Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

Abstract

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

Keywords: Nrf1; aspartic protease; cancer biology; cell biology; human; ubiquitin; ubiquitin-binding protein; ubl doman.

Figure 1.. Recovery of proteasome activity is DDI2 independent.

(a) Expression of DDI2 in the CRISPR-generated clones of HAP1 cells used in this work was analyzed by western blot. (b) The experimental setup used in this study. Cells were pulse treated with bortezomib (Btz) or carfilzomib (Cfz) for 1 hr, then cultured in drug-free media for times indicated and analyzed as described. (c) The viability of wt- and DDI2 KO clones of HAP1 cells was measured using CellTiter-Glo, and the inhibition of β5 sites was measured with the Proteasome-Glo assay at times indicated; n=2–5. (d) Knockout of DDI2 inhibits the Nrf1 processing. Western blots of Btz-treated HAP1 cells. The sample in the first lane is wt cells treated with VCP/p97 inhibitor CB-5083 immediately after removal of Btz. VCP inhibitors blocks Nrf1 processing (Radhakrishnan et al., 2014; Sha and Goldberg, 2014; Anderson et al., 2015). (e) MDA-MB-231 and SUM149 cells were analyzed by western blot 72 hr after transfection with DDI2 siRNAs (f) Theβ5 activity in siRNA-transfected SUM149 and MDA-MB-231 was measured using Suc-LLVY-AMC immediately and 18 hr after treatment with 100 nM Btz; n=3. (g) β5 activity was measured in HCT-116 cells with the Proteasome-Glo assay immediately and 18 hr after treatment with PIs; n=2.

Figure 1—figure supplement 1.. The proteasome activity of the samples used in Figure 1d was measured with Suc-LLVY-AMC; n=9.

Figure 1—figure supplement 2.. Comparison of proteasome activity and proteasome inhibitor (PI) sensitivity between HAP1, MDA-MB-231, and SUM149 cells.

(a) Cells were treated with PIs for 1 hr, media was shaken off, and cells were cultured in an inhibitor-free fresh media for 48 hr when Alamar Blue assay was performed; n=3–4. See Figure 1 in Weyburne et al., 2017 for a comparison of SUM149 and MDA-MB-231 cells. (b) The β5 proteasome activity was measured using Suc-LLVY-AMC in the cell extracts of untreated cells; n=2-8.

Figure 2.. Proteasome activity recovers before upregulation of proteasome gene expression.

Wt-HAP1 cells were pulse-treated with bortezomib (Btz) (100 nM), cultured in a drug-free medium, and analyzed at indicated times. (a) β5 activity was measured using Proteasome-Glo and normalized first to CellTiter-Glo viability data and then to proteasome activity in the mock-treated samples; n=2–5. (b) In a parallel experiment, the mRNA was isolated, and the expression of proteasome genes was quantified using quantitative RT-PCR; n=3. Results of the t-test at 8 hr are in parenthesis.

Figure 3.. The recovery of proteasome activity requires protein synthesis.

(a) Wt-HAP1 and DDI2 KO cells were treated for 1 hr at indicated concentrations of bortezomib (Btz) and carfilzomib (Cfz) and then cultured in a drug-free media in the absence (solid lines) or presence (dashed lines) of cycloheximide (CHX). The β5 activity was measured using Proteasome-Glo and normalized first to cell viability, which was determined in a parallel experiment using CellTiter-Glo, and then to untreated controls; n=3–4. (b) All proteasome mRNAs are actively translated. mRNA isolated from untreated wt-HAP1 cells were analyzed by polysome profiling. The combined mRNAs in the 80 S and polysomal fractions as a % of the total is shown; n=2.

Figure 3—figure supplement 1.. Translation of catalytic subunits is not altered after treatment with inhibitors.

Cells were treated with bortezomib (Btz) for 1 hr, and then cultured in drug-free media. harvested at indicated times and analyzed by polysome profiling and qPCR as in Figure 3b; n=2.

Figure 4.. Escape from rapid degradation of nascent subunits can explain rapid recovery of proteasome activity.

(a) Turnover of proteasome subunit in human RPE-1 cells was measured by quantitative mass-spectrometry following 1 hr labeling with heavy isotopes. Data taken from Table S4 in McShane et al., 2016; n=2-3. (b) Proposed model of how nascent proteasome subunits are partitioned between assembly and degradation.