Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome
- Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, University of Groningen, Netherlands
- Laboratory of Cellular and Structural Biology, The Rockefeller University, United States
- European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Netherlands
- Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Netherlands
Figures
Figure 1 with 1 supplement
Protein aggregation is increased following a functional loss of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and upon topoisomerase poisoning.
See also Figure 1—figure supplement 1. (A) In-gel Coomassie staining of indicated fractions of cell extracts of WT and ATM KO U2OS cells. The relative amounts of each fraction loaded are indicated. …
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Figure 1—source data 1
Data from Figure 1A.
Raw Coomassie-stained SDS-PAGE gel with cropped parts indicated in red.
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Figure 1—source data 2
Data from Figure 1C.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
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Figure 1—source data 3
Data from Figure 1E.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
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Figure 1—source data 4
Data from Figure 1G.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig1-data4-v2.jpg
Figure 1—figure supplement 1
Aggregation is increased in cells lacking ataxia telangiectasia mutated (ATM).
Western blot of U2OS wild-type and ATM KO cells, probed using the indicated antibodies. (B) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293 wild-type and ATM KO …
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Figure 1—figure supplement 1—source data 1
Data from Figure 1—figure supplement 1A.
Raw Western blot images with antibodies annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig1-figsupp1-data1-v2.jpg
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Figure 1—figure supplement 1—source data 2
Data from Figure 1—figure supplement 1B.
Raw Coomassie- and silver-stained SDS-PAGE gels and Western blot images with antibodies annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig1-figsupp1-data2-v2.jpg
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Figure 1—figure supplement 1—source data 3
Data from Figure 1—figure supplement 1E.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
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Figure 1—figure supplement 1—source data 4
Data from Figure 1—figure supplement 1F.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig1-figsupp1-data4-v2.jpg
Figure 2 with 1 supplement
Camptothecin (CPT) and ataxia telangiectasia mutated (ATM) loss drives aggregation in a cell-type-dependent manner.
See also Figure 2—figure supplement 1. (A) Volcano plot of label-free quantification (LFQ) MS/MS analysis of the aggregated fractions of DMSO and CPT-treated HEK293T cells. n = 4. Only proteins …
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Figure 2—source data 1
Data from Figure 2D.
Raw Western blot images with each antibody annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig2-data1-v2.jpg
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Figure 2—source data 2
Data from Figure 2G.
Raw Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig2-data2-v2.jpg
Figure 2—figure supplement 1
GO term analyses of the aggregation triggered by camptothecin (CPT) and ataxia telangiectasia mutated (ATM) loss.
(A) Experimental outline. All samples (whole-cell lysate [WCL], 1% SDS insoluble proteins, and RNA) were generated in parallel from the same cells in four independent repeats for each experiment. (B)…
Figure 3 with 2 supplements
Proteins that aggregate after topoisomerase I poisoning are supersaturated and prone to engage in liquid-liquid phase separation (LLPS).
See also Figure 3—figure supplements 1 and 2. (A) TANGO scores of HEK293T whole-cell lysate (WCL), nonaggregated proteins (NIA), and aggregated fractions. (B) Protein abundance of HEK293T WCL, …
Figure 3—figure supplement 1
Proteins that aggregate after camptothecin treatment and ataxia telangiectasia mutated (ATM) loss represent a vulnerable subfraction of the proteome.
(A) CamSol-intrinsic (in)solubility scores of complete whole-cell lysate (WCL), nonaggregated proteins (NIA), and aggregated fractions in HEK293T cells. Dotted line indicates the theoretical …
Figure 3—figure supplement 2
GO term analyses of RNAseq data.
GO term analysis of the differentially expressed genes (–1 > log2FC > 1) RNAseq data of the indicated cell line after the indicated treatment. Upper panels: upregulated processes are shown. Lower …
Figure 4 with 2 supplements
The cell-intrinsic aggregation threshold is lowered upon targeting ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA topoisomerases.
See also Figure 4—figure supplements 1 and 2. (A) Overlap between RNA-sequencing analysis and label-free quantification (LFQ) MS/MS analysis for whole-cell lysate (WCL) and aggregated (Agg.) protein …
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Figure 4—source data 1
Data from Figure 4D.
Raw autoradiograms and Coomassie- and silver-stained SDS-PAGE gels with cropped parts indicated in red.
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Figure 4—source data 2
Data from Figure 4F.
Raw filter trap images with the cropped parts indicated in red.
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Figure 4—source data 3
Data from Figure 4G.
Raw filter trap image with the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig4-data3-v2.jpg
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Figure 4—source data 4
Data from Figure 4H.
Raw Western blot images with each antibody annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig4-data4-v2.jpg
Figure 4—figure supplement 1
Increased aggregation triggered by camptothecin treatment and ataxia telangiectasia mutated (ATM) loss overlaps with that occurring in various proteinopathies.
(A) Overlap between RNA-sequencing analysis and label-free quantification (LFQ) MS/MS analysis for whole-cell lysate (WCL) and aggregated (Agg.) protein fractions in U2OS cells. Only significant …
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Figure 4—figure supplement 1—source data 1
Data from Figure 4—figure supplement 1F.
Raw Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig4-figsupp1-data1-v2.jpg
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Figure 4—figure supplement 1—source data 2
Data from Figure 4—figure supplement 1G.
Raw Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig4-figsupp1-data2-v2.jpg
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Figure 4—figure supplement 1—source data 3
Data from Figure 4—figure supplement 1H.
Raw Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig4-figsupp1-data3-v2.jpg
Figure 4—figure supplement 2
Proteins that aggregate in HEK293T treated with camptothecin (CPT) are linked to various proteinopathies.
Relative occurrences of proteins identified as aggregating in various proteinopathy (model) datasets in the HEK293T NIA and CPT-induced aggregation fractions. See also Figure 4E. Proteins marked …
Figure 5 with 2 supplements
The lowered aggregation threshold caused by topoisomerase poisoning or a loss of ataxia telangiectasia mutated (ATM) is accompanied by a rewiring and aggregation of known interacting (co)chaperones.
See also Figure 5—figure supplements 1 and 2. (A) Presence of (co)chaperones in the aggregated protein fractions in HEK293T cells. Left panel: Venn diagram showing the overlap in aggregating …
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Figure 5—source data 1
Data from Figure 5G.
Raw Western blot images with each antibody annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig5-data1-v2.jpg
Figure 5—figure supplement 1
Chaperone systems are rewired in line with the presence of chaperone clients in aggregates induced by camptothecin or ataxia telangiectasia mutated (ATM) loss.
(A–D) Volcano plots showing the presence of (co)chaperones in SDS-insoluble fractions as identified via MS/MS analysis for HEK293T ATM vs. DMSO, HEK293T camptothecin (CPT) vs. DMSO, U2OS ATM KO vs. …
Figure 5—figure supplement 2
Heatmaps of chaperone interactions of aggregating fractions.
Complete overview of (co)chaperone interactions with the indicated fractions identified in this study as logged in BioGRID, per (co)chaperone. Darker colors represent a higher percentage of proteins …
Figure 6 with 2 supplements
Protein aggregation triggered by genotoxic stress is amenable to modulation by chaperones.
See also Figure 6—figure supplements 1 and 2. (A) Western blot of whole-cell lysate (WCL) and aggregated proteins isolated from HEK293T cells treated transiently with DMSO or camptothecin (CPT), …
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Figure 6—source data 1
Data from Figure 6A.
Raw Western blot images with each antibody annotated and cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-data1-v2.jpg
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Figure 6—source data 2
Data from Figure 6B.
Raw Coomassie- and silver-stained SDS-PAGE gels and Western blot images with cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-data2-v2.jpg
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Figure 6—source data 3
Data from Figure 6H.
Raw Coomassie- and silver-stained SDS-PAGE gels and Western blot images with cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-data3-v2.jpg
Figure 6—figure supplement 1
HSPB5 alleviates protein aggregation triggered by a loss of ataxia telangiectasia mutated (ATM) in U2OS cells independent of overt DNA repair capacity changes.
(A) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293T cells treated transiently with DMSO or camptothecin (CPT), followed by treatment with the VER-155008 HSP70 …
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Figure 6—figure supplement 1—source data 1
Data from Figure 6—figure supplement 1A.
Raw Coomassie- and silver-stained SDS-PAGE gels with the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-figsupp1-data1-v2.jpg
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Figure 6—figure supplement 1—source data 2
Data from Figure 6—figure supplement 1B.
Raw Coomassie-stained SDS-PAGE gels with the cropped parts indicated in red. Raw Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-figsupp1-data2-v2.zip
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Figure 6—figure supplement 1—source data 3
Data from Figure 6—figure supplement 1C.
Raw Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-figsupp1-data3-v2.jpg
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Figure 6—figure supplement 1—source data 4
Data from Figure 6—figure supplement 1G.
Raw Coomassie- and silver-stained SDS-PAGE gels and Western blot images with the antibodies annotated and the cropped parts indicated in red.
- https://cdn.elifesciences.org/articles/70726/elife-70726-fig6-figsupp1-data4-v2.jpg
Figure 6—figure supplement 2
HSPB5, HSP70, and HSP90 do not (re-)localize to DNA damage sites.
(A–C) Representative immunofluorescence pictures of U2OS cells treated with camptothecin (CPT) or gamma-irradiation, stained with anti-HSPB5, anti-HSP70, or anti-HSP90, respectively (in red), …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HEK293T | ATCC | CRL-3216 | |
| Cell line (human) | HEK293 | ATCC | CRL-1573 | |
| Cell line (human) | HEK293 ATM KO | This study | See ‘Mammalian cell culture’ | |
| Cell line (human) | HEK293 HTT Q71-GFP | PMID:20159555 | See ‘Mammalian cell culture’ | |
| Cell line (human) | HEK293 luciferase-GFP | PMID:21231916 | See ‘Mammalian cell culture’ | |
| Cell line (human) | HEK293+ HSPB5 | This study | See ‘Mammalian cell culture’ | |
| Cell line (human) | U2OS | ATCC | HTB-96 | |
| Cell line (human) | U2OS ATM KO | This study | See ‘Mammalian cell culture’ | |
| Cell line (human) | U2OS + HSPB5 | This study | See ‘Mammalian cell culture’ | |
| Cell line (human) | U2OS ATM KO + HSPB5 | This study | See ‘Mammalian cell culture’ | |
| Cell line (human) | Phoenix-Ampho | ATCC | RRID:CVCL_H716 | Retrovirus packaging cell line |
| Antibody | GFP (mouse, monoclonal) | Takara Bio Clontech | 632380 | WB (1:5000) |
| Antibody | ATM (mouse, monoclonal) | Santa Cruz | Sc-23921 | WB (1:200) |
| Antibody | HSPB5 (mouse, monoclonal) | StressMarq | SMC-159 | WB (1:2000) |
| Antibody | HSPB5 (mouse, monoclonal) | StressMarq | SMC-165 | IF (1:200) |
| Antibody | GAPDH (mouse, monoclonal) | Fitzgerald | 10R-G109a | WB (1:10,000) |
| Antibody | TUB (mouse, monoclonal) | Sigma-Aldrich | T5138 | WB (1:4000) |
| Antibody | HDAC1 (mouse, monoclonal) | DSHB | PCPR-HDAC1-2E12 | WB (0.5 μg/ml) |
| Antibody | MCM7 (Mmouse, monoclonal) | Santa Cruz | 47DC141 | WB (1:100) |
| Antibody | TUBA1A (mouse, monoclonal) | Sigma-Aldrich | T5168 | WB (1:2000) |
| Antibody | FUS (mouse, monoclonal) | Santa Cruz | Sc-47711 | IF (1:200) |
| Antibody | 53BP1 (rabbit, monoclonal) | Santa Cruz | Sc-22760 | IF (1:150) |
| Antibody | 53BP1 (rabbit, monoclonal) | Bethyl | A300-272A | IF (1:500) |
| Antibody | HSP70 (mouse, monoclonal) | StressMarq | SMC-104A | IF (1:100) |
| Antibody | HSP90 (mouse, monoclonal) | StressMarq | SMC-149 | IF (1:100) |
| Recombinant DNA reagent | pQCXIN–HSPB5 (plasmid) | PMID:20843828 | ||
| Recombinant DNA reagent | ATM CRISPR/Cas9 KO (plasmid) | Santa Cruz | sc-400192 | |
| Recombinant DNA reagent | ATM HDR (plasmid) | Santa Cruz | sc-400192-HDR | |
| Sequence-based reagent | HEK293Q71F(forward primer) | This study | PCR primer | GAGTCCCTCAAGTCCTTCC |
| Sequence-based reagent | HEK293Q71R(reverse primer) | This study | PCR primer | AAACGGGCCCTCTAGACTC |
| Commercial assay or kit | Silver stain kit | Pierce (Thermo Scientific) | 24612 | |
| Commercial assay or kit | Allprep DNA/RNA isolation mini kit | QIAGEN | 80004 | |
| Commercial assay or kit | S-trap micro | Protifi | K02-micro-10 | |
| Commercial assay or kit | Masterpure Complete DNA and RNA purificiation kit | Epicentre (supplied through Lucigen) | MC85200 | |
| Commercial assay or kit | QuantSeq 3’ mRNA-Seq library prep kit (FWD) | Lexogen | 015.96 | |
| Chemical compound, drug | Camptothecin | Selleckchem | S1288 | See Table 1 |
| Chemical compound, drug | Etoposide | Sigma-Aldrich | E1383 | See Table 1 |
| Chemical compound, drug | TDP1 inhibitor | Merck | 532177 | See Table 1 |
| Chemical compound, drug | KU-55933 (ATM inhibitor) | Selleckchem | S1092 | See Table 1 |
| Chemical compound, drug | KU 0058948 (PARP inhibitor) | Axon Medchem | 2001 | See Table 1 |
| Chemical compound, drug | VE-821 | Axon Medchem | 1893 | See Table 1 |
| Chemical compound, drug | VER-155008 | Axon Medchem | 1608 | (10 μM) |
| Chemical compound, drug | [S35]Met/cys | Hartmann Analytic | IS-103 | (10 μCi/ml) |
| Chemical compound, drug | ProteoStat | Enzo Life Sciences | ENZ-51023-KP050 | |
| Chemical compound, drug | AmpliTaq Gold Fast PCR mix | Applied Biosystems (supplied through Thermo Fisher) | 4390937 | |
| Software, algorithm | Prism | GraphPad | ||
| Software, algorithm | Illustrator 2021 | Adobe | ||
| Software, algorithm | TANGO | PMID:15361882 | ||
| Software, algorithm | CamSol Intrinsic | PMID:25451785 | ||
| Software, algorithm | catGRANULE | PMID:23222640 | ||
| Software, algorithm | PScore | PMID:29424691 | ||
| Software, algorithm | MaxQuant | PMID:19029910 | ||
| Software, algorithm | Lexogen QuantSeq 2.3.1 FWD UMI | BlueBee genomics (Illumina) | ||
| Software, algorithm | edgeR | PMID:19910308 | ||
| Software, algorithm | Cytoscape (in Python) | PMID:31477170 | ||
| Software, algorithm | Metascape (webserver) | PMID:30944313 | ||
| Software, algorithm | ImageJ (Fiji) | PMID:22930824 | ||
| Other | DMEM without methionine/cysteine | Gibco (supplied through Thermo Fisher) | 21013024 |
Table 1
Genotoxic drugs used in this study.
| Drug | Target | Concentration |
|---|---|---|
| Camptothecin | TOP1 | 20–600 nM. Figure 1C: 100 nM; Figure 1E: 20–100 nM; Figure 1G: 40 nM; Figure 1—figure supplement 1E: 200–600 nM; Figure 1—figure supplement 1F: 40 nM; MS/MS HEK293T: 100 nM; MS/MS U2OS: 400 nM; Figure 4F: 100 nM; Figures 5G, 6A: 40 nM; Figure 6—figure supplement 1A: 40 nM; Figure 6H: 400 nM; Figure 6—figure supplement 1G: 400 nM; Figure 6—figure supplement 2A–C: 400 nM. |
| CD00509 | TDP1 | 4 µM |
| Etoposide | TOP2 | Figure 1C: 3 µM; Figure 1E: 0.6–3 µM; Figure 4F: 3 µM. |
| Ku-55933 | ATM | Everywhere 9 µM, except in Figure 4—figure supplement 1H: 3, 6, or 9 µM, and in Figure 5G: 13.5 µM |
| Ku-58948 | PARP1-3 | 4 µM |
| VE-821 | ATR | 3 µM |
Table 2
Online databases used.
| Analysis | Tool/database | Source/weblink |
|---|---|---|
| Supersaturation | Supersaturation database | Ciryam et al., 2013 |
| Heat-sensitive proteins | Heat-sensitive protein database | Mymrikov et al., 2017 |
| Stress-granule constituents | RNA granule database | https://rnagranuledb.lunenfeld.ca |
| (Co)chaperone interactions | HSPA1A/HSPA8 client database | Ryu et al., 2020 |
| BioGRID PPI database | https://thebiogrid.org |
Additional files
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Supplementary file 1
MS/MS datasets of aggregated and whole-cell lysate (WCL) protein fractions.
- https://cdn.elifesciences.org/articles/70726/elife-70726-supp1-v2.xlsx
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Supplementary file 2
RNA-sequencing differential expression analyses.
- https://cdn.elifesciences.org/articles/70726/elife-70726-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70726/elife-70726-transrepform1-v2.docx
