Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1
Abstract
:1. Introduction
2. Materials and Methods
2.1. Prediction of Intrinsically Disordered Regions
2.2. Protein Sequence Alignment
2.3. Yeast Growth Assays and Microscopy
2.4. Cell Lines and Culture Conditions
2.5. Cell Viability Assays
2.6. Fluorescence and Immunofluorescence Microscopy
2.7. RNA Isolation and Quantitative Reverse Transcription PCR (RT-qPCR)
2.8. Protein Expression and Purification
2.9. SDS-PAGE and Coomassie Blue Gel Staining
2.10. SDD-AGE (Semi-Denaturing Detergent Agarose Gel Electrophoresis)
2.11. Combined SDD-AGE and Fractionation Assay
2.12. Statistical Analysis
3. Results
3.1. Analysis of Nrf2
3.1.1. Nrf2 Is Intrinsically Disordered and Keap1′s High Cysteine Content Is Evolutionarily Conserved
3.1.2. Oxidative Stress and Nrf2 and Keap1 Expression in Yeast
3.1.3. Nrf2 Forms Protein Inclusions under High Oxidative Stress Conditions in HeLa Cells
3.1.4. Nrf2 Protein Inclusions Are Not Artifacts, Are Preventable by Certain Antioxidants, and May Remain Functional
3.2. Analysis of Keap1
3.2.1. Keap1 Forms Protein Inclusions under High Oxidative Stress Conditions in HeLa Cells
3.2.2. Keap1 Protein Inclusions Are Not Artifacts and Are Not Preventable by Pretreatment with Certain Antioxidants
3.2.3. Keap1 Forms Oxidative Stress-Induced Protein Inclusions in Breast Cancer Cell Lines
3.3. Analysis of Nrf2 and Keap1 Purified Protein
Purified Proteins for Nrf2 and Keap1 Aggregate upon Exposure to Oxidative Stress
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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mRNA Probe | NCBI Gene Accession Number | Primer Sequences (Forward and Reverse, 5′ to 3′) |
---|---|---|
NFE2L2 | AC079305 | F: GCCCAATGTGAGAACACACC R: TGTGAGATGAGCCTCCAAGC |
HMOX1 | AY460337 | F: CCCCAACGAAAAGCACATCC R: AGACAGCTGCCACATTAGGG |
NQO1 | AH005427 | F: TGGAAGAAACGCCTGGAGAAT R: CTGGTTGTCAGTTGGGATGG |
TXN | AF548001 | F: ATTGTGACCAGCACCTACGG R: CATGGTGGAGTTGTCCCGAA |
RPLP0 | AC004263 | F: CCTCATATCCGGGGGAATGTG R: GCAGCAGCTGGCACCTTATTG |
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Ngo, V.; Karunatilleke, N.C.; Brickenden, A.; Choy, W.-Y.; Duennwald, M.L. Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1. Antioxidants 2022, 11, 243. https://doi.org/10.3390/antiox11020243
Ngo V, Karunatilleke NC, Brickenden A, Choy W-Y, Duennwald ML. Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1. Antioxidants. 2022; 11(2):243. https://doi.org/10.3390/antiox11020243
Chicago/Turabian StyleNgo, Vy, Nadun C. Karunatilleke, Anne Brickenden, Wing-Yiu Choy, and Martin L. Duennwald. 2022. "Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1" Antioxidants 11, no. 2: 243. https://doi.org/10.3390/antiox11020243
APA StyleNgo, V., Karunatilleke, N. C., Brickenden, A., Choy, W.-Y., & Duennwald, M. L. (2022). Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1. Antioxidants, 11(2), 243. https://doi.org/10.3390/antiox11020243