HA-CyclinB1-NT as described in Fig. 5a. Post-translation of this chimeric protein, NS3 tag is removed by auto cleavage and allow HA-CyclinB1-NT to be degraded by proteasome.
Expression of K0 mutant of HA-CyclinB1-NT in HEK293 cells When the K0 mutant of HA-cyclinB1-NT was expressed with the NS3-tag we could not detect its protein levels due to rapid degradation by proteasome (Supplementary Fig. 11b).
In order to track its rate of degradation we follow a "bortezomib pulse-chase" experiment where we first accumulated K0-HA-CyclinB1-NT proteins by 6 h treatment of bortezomib (500nM) then chased the rate of CyclinB1 degradation after removal of bortezomib from media. We did also confirm that inhibition of proteasome by bortezomib in cells is reversible and by 6h 50% of proteasome activity was restored. (Supplementary Fig. 11c & d).