![Defective immunoproteasome assembly in IFN-stimulated PSMB10 G209R skin fibroblasts. (A) Primary neonatal skin fibroblasts isolated from the patient with the PSMB10 G209R mutation and age-matched neonatal primary PSMB10 WT skin fibroblasts were treated with 75 U/ml IFNγ for 98 h for in-depth MS analysis of purified proteasome complexes (scheme was prepared using BioRender). (B) TD-MS map (using VisioProtMS) of immunopurified 20S from cross-linked IFNγ-treated PSMB10 G209R cells showing 77% incorporated PSMB10 WT [amino acid sequence 40–273] and 23% PSMB10 G209R [amino acid sequence 40–273] proteoforms at 24.65 and 24.75 Da, respectively, while PSMB10 WT cells contain only the WT form. The 99 Da increase in the mass corresponds to the G-to-R mutation. (C) Relative proteasome activities in cell lysates of IFNγ-treated cells using specific AMC-fluorescently labeled substrates for the CT-L, T-L, and C-L activities (n = 4, statistics based on t test, P value: * <0.05, ** <0.01). (D) BU-MS/MS analysis of immunopurified 20S from cross-linked IFNγ-treated PSMB10 WT and PSMB10 G209R skin fibroblasts showing the identified PSMB10 peptides corresponding to the amino acid sequence [181–219] for PSMB10 WT (top) and [181–209] for PSMB10 G209R (bottom) matching the MS/MS spectra with the identified b (in blue) and y (in red) fragment ions. (E–H) BU-MS/MS analysis of immunopurified 20S from PSMB10 WT and PSMB10 G209R cells showing the relative stoichiometries of (E) the 20S catalytic subunits, (F) the 20S-bound chaperones after normalization of MS intensities to the total amount of 20S noncatalytic subunits, (G) the relative MS intensities of total 20S, assembled 20S, and assembling 20S, which were estimated thanks to the stoichiometry of the 20S-bound PAC1-PAC2, and (H) the 20S-bound activators (n = 4, statistics based on t test, P value: * <0.05, ** <0.01, *** <0.001, **** <0.0001). CT-L activity, chymotrypsin-like activity; T-L activity, trypsin-like activity; C-L activity, caspase-like activity. Refer to the image caption for details.](https://cdn.rupress.org/rup/content_public/journal/jhi/2/3/10.70962_jhi.20250129/4/jhi_20250129_fig5.png?Expires=1778913765&Signature=BPeU6j6PNqc-aFQ685zmziWQ1IVGCT0-qkZwY0whjNYstBYicv0e8SWxJfgUk--nAZh0u4YB~xcinpNmlNXVdfqag5KJL9p0G1-x10S1egU1bQyAdxihA-ulIGr29AIlfwIR2kObI8qrEm7Vg3Umm62Y3FXAz5ZhP2Plh8KnBqc4u1AnGfP1BYcAfUWQD0Bk60L4c89tDcEDeZIoKHQDjkS446zoUgtBJp3r97bEaBLpPl-4tyRNjZ~45dKeelbAKrADlN44ssuo2Hwf7C4Jnfbjn1UIF8CDvGMpTt3NwFORqxSq3nn3sdcaN376NJZssPnRxk4ReI-AYVNl1zWC5Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Panel A: A schematic diagram outlines the experimental workflow for mass spectrometry analysis of proteasome complexes in P S M B 10 W T and P S M B 10 G 209 R skin fibroblasts. Panel B: A top-down mass spectrometry (T D-M S) map displays the incorporation of P S M B 10 W T and P S M B 10 G 209 R proteoforms in immunopurified 20 S from crosslinked I F N-treated cells, showing peaks at 24.65 D a and 24.75 D a respectively. Panel C: A vertical bar graph compares relative proteasome activities for chymotrypsin-like (C T-L), trypsin-like (T-L), and caspase-like (C-L) activities in cell lysates of I F N-treated cells. Panel D: Bottom-up mass spectrometry (B U-M S slash M S) analysis shows identified P S M B 10 peptides corresponding to amino acid sequences for P S M B 10 W T and P S M B 10 G 209 R, with matching M S slash M S spectra and identified b- and y-fragment ions. Panel E: A vertical bar graph illustrates the relative stoichiometries of 20 S catalytic subunits in P S M B 10 W T and P S M B 10 G 209 R cells. Panel F: Another vertical bar graph shows the relative stoichiometries of 20 S-bound chaperones after normalization of M S intensities to the total amount of 20 S non-catalytic subunits. Panel G: A vertical bar graph depicts the relative M S intensities of total 20 S, assembled 20 S, and assembling 20 S estimated using the stoichiometry of the 20 S-bound P A C 1-P A C 2. Panel H: A final vertical bar graph presents the relative stoichiometries of 20 S-bound activators in P S M B 10 W T and P S M B 10 G 209 R cells.
Defective immunoproteasome assembly in IFN-stimulated PSMB10 G209R skin fibroblasts. (A) Primary neonatal skin fibroblasts isolated from the patient with the PSMB10 G209R mutation and age-matched neonatal primary PSMB10 WT skin fibroblasts were treated with 75 U/ml IFNγ for 98 h for in-depth MS analysis of purified proteasome complexes (scheme was prepared using BioRender). (B) TD-MS map (using VisioProtMS) of immunopurified 20S from cross-linked IFNγ-treated PSMB10 G209R cells showing 77% incorporated PSMB10 WT [amino acid sequence 40–273] and 23% PSMB10 G209R [amino acid sequence 40–273] proteoforms at 24.65 and 24.75 Da, respectively, while PSMB10 WT cells contain only the WT form. The 99 Da increase in the mass corresponds to the G-to-R mutation. (C) Relative proteasome activities in cell lysates of IFNγ-treated cells using specific AMC-fluorescently labeled substrates for the CT-L, T-L, and C-L activities (n = 4, statistics based on t test, P value: * <0.05, ** <0.01). (D) BU-MS/MS analysis of immunopurified 20S from cross-linked IFNγ-treated PSMB10 WT and PSMB10 G209R skin fibroblasts showing the identified PSMB10 peptides corresponding to the amino acid sequence [181–219] for PSMB10 WT (top) and [181–209] for PSMB10 G209R (bottom) matching the MS/MS spectra with the identified b (in blue) and y (in red) fragment ions. (E–H) BU-MS/MS analysis of immunopurified 20S from PSMB10 WT and PSMB10 G209R cells showing the relative stoichiometries of (E) the 20S catalytic subunits, (F) the 20S-bound chaperones after normalization of MS intensities to the total amount of 20S noncatalytic subunits, (G) the relative MS intensities of total 20S, assembled 20S, and assembling 20S, which were estimated thanks to the stoichiometry of the 20S-bound PAC1-PAC2, and (H) the 20S-bound activators (n = 4, statistics based on t test, P value: * <0.05, ** <0.01, *** <0.001, **** <0.0001). CT-L activity, chymotrypsin-like activity; T-L activity, trypsin-like activity; C-L activity, caspase-like activity.