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A high-throughput microscopy method to find novel p97 inhibitors
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Karolinska Institutet, Institutionen för medicinsk biokemi och biofysik.
Karolinska Institutet, Institutionen för medicinsk biokemi och biofysik.
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(English)Manuscript (preprint) (Other academic)
Keyword [en]
p97, proteasome, proteasome inhibition, high-throughput screening
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-43698OAI: oai:DiVA.org:su-43698DiVA: diva2:359100
Available from: 2010-10-26 Created: 2010-10-26 Last updated: 2011-10-28Bibliographically approved
In thesis
1. The p97 ATPase and the Drosophila Proteasome: Protein Unfolding and Regulation
Open this publication in new window or tab >>The p97 ATPase and the Drosophila Proteasome: Protein Unfolding and Regulation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2010. 69 p.
Keyword
proteasome, ubiquitin, p97, unfolding, regulation, RNAi screen, high-throughput screen, cnc, basic leucine zipper, transcription factors, proteasome inhibition
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43778 (URN)978-91-7447-167-0 (ISBN)
Public defence
2010-11-25, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
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Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.Available from: 2010-11-03 Created: 2010-10-27 Last updated: 2011-10-30Bibliographically approved

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