Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation
Abstract
:1. Introduction
2. Results
2.1. p62-H2 Protein Was Present at a Slightly Higher Level than p62-H1 in Human Liver
2.2. p62-H2 Formed Larger Aggregates than p62-H1 in CHO-K1 Cells
2.3. β-Sheet-Conformation Was Predominantly Associated with p62-H2 and K8 Co-Aggregates
2.4. Overexpressed Ubiquitin Accumulated in Co-Aggregates of Keratins with Both p62 Isoforms
2.5. p62-H1 and p62-H2 did Not Co-Localize with the Endogenous Keratin Intermediate Filament Cytoskeleton of PLC/PRF/5 Cells
2.6. Overexpressed p62-H2 Predominantly Accumulates in a Diffuse Fashion in the Cytoplasm of Fibroblasts
2.7. p62-H2 Predominantly Accumulated in IHBs and Hybrid Inclusions of Human HCC
3. Discussion
4. Materials and Methods
4.1. Cloning
4.2. Quantitative Reverse Transcriptase-Polymerase Chain Reaction Analysis (qRT-PCR) of Human Liver Samples
4.3. Western Blot Analysis
4.4. Cell Culture
4.4.1. Transfection
4.4.2. Immunocytochemistry of Transfected Cells
4.4.3. Scoring of p62 Aggregate Size
4.4.4. Confocal Co-Localization and Particle Analysis
4.4.5. FRET Microscopy and Analysis
4.4.6. Triple Co-Localization of K8/18 and p62 with LCO/Ubiquitin
4.4.7. Immunostaining of Tissues
4.4.8. Statistical Analysis
5. Conclusions
- p62-H1 and p62-H2 isoforms have different properties in protein aggregation;
- p62-H2 forms larger aggregates in CHO-K1 and PLC/PRF/5 cells and is present in IHBs in human liver diseases;
- p62-H1 forms smaller aggregates in vitro and is present together with p62-H2 in MDBs in human liver disease;
- p62-H2 and p62-H1 differ in their ability to acquire cross-β-sheet conformation in protein aggregates.
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
p62-H1 | Human p62 isoform-1 |
p62-H2 | Human p62 isoform-2 |
p62ΔSH2 | SH2 region deleted p62 |
MEF | Mouse embryonic fibroblasts |
p62WT MEFs | p62 wild-type mouse embryonic fibroblasts |
p62KO MEFs | p62 total knockout mouse embryonic fibroblasts |
keratin 8 | K8 |
keratin 18 | K18 |
MDBs | Mallory–Denk bodies |
IHBs | Intracytoplasmic hyaline bodies |
PB1 | Phox and Bhem1p |
LIR | LC3 interacting region |
UBA | Ubiquitin associated |
TRIM5α | Tripartite motif-containing protein 5 alpha |
FRET | Förster resonance energy transfer |
p62 TV1 | p62 transcript variant 1 |
p62 TV123 | p62 variants 1, 2, and 3 |
RIPA | Radioimmunoprecipitation assay |
FBS | Fetal bovine serum |
LCOs | Luminescent conjugated oligothiophenes |
Appendix A
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Somlapura, M.; Gottschalk, B.; Lahiri, P.; Kufferath, I.; Pabst, D.; Rülicke, T.; Graier, W.F.; Denk, H.; Zatloukal, K. Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation. Int. J. Mol. Sci. 2021, 22, 6227. https://doi.org/10.3390/ijms22126227
Somlapura M, Gottschalk B, Lahiri P, Kufferath I, Pabst D, Rülicke T, Graier WF, Denk H, Zatloukal K. Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation. International Journal of Molecular Sciences. 2021; 22(12):6227. https://doi.org/10.3390/ijms22126227
Chicago/Turabian StyleSomlapura, Meghana, Benjamin Gottschalk, Pooja Lahiri, Iris Kufferath, Daniela Pabst, Thomas Rülicke, Wolfgang F. Graier, Helmut Denk, and Kurt Zatloukal. 2021. "Different Roles of p62 (SQSTM1) Isoforms in Keratin-Related Protein Aggregation" International Journal of Molecular Sciences 22, no. 12: 6227. https://doi.org/10.3390/ijms22126227