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

  1. Ibtisam Ibtisam
  2. Alexei F Kisselev  Is a corresponding author
  1. Department of Drug Discovery and Development, Harrison College of Pharmacy, Auburn University, United States
4 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
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—source data 1

PDF file containing Figure 1a and original full-size western blot membranes (anti-DDI2, anti-GAPDH) with molecular weight markers.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data1-v1.zip
Figure 1—source data 2

Excel file containing data for Figure 1c.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data2-v1.xlsx
Figure 1—source data 3

PDF file containing Figure 1d and original full-size western blot membranes (anti-Nrf1, anti-DDI2, anti-β-actin) with molecular weight markers.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data3-v1.zip
Figure 1—source data 4

PDF file containing Figure 1e and full-size western blot membranes (anti-DDI2, anti-β-actin).

Additional lanes demonstrate that the knockdown of DDI2 is maintained throughout the experiment.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data4-v1.zip
Figure 1—source data 5

Excel file containing data and statistical analysis for Figure 1f.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data5-v1.xlsx
Figure 1—source data 6

Excel file containing data for Figure 1g.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-data6-v1.xls
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 1—source data 1

Excel file containing data and statistical analysis.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-figsupp1-data1-v1.xlsx
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 1—figure supplement 2—source data 1

Excel file containing data for both panels.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig1-figsupp2-data1-v1.xlsx
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 2—source data 1

Prism file containing data and statistical analysis for both panels.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
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.

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.

Figure 4—source data 1

Prism file containing data from Table S4 in McShane et al., 2016 that was used to create figure.

https://cdn.elifesciences.org/articles/91678/elife-91678-fig4-data1-v1.txt

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HAP1-wtHorizon DiscoveryRRID:CVCL_Y019, Cat # C631Parenteral cell line (clone 631) for
DDI2 KO cells below. https://horizondiscovery.com/en/engineered-cell-lines/products/hap1-parental-cell-lines
Cell line (Homo sapiens)HAP1-DDI2 KO, clone 010Horizon DiscoveryCat # HZGHC000396c010Generated by CRISPR using
gRNA:AATAGCTATGGAAGAGGCTC;
41 bp deletion; https://horizondiscovery.com/en/search?searchterm=HZGHC000396c010,
Cell line (Homo sapiens)HAP1-DDI2 KO, clone 023Horizon DiscoveryCalatogue # HZGHC000182c023Generated by CRISPR using
gRNA:GCTCGAAGTCGGCGTCGACC;
1 bp insertion; https://horizondiscovery.com/en/search?searchterm=HZGHC000182c023
Cell line (Homo sapiens)HAP1-DDI2 KO, clone 006Horizon DiscoveryCat # HZGHC000182c006Genertaed by CRISPR using
gRNA GCTCGAAGTCGGCGTCGACC;
4 bp deletion; https://horizondiscovery.com/en/search?searchterm=HZGHC000182c006
Cell line (Homo sapiens)MDA-MB-231ATCCCat# HTB-26https://www.atcc.org/products/htb-26#detailed-product-information
Cell line (Homo sapiens)SUM149BioIVTRRID:CVCL_3422
Cell line (Homo sapiens)HCT-11, wthttps://doi.org/10.7554/eLife.18357RRID:CVCL_0291A matching wt clone to a mutant below,
provided by Murata laboratory
cell line (Homo sapiens)HCT-116, DDI2--D252Nhttps://doi.org/10.7554/eLife.18357Contains CRISPR-generated D252N mutation
in the active site of DDI2, provided by Murata laboratory
Transfected construct (Homo sapiens)DDI2 siRNA10Horizon Discovery - DharmaconJ-032713-10-0050Sequences: GGACAUGCUUAAACGGCAC
Transfected construct (Homo sapiens)DDI2 siRNA12Horizon Discovery - DharmaconJ-032713-12-0050Sequence: CAAGAAAGGAUUCGUCUGU
Transfected construct (Homo sapiens)Non-targeting pool siRNAHorizon Discovery - DharmaconD-001810-10-20Sequences: UGGUUUACAUGUCGACUAA,
UGGUUUACAUGUUGUGUGA,
UGGUUUACAUGUUUUCUGA,
UGGUUUACAUGUUUUCCUA
AntibodyAnti-TCF11/NRF1 D5B10 (rabbit mAb)Cell SignalingCat# 8052 SWB (1:500)
AntibodyAnti-GAPDH D4C6R (mouse mAb)Cell SignalingCat# 97166WB (1:1000)
AntibodyAnti-β-actin 8H10D10 (mouse mAb)Cell SignalingCat #3700WB (1:1000)
AntibodyAnti-DDI2 (rabbit pAb)Bethyl LaboratoriesCat# A304-629AWB (1:5000)
AntibodyAnti-rabbit IgG, HRP-linked (goat)Cell SignalingCat#7074WB (1:1000)
AntibodyAnti-mouse IgG, HRP-linked (goat)Cell SignalingCat#7076 P2WB (1:1000)
AntibodyGoat anti-Rabbit IgG, Alexa Fluor Plus 647Thermofisher - InvitrogenCat#A32733WB (1:3500)
AntibodyGoat anti-Rabbit IgG, Alexa Fluor 680Thermofisher - InvitrogenCat#A-21076WB (1:3500)
AntibodyIRDye 800CW Goat anti-Mouse IgGLI-CORCat#926–32210WB (1:3500)
Commercial assay or kitDharmaFECT 1Horizon Discovery - DharmaconT-2001–03Transfection reagent for MDA-
MB-231 and SUM-149 cells
Commercial assay or kitProteasome-Glo AssayPromegaG8622Assay for Chymotrypsin-like
Commercial assay or kitCellTiter-Glo AssayPromegaG7572Assay for Cell Viability
Commercial assay or kitPierce Coomassie Plus (Bradford) AssayThermoFisher - Life Technologies23238Assay for Protein Quantification
Commercial assay or kitTRIzol ReagentThermoFisher - Life Technologies15596018RNA Isolation
Commercial assay or kitHigh-Capacity cDNA Reverse Transcription kitThermofisher - Applied Biosystems4368814
Commercial assay or kit2 x SYBR Green Bimake qPCR Master MixSelleckchem - BimakeB21203
Commercial assay or kitRNasin Plus Ribonuclease InhibitorPromegaN2615
Chemical compound, drugBortezomibLC LaboratoriesAS# 179324-69-7, Cat# B-1408Proteasome Inhibitor,
Chemical compound, drugCarfilzomibLC LaboratoriesCAS# 868540-17-4, Cat# C-3022Proteasome Inhibitors,
Chemical compound, drugCB-5083Cayman ChemicalsCAS# 1542705-92-9, Cat# 19311p97 inhibitor,
Chemical compound, drugCHAPS (3-((3-cholamidopropyl)
dimethylammonio)–1-propanesulfonate)
Thermo ScientificCAS# 331717-45-4, Cat # 28300Detergent
Chemical compound, drugCycloheximideSigma-AldrichCAS# 66-81-9, Cat #C1988Protein Synthesis Inhibitor,
Chemical compound, drugDigitoninGoldBioCAS# 11024-24-1, Cat# D-180–250Detergent
Chemical compound, drugPhosSTOPRocheCat# 4906837001Mixture of Phosphatase Inhibitors
Chemical compound, drugSuc-LLVY-AMCBachemCAS# 94367-21-2, Cat # 4011369Proteasome substrate
Chemical compound, drugResazurin sodium saltSigma-AldrichCAS# 62758-13-8, Cat#R7017Alamar Blue Viability Assay
Software, algorithmPRISMGraphPadversion 10

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  1. Ibtisam Ibtisam
  2. Alexei F Kisselev
(2024)
Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2
eLife 12:RP91678.
https://doi.org/10.7554/eLife.91678.3