Extended Data Fig. 7: PSME4 modulates proteasome activity and HLA maturation.
a-c, Proteasome activity assays using fluorogenic substrates LLE-bNA (caspase, β1;a), nLPnLD-AMC (caspase, β1;b), or LLVY-AMC (chymotryptic, β5;c) for 3.5 hours. Relative fluorescence unit (RFU) of the substrate is shown across the 3.5 hours of the experiment (left) or at the endpoint (right). Recombinant PSME4 in different ratios, as indicated, was added to A549 lysates (one-way ANOVA; **P = 0.0022, **** P < 0.0001; bars indicate mean ± s.d.; n = 3 independent samples). d-h, Proteasome activity assays using the nLPnLD-AMC (caspase, β1; d), RLR-AMC (tryptic, β2/ β2i; e), PAL-AMC (chymotryptic, β1i; f), LLVY-AMC (chymotryptic, β5/ β5i; g) or ANW-AMC (chymotryptic, β5i; h) substrates. Relative fluorescence unit (RFU) of the substrate is shown across the 3.5 hours of the experiment (left) or at the endpoint (right). A549 lysates were treated with TNFα and IFNγ (T + I; red) or untreated (UT; black) and recombinant PSME4 was added to the lysate where indicated (two-sided paired student’s T-test) Red squares indicate the portion of the Figure reproduced in Fig. 3 (n = 4 independent samples; bars indicate mean ± s.d.). i, The MFI of HLA on the cell surface was monitored using flow cytometry following acid stripping. PSME4 depletion (shPSME4) increases levels of HLA on the cell surface starting from 2 hours after stripping (2-way-ANOVA ****P ≤ 0.0001, n = 3 independent samples; bars indicate mean ± s.d.). j, The percentage of HLA-high cells was monitored using flow cytometry following acid stripping. PSME4 overexpression (PSME4 OE) decreases levels of HLA on the cell surface starting from 4 hours after stripping. (2-way-ANOVA **P = 0.0033, n = 3 independent samples; bars indicate mean ± s.d.). k,l, The percent of cells with high levels of HLA on the surface is significantly decreased following PSME4 overexpression (k; n = 3 independent samples per condition). By contrast, the intracellular levels of HLA are significantly increased upon PSME4 overexpression (l; bars indicate mean ± s.d.).
