On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation
Abstract
:1. Introduction
2. Results
2.1. Heat-Mediated Degradation of BRCA2 and Modulation of HR
2.2. Various Inhibitors Affect Heat-Mediated BRCA2 Degradation
2.3. BRCA2 Stability Is Dependent on HSP90
2.4. Searching for the Proteins that Mediate Degradation of BRCA2 upon Hyperthermia
2.5. Heat-Mediated BRCA2 Degradation from the Protein’s Perspective
2.6. Semi-Quantitative Mass Spectrometry Analysis Identifies Putative BRCA2-Interactors upon Hyperthermia
2.7. Oxidative Stress Induces BRCA2 Degradation
3. Discussion
3.1. Heat-Mediated Inhibition of HR from an Evolutionary Perspective
3.2. BRCA2 Protein Stability, HSP90, and Cycloheximide
3.3. HSP90 Is an Attractive Target to Modulate Heat-Induced BRCA2 Degradation
3.4. Ubiquitination May Not Be Required for Heat-Mediated BRCA2-Degradation by the Proteasome
3.5. BRCA2 and the Oxidative Stress Response
4. Materials and Methods
4.1. Cell Culture
4.2. Generation of Constructs and Cell Lines
4.3. Hyperthermia Treatment
4.4. Chemical Agents and UV Irradiation
4.5. siRNA Transfection
4.6. Immunoprecipitation
4.7. Cell Fractionation, Lysis, and Immunoblotting
4.8. Immunofluorescent Staining and Analysis of RAD51-Foci
4.9. Antibodies
4.10. SILAC-Based Mass Spectrometry
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Protein | Peptides | Coverage | Maxquant Pep | 20 Min HT | 60 Min HT | ||
---|---|---|---|---|---|---|---|
Exp 1 | Exp 2 | Exp 1 | Exp 2 | ||||
BRCA2 | 211 | 65 | 0.0E + 00 | 1.20 | 0.90 | 0.28 | 0.53 |
PALB2 | 36 | 44.5 | 0.0E + 00 | 1.49 | 0.92 | 0.30 | 0.37 |
RAD51 | 16 | 57.5 | 0.0E + 00 | 1.24 | 0.57 | 0.18 | 0.22 |
BRCA1 | 19 | 12.9 | 2.5E − 58 | 1.25 | 1.39 | 0.15 | 0.83 |
KEAP1 | 21 | 39.6 | 3.5E − 164 | 1.42 | 1.02 | 0.29 | 0.42 |
MORF4L1 | 17 | 61.9 | 1.1E − 271 | 1.44 | 0.89 | 0.29 | 0.42 |
MORF4L2 | 11 | 43.1 | 2.2E − 78 | 1.47 | 0.79 | 0.71 | 0.49 |
Ubiquitin | 8 | 56.4 | 8.3E − 87 | 3.42 | 4.60 | 4.06 | 3.54 |
HSPB1 | 10 | 67.9 | 4.3E − 69 | 1.45 | 2.26 | 3.19 | 1.87 |
USP28 | 6 | 6 | 1.0E − 12 | 1.61 | 3.75 | 1.00 | 2.74 |
% | SILAC ratio treated to untreated |
Name | BRCA2-region (Amino Acid) | Molecular Weight (kDa) | Vector | Pro-Motor | Code/Ref |
---|---|---|---|---|---|
FLAG-BRCA2 | Met 1–Ile 3418 | 382 | pGb-LPL | CAG | pAZ148 |
GFP-BRCA2 | Met 1–Ile 3418 | 406 | pGb-LPL | CAG | pAZ114 [26] |
BRCA2-N-GFP | Met 1–Thr 939 | 131 | pGb-LPL | CAG | pAZ108 |
BRCA2-M-GFP | Gln 940–Glu 2198 | 167 | pGb-LPL | CAG | pAZ109 |
BRCA2-C-GFP | Thr 2199–Ile 3418 | 162 | pGb-LPL | CAG | pAZ110 |
FLAG-BRCA2-N | Met 1–Thr 939 | 107 | pGb-LPL | CMV | pAZ97 |
FLAG-BRCA2-M | Gln 940–Glu 2198 | 123 | pGb-LPL | CMV | pAZ98 |
FLAG-BRCA2-C | Thr 2199–Ile 3418 | 118 | pGb-LPL | CMV | pAZ104 |
FLAG-BRCA2-ΔM | Met 1–Thr 939 Thr 2199–Ile 3418 | 250 | pGb-LPL | CAG | pAZ253 |
Clover-HSP90 | n.a. | 109 | pGb-LPL | CAG | n.a. |
FLAG-HSP90 | n.a. | 82 | pGb-LPL | CMV | [99] |
Set | Name | Sequence |
---|---|---|
1F | GA15N-F | 5’-GCTCCTGGGCAACGTGCCTCGAGATGCCTATTGGATCCAAA GAGAGGCCAAC-3’ |
1R | GA15N-R | 5’-TTGCTCACCATGGTGGCCTCGAGGGTTGCTTGTTTATCACCT GTGT-3’ |
2F | GA15M-F | 5’-GCTCCTGGGCAACGTGCCTCGAGATGCAAGTGTCAATTAAAA AAGATTTGGTTTATGTTCTTGC-3’ |
2R | GA15M-R | 5’-TTGCTCACCATGGTGGCCTCGAAAGTTTCAGTTTTACCAATTT CCATTTTTACGTT-3’ |
3F | GA15C-F | 5’-GCTCCTGGGCAACGTGCCTCGAGATGACTTTTTCTGATGTTC CTGTGAAAACAAATATAGAAG-3’ |
3R | GA15C-R | 5’-TTGCTCACCATGGTGGCCTCGAGGATATATTTTTTAGTTGTAA TTGTGTCCTGCTTATTTTTCTCACA-3’ |
4F | BRCA2-Nterm-F | 5’-CAAGGATGACGACGACAAGAGCCCTATTGGATCCAAAGAGA GGC-3’ |
4R | BRCA2-Nterm-R | 5’-GCTGATTATGATCTAGAGTCAGGTTGCTTGTTTATCACCTGT GTCT-3’ |
5F | GA04M-F | 5’-CAAGGATGACGACGACAAGAGATCTACCCAAGTGTCAATTA AAAAAGATTTGGTTTATGT-3’ |
5R | GA04M-R | 5’-GCTGATTATGATCTAGAGTCAGATCTTTTCAGTTTTACCAATT TCCATTTTTACGTTTTTAGGT-3’ |
6F | GA04C-F | 5’-CAAGGATGACGACGACAAGAGATCTACTTTTTCTGATGTTCC TGTGAAAACAAATATAGAAG-3’ |
6R | GA04C-R | 5’-GCTGATTATGATCTAGAGTCAGATCTTTAGATATATTTTTTAGT TGTAA TTGTGTCCTGCTTATTTTTCTCACAT-3’ |
7F | B2flFLAG-F1 | 5’-CCTGGGCAACGTGCCGATTATAAAGACCACGATGGAGACT ATAAAGATCATGACATTGACT-3’ |
7R | B2flFLAG-R2 | 5’-CACTGTCCTTCCTGCAGGCATGACAGAGAA-3’ |
8F | B2intdel-GA-F1 | 5’-TTCTCTGTCATGCCTGCAGGAAGGAC-3’ |
8R | B2intdel-GA-R1 | 5’-CAACATTTAAGTTATTTGATAATTTGGTTGCTTGTTTATCACC TGTGTCT-3’ |
Name | Reference/Suppliers |
---|---|
Bafilomycin | Sigma-Aldrich |
NMS-873 | Selleckchem |
Ganetespib | Syntha Pharmaceuticals |
MG132 | Merck Millipore |
Cycloheximide | Sigma-Aldrich |
MLN4924 | MedChem Express |
PYR-41 | Calbiochem |
NAC | Sigma-Aldrich |
Ascorbic Acid | Sigma-Aldrich |
DTT | Sigma-Aldrich |
H2O2 | Sigma-Aldrich |
t-BHP | Sigma-Aldrich |
Rotenone | MP Biomedicals |
siRNA | SenseSequence | Reference/Suppliers |
---|---|---|
Luc | CGUACGCGGAAUACUUCGA | Thermo Scientific |
siAKTIP#1 | GAAUUUACCUUGGUUGUGA | [37] |
siAKTIP#2 | AGAAAACAGUGGCGACUUA | [37] |
siBIRC6#1 | UCAUUGCCUUACUCACAUA | [37] |
siBIRC6#2 | GGUCAAAGAUCACUUAGUA | [37] |
siTSG101#1 | AGUAGCCGAGGUUGAUAAA | [37] |
siTSG101#2 | AAACUGAGAUGGCGGAUGA | [37] |
siUBE2A#1 | UGAUGUGUCUUCCAUUCUA | [37] |
siUBE2A#2 | GAUGAACCCAAUCCCAAUA | [37] |
siUBE2B#1 | AUAGACAACUGGUCUGUUA | [37] |
siUBE2B#2 | UUGGACCAGAAGGGACACC | [37] |
siUBE2C#1 | GCAAGAAACCUACUCAAAG | [37] |
siUBE2C#2 | UAAAUUAAGCCUCGGUUGA | [37] |
siUBE2D1#1 | UACUGUAUGUGUUGUCUAA | [37] |
siUBE2D1#2 | CAACAGACAUGCAAGAGAA | [37] |
siUBE2D2#1 | CAGUAAUGGCAGCAUUUGU | [37] |
siUBE2D2#2 | CCAACCAGAUUAAACUCUA | [37] |
siUBE2D3#1 | UGAUGUAAAGUUCGAAAGA | [37] |
siUBE2D3#2 | CCACAAUUAUGGGACCUAA | [37] |
siUBE2D4#1 | CAGCGUUGACUGUGUCAAA | [37] |
siUBE2D4#2 | GGAAUUAACCGACUUGCAG | [37] |
siUBE2E1#1 | GCGAUAACAUCUAUGAAUG | [37] |
siUBE2E1#2 | GGUGUAUUCUUUCUCGAUA | [37] |
siUBE2E2#1 | ACUUGAAAGAUUUGGGAUU | [37] |
siUBE2E2#2 | UCACCAGACUAUCCGUUUA | [37] |
siUBE2E3#1 | GCAUAGCCACUCAGUAUUU | [37] |
siUBE2E3#2 | GCUAAGUUAUCCACUAGUG | [37] |
siUBE2F#1 | GGAAUAAAGUGGAUGACUA | [37] |
siUBE2F#2 | CAACAUAAAUACAGCAAGA | [37] |
siUBE2G1#1 | UGUUGAUGCUGCGAAAGAA | [37] |
siUBE2G1#2 | GGGAAGAUAAGUAUGGUUA | [37] |
siUBE2G2#1 | AUGAUGACUUAAUGUCGAA | [37] |
siUBE2G2#2 | UGACGAAAGUGGAGCUAAC | [37] |
siUBE2H#1 | CGAGAGUAAACAUGAGGUU | [37] |
siUBE2H#2 | CUACUGAACUGUCGAAGGA | [37] |
siUBE2J1#1 | GAACUGGCUAGGCAAAUAA | [37] |
siUBE2J1#2 | GAAAGAAGCGGCAGAAUUG | [37] |
siUBE2J2#1 | GAAGGUGGCUAUUAUCAUG | [37] |
siUBE2J2#2 | GCACAAGACGAACUCAGUA | [37] |
siUBE2K#1 | CUCUCCGCACGGUAUUAUU | [37] |
siUBE2K#2 | GAAUCAAGCGGGAGUUCAA | [37] |
siUBE2L3#1 | UGAAGAGUUUACAAAGAAA | [37] |
siUBE2L3#2 | GGGCUGACCUAGCUGAAGA | [37] |
siUBE2L6#1 | UGAUCAAAUUCACAACCAA | [37] |
siUBE2L6#2 | UCAAUGUGCUGGUGAAUAG | [37] |
siUBE2M#1 | AGCCAGUCCUUACGAUAAA | [37] |
siUBE2M#2 | GAUGAGGGCUUCUACAAGA | [37] |
siUBE2N#1 | GCGGAGCAGUGGAAGACCA | [37] |
siUBE2N#2 | CUAUCUAGCUUGUGUGUCA | [37] |
siUBE2NL#1 | AAACGUGAACUAUUACUUG | [37] |
siUBE2NL#2 | GACAAGUUGGAAAGAAUAA | [37] |
siUBE2O#1 | ACAUCGACUGUGCCGUCAA | [37] |
siUBE2O#2 | GGGACUACAUUGCCUAUGA | [37] |
siUBE2Q1#1 | UCAUCUCCGACCUGUGUAA | [37] |
siUBE2Q1#2 | GAAAGGGAAUACUCUGCUA | [37] |
siUBE2Q2#1 | UACAGAUCACAGAGUUAUA | [37] |
siUBE2Q2#2 | GUAUGGAACUUCUCACAAA | [37] |
siUBE2R1#1 | CGCAGAACGUCAGGACCAU | [37] |
siUBE2R1#2 | GGAAGUGGAAAGAGAGCAA | [37] |
siUBE2R2#1 | UGUGAGGACUAUCCUAUUA | [37] |
siUBE2R2#2 | CCACAACCCUGGCGGAAUA | [37] |
siUBE2S#1 | AUGGCGAGAUCUGCGUCAA | [37] |
siUBE2S#2 | ACAAGGAGGUGACGACACU | [37] |
siUBE2T#1 | AGAGAGAGCUGCACAUGUU | [37] |
siUBE2T#2 | CCUGCGAGCUCAAAUAUUA | [37] |
siUBE2U#1 | ACAGGCCAUUACAAAUGAA | [37] |
siUBE2U#2 | GAAGUGGAAUACAAACUAU | [37] |
siUBE2V1#1 | GGACAGUGUUACAGCAAUU | [37] |
siUBE2V1#2 | GUGGAUGCAUACCGAAAUA | [37] |
siUBE2V2#1 | AGUUGUACUUCAAGAGCUA | [37] |
siUBE2V2#2 | GUUAAAGUUCCUCGUAAUU | [37] |
siUBE2W#1 | CGACCACCGGAUAAUUCUU | [37] |
siUBE2W#2 | GCGAACAUGUAACAAGAAU | [37] |
siUBE2Z#1 | AUGUUCGUUGUACCUGAUA | [37] |
siUBE2Z#2 | GGGAAAGUCUGCUUGAGUA | [37] |
siUEV3#1 | CGAUGGACCUUGAAAUCUU | [37] |
siUEV3#2 | AGAAAGACCUGCUGAAUUU | [37] |
UBC9 | GGGAUUGGUUUGGCAAGAA | [39] |
CHIP#1 | GCGCUCUUCGAAUCGCGAAGA | Invitrogen |
CHIP#2 | UGCCGCCACUAUCUGUGUAAU | Invitrogen |
PIAS1 | GGAUCAUUCUAGAGCUUUA | [43] |
PIAS2 | CUUGAAUAUUACAUCUUUA | [43] |
PIAS3 | CCCUGAUGUCACCAUGAAA | [43] |
PIAS4 | GGAGUAAGAGUGGACUGAA | [43] |
RNF4#1 | GAAUGGACGUCUCAUCGUU | [39] |
RNF4#2 | GACAGAGACGUAUAUGUGA | Thermo Scientific |
RNF111 | GGAUAUUAAUGCAGAGGAA | [39] |
Host | Epitope | Dilutions Used | Reference/Suppliers |
---|---|---|---|
Mouse | BRCA2 (OP95) | WB 1:1000 | EMD Millipore |
Mouse | FLAG (M2) | WB 1:1000–1:5000 | Sigma-Aldrich |
Mouse | ORC2 (68348) | WB 1:1000 | Abcam |
Rabbit | Brca2 (27976) | WB 1:500 | Abcam |
Mouse | GFP (clones 7.1 and 13.1) | WB 1:1000–1:5000 | Sigma-Aldrich |
Mouse | PARP-1 (C2-10) | WB 1:5000 | Enzo Lifesciences |
Mouse | BRCA1 (OP92) | WB 1:250 | EMD Millipore |
Rabbit | Cyclin A (C-19) | WB 1:5000 | Santa Cruz |
Goat | RAD54 (D-18) | WB 1:1000 | Santa Cruz |
Rabbit | RAD51 (2307) | WB 1:10,000 | Home-made [100] |
Rabbit | RAD51 (2308) | IF 1:10,000 | Home-made [100] |
Mouse | GRB2 | WB 1:1000 | BD Pharmingen |
Mouse | HSP90 (AC88, 13492) | WB 1:5000 | Abcam |
Rabbit | CDC37 (3618S) | WB 1:1000 | Cell Signaling |
Goat | UBC9 (N-15) | WB 1:1000 | Santa Cruz |
Rabbit | CHIP (PA1-015) | WB 1:1000 | Thermo Scientific |
Sheep | Peroxidase anti-Mouse IgG (H+L) | WB 1:2000 | Jackson Immunoresearch |
Donkey | Peroxidase anti-Rabbit IgG (H+L) | WB 1:2000 | Jackson Immunoresearch |
Donkey | Peroxidase anti-Goat IgG (H+L) | WB 1:2000 | Jackson Immunoresearch |
Goat | Alexa-Fluor-594 (red) | IF 1:1000 | Thermo Scientific |
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van den Tempel, N.; Zelensky, A.N.; Odijk, H.; Laffeber, C.; Schmidt, C.K.; Brandsma, I.; Demmers, J.; Krawczyk, P.M.; Kanaar, R. On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation. Cancers 2019, 11, 97. https://doi.org/10.3390/cancers11010097
van den Tempel N, Zelensky AN, Odijk H, Laffeber C, Schmidt CK, Brandsma I, Demmers J, Krawczyk PM, Kanaar R. On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation. Cancers. 2019; 11(1):97. https://doi.org/10.3390/cancers11010097
Chicago/Turabian Stylevan den Tempel, Nathalie, Alex N. Zelensky, Hanny Odijk, Charlie Laffeber, Christine K. Schmidt, Inger Brandsma, Jeroen Demmers, Przemek M. Krawczyk, and Roland Kanaar. 2019. "On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation" Cancers 11, no. 1: 97. https://doi.org/10.3390/cancers11010097