The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Cell Culture
2.2. Yeast Strains, Media, and Growth Conditions
2.3. Yeast Phenotype Analysis
2.4. Plasmids Constructs
2.5. Immunofluorescence and Image Analysis
2.6. Bacterial Expression and GST Pull-Down Assay
2.7. Filter Trap Assay from Human Neuroblastoma Cells
2.8. Filter Retardation Assay and Gradient Gel Analysis from Yeast Cells
2.9. RNA Extraction and cDNA Reverse Transcription
2.10. Quantitative Real-Time PCR
2.11. SDS-PAGE and Western Blot
2.12. Purification of Proteasome Complexes
2.13. Proteasome (20S CP) Activity Assay
2.14. In Vitro Protein Degradation Assay
2.15. Peptidomics Analysis
2.16. Statistical Analysis
3. Results
3.1. Blm10-Proteasomes Antagonize the Toxicity of Mutant Huntingtin Expression in Yeast
3.2. Endogenous PA200 Colocalizes with Wild Type (wt) and Mutant N-Htt in Human SH SY5Y Cells
3.3. PA200 Interacts with wt and Mutant N-Htt In Vitro
3.4. Loss of Mammalian PA200 Results in Increased Toxic N-Htt Aggregate Size and Number in Human Neuroblastoma Cells
3.5. Blm10/PA200 Contribute to the Degradation of wt and Mutant N-Htt In Vitro
3.6. Blm10 Enhances the Ability of the Proteasome to Cut within Expanded polyQ Repeats
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
HD | Huntington’s disease |
CP | Core particle |
N-Htt | N-terminal huntingtin fragments |
Blm10 | Bleomycin resistance protein BLM10 |
PA200 | Proteasome activator 200 |
GST | Glutathione S-transferase |
IB | Inclusion body |
GFP | Green fluorescent protein |
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Strains | Genotypes | Ref. |
---|---|---|
BY4742 | MATα his3∆1 leu2∆0 lys2∆0 ura3∆0 | [36] |
yMS122 | MATa his3 Δ1 leu2Δ0 met15Δ0 ura3Δ0 PRE1tevProA (HIS3) (KanMX)GAL1pHA3BLM10 | [27] |
yMS131 | MATα his3∆1 leu2∆0 met15∆0 ura3∆0 blm10∆::NatMX | [39] |
yMS268 | MATα his3∆1 leu2∆0 met15∆0 lys2∆0 ura3∆0 | [39] |
yMS285 | MATα his3∆1 leu2∆0 ura3∆0 rpn4∆::KanMX | [31] |
yMS476 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 PRE1TEVProA(HIS3) | [29] |
yMS1371 | MATa his3∆1 leu2∆0 met15∆0 ura3∆0+ pYES2-Htt25Q-CFP | [40] |
yMS1372 | MATa his3 ∆1 leu2Δ0 lys2Δ0 ura3Δ0 rpn4Δ::HphMX+ pYES2-Htt25Q-CFP | This study |
yMS1377 | MATa his3∆1 leu2∆0 met15∆0 ura3∆0+ pYES2-Htt103Q-CFP | [40] |
yMS1378 | MATa his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 rpn4Δ::HphMX+ pYES2-Htt103Q-CFP | This study |
yMS1392 | MATa MATα his3∆1 leu2∆0 lys2∆0 ura3∆0 blm10∆::NatMX+ pYES2-Htt25Q-CFP | This study |
yMS1393 | MATa MATα his3∆1 leu2∆0 lys2∆0 ura3∆0 blm10∆::NatMX+ pYES2-Htt103Q-CFP | This study |
Primary Antibody (Source) | Catalog No. | Host | Dilution | Method |
Anti-HA-Tag (Cell Signaling Technology, Danvers, MA, USA) | 3724S | Rabbit | 1:1000 | Immunoblot |
Immunofluorescence | ||||
Anti-HA-Tag (Cell Signaling Technology, Danvers, MA, USA) | 2367 | Mouse | 1:1000 | Immunoblot |
1:500 | Immunofluorescence | |||
PSME4/PA200 (Novus Biologicals, Littleton, CO, USA) | NBP2-22236 | Rabbit | 1:2000 | Immunoblot |
1:1000 | Immunofluorescence | |||
Anti-GFP (Clontech, Mountain View, CA, USA) | 632592 | Rabbit | 1:1000 | Immunoblot |
Anti-GST Tag (Upstate Biotechnology, Lake Placid, NY, USA) | 05-311 | Mouse | 1:1000 | Immunoblot |
β1 subunit of Proteasome 20S (human) (Enzo Biochem, Farmingdale, NY, USA). | BML-PW8140-0100 | Mouse | 1:1000 | Immunoblot |
Proteasome 20S (Yeast) core subunits (Enzo Biochem, Farmingdale, NY, USA) | BML-PW9355-0100 | Rabbite | 1:1000 | Immunoblot |
Actin (Santa Cruz Biotechnology, Dallas, TX, USA) | Sc-1616 | Goat | 1:10,000 | Immunoblot |
Secondary Antibody (Source) | Catalog No. | Host | Dilution | Method |
Alexa fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA) | A11005 | Mouse | 1:1000 | Immunofluorescence |
A31631 | Rabbit | |||
Alexa fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA) | A11001 | Mouse | 1:1000 | Immunofluorescence |
A11034 | Rabbit | |||
IRDye 800CW (LI-COR, Lincoln, NE, USA) | 926-32211 | Rabbit | 1:10,000 | Immunoblot |
IRDye 680RD (LI-COR, Lincoln, NE, USA) | 926-68072 | Mouse | 1:5000 | Immunoblot |
Anti-Mouse IgG Antibody, HRP conjugate | A9044 | Rabbit | 1:3000 | Immunoblot |
Anti-Goat IgG Antibody, HRP conjugate | A8919 | Rabbit | 1:3000 | Immunoblot |
Anti-Rabbit IgG Antibody, HRP conjugate | A0545 | Goat | 1:3000 | Immunoblot |
Gene | Forward Primer | Reverse Primer | |
---|---|---|---|
PMSE4(PA200) | Human | 5′-ATGGAGAGTGCCTGAACTATTG-3′ | 5′-GTAGGTCAGCACACTTCCTATTC-3′ |
GAPDH | Human | 5′-GAGTCAACGGATTTGGTCGT-3′ | 5′-GATCTCGCTCCTGGAAGATG-3′ |
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Aladdin, A.; Yao, Y.; Yang, C.; Kahlert, G.; Ghani, M.; Király, N.; Boratkó, A.; Uray, K.; Dittmar, G.; Tar, K. The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin. Biomolecules 2020, 10, 1581. https://doi.org/10.3390/biom10111581
Aladdin A, Yao Y, Yang C, Kahlert G, Ghani M, Király N, Boratkó A, Uray K, Dittmar G, Tar K. The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin. Biomolecules. 2020; 10(11):1581. https://doi.org/10.3390/biom10111581
Chicago/Turabian StyleAladdin, Azzam, Yanhua Yao, Ciyu Yang, Günther Kahlert, Marvi Ghani, Nikolett Király, Anita Boratkó, Karen Uray, Gunnar Dittmar, and Krisztina Tar. 2020. "The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin" Biomolecules 10, no. 11: 1581. https://doi.org/10.3390/biom10111581