Abstract
PA28γ is a nuclear activator of the 20S proteasome that, unlike the 19S regulatory particle, stimulates hydrolysis of several substrates in an ATP- and ubiquitin-independent manner and whose exact biological functions and molecular mechanism of action still remain elusive. In an effort to shed light on these important issues, we investigated the stimulatory effect of PA28γ on the hydrolysis of different fluorogenic peptides and folded or denatured full-length proteins by the 20S proteasome. Importantly, PA28γ was found to dramatically enhance breakdown rates by 20S proteasomes of several naturally or artificially unstructured proteins, but not of their native, folded counterparts. Furthermore, these data were corroborated by experiments in cell lines with a nucleus-tagged myelin basic protein. Finally, mass spectrometry analysis of the products generated during proteasomal degradation of two proteins demonstrated that PA28γ does not increase, but rather decreases, the variability of peptides that are potentially suitable for MHC class I antigen presentation. These unexpected findings indicate that global stimulation of the degradation of unfolded proteins may represent a more general feature of PA28γ and suggests that this proteasomal activator might play a broader role in the pathway of protein degradation than previously believed.
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MS data as raw files, peptides identified with relative intensities and search parameters have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD029248.
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We thank Francesco Turci and Francesco Ferrini for help in preparing figures, Patrick Moore for assistance in preparation of the manuscript and Massimo Coletta for insightful discussions.
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J-YAF acquisition, analysis and interpretation of data; FC acquisition, analysis and interpretation of data; JL acquisition, analysis and interpretation of data; AC acquisition, analysis and interpretation of data, AB analysis and interpretation of data AS, analysis and interpretation of data; OC analysis and interpretation of data; PC conception and design, acquisition of data, analysis and interpretation of data, drafting the article.
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18_2021_4045_MOESM3_ESM.tif
Supplementary file3 Supplementary Fig. 1 Purified PA28γ and PA28αβ elute from Sephacryl S-200 gel filtration column as a complex of apparent molecular weight of about 200 kDa. ̴150 µg of the final preparation of PA28γ A and PA28αβ B were analyzed by size exclusion chromatography on a calibrated Sephacryl S-200 column. The final preparation of PA28αβ consists of a heteroheptameric complex containing both the α and β subunits C and is able to strongly enhance the chymotryptic activity of 20S proteasome at picomolar concentrations D (TIF 144 KB)
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Supplementary file4 Supplementary Fig. 2 Effects of increasing concentrations of PA28γ on the hydrolysis of different fluorogenic substrates by 20S peptidase activities. Proteasome chymotrypsin-like A, tryptic-like B, and caspase-like C activities were probed with the indicated fluorogenic peptides in the presence of increasing concentrations of PA28γ and expressed as fold activation compared to the activity of 20S alone. R2 ≥ 0.9 in all cases (TIF 135 KB)
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Supplementary file5 Supplementary Fig. 3 PA28γ is unable to stimulate proteasomal degradation of two tryptic substrates. A Hydrolysis rates of 100 µM Z-LR-amc and Z-FR-amc were assessed in the presence of increasing concentration of PA28γ and displayed as in Figure 1. B Specific activities of 20S proteasomes were assessed at 100 and 250 µM concentrations of each substrate. * P <0.05 (TIF 85 KB)
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Supplementary file6 Supplementary Fig. 4 Absolute dependence on proteasome proteolytic activity of β-casein hydrolysis. NH2 generation was completely absent when the substrate was incubated alone A or with only PA28γ B (TIF 96 KB)
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Supplementary file7 Supplementary Fig. 5 Calibration curve for the polyhydroxyethyl aspartamide size exclusion column using fluorescamine-derivatized amino acid and peptide molecular weight standards. The typical peak width of these amino acids and peptides was 0.7 min (TIF 86 KB)
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Supplementary file8 Supplementary Fig. 6 Absolute dependence on proteasome proteolytic activity of MBP, IGF-1, and α-lactalbumin hydrolysis. NH2 generation was completely absent when the substrates were incubated alone (left panels) or with only PA28γ (right panels) (TIF 147 KB)
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Supplementary file9 Supplementary Fig. 7 Absolute dependence on the proteasome proteolytic activity of MBP, IGF-1, and α-lactalbumin hydrolysis. MBP A, IGF-1 B, and α-lactalbumin C were incubated alone or in the presence of PA28γ-20S proteasomes inhibited by 20 μM epoxomicin and analyzed as in Figure 4 (TIF 203 KB)
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Supplementary file10 Supplementary Fig. 8 Relative frequencies of amino acids surrounding the peptide bonds preferentially hydrolyzed by the 20S and PA28γ-20S proteasomes. Logos sequences were generated using WebLogo 3 (available at http://weblogo.threeplusone.com/), and refer to the cleavage sequences (Positions from P4 to P4 ') of the peptides generated in greater amounts by PA28γ-20S (left) and 20S proteasome (right) during the hydrolysis of IGF-1 and MBP. The colors of amino acids are based on their chemical properties: Polar (G, S, T, Y, C) green, Neutral (Q,N) purple, Basic (K,R,H) blue, Acidic (D,E) red, Hydrophobic (A,V,L,I,P,W,F,M) black (TIF 247 KB)
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Supplementary file11 Supplementary Fig. 9 Knockout of PA28γ results in accumulation of MBP in cells. The expression of nucleus-targeted MBP was assessed with a specific antibody by western blotting in A375 and A375 Crispr melanoma cells, and β-actin was used as a loading control. At 24 hours post-transfection, accumulation of MBP can be noted which becomes increasingly evident after 48 hours (TIF 88 KB)
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Frayssinhes, JY.A., Cerruti, F., Laulin, J. et al. PA28γ–20S proteasome is a proteolytic complex committed to degrade unfolded proteins. Cell. Mol. Life Sci. 79, 45 (2022). https://doi.org/10.1007/s00018-021-04045-9
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DOI: https://doi.org/10.1007/s00018-021-04045-9