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The p97 ATPase and the Drosophila Proteasome: Protein Unfolding and Regulation
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Patrick Young)
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 [en]
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: urn:nbn:se:su:diva-43778ISBN: 978-91-7447-167-0 (print)OAI: oai:DiVA.org:su-43778DiVA: diva2:359378
Public defence
2010-11-25, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
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
List of papers
1. A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation
Open this publication in new window or tab >>A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation
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2009 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 394, no 4, 732-46 p.Article in journal (Refereed) Published
Abstract [en]

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97(Ufd1/Npl4) complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.

Keyword
Animals, Antineoplastic Agents/*pharmacology, Cell Survival, DNA Damage, DNA Repair, DNA Repair Enzymes, Humans, Medical Oncology/methods/trends, Models; Biological, Neoplasms/*therapy, Treatment Outcome
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-31936 (URN)10.1016/j.jmb.2009.09.050 (DOI)000272602900014 ()19782090 (PubMedID)
Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved
2. A high-throughput microscopy method to find novel p97 inhibitors
Open this publication in new window or tab >>A high-throughput microscopy method to find novel p97 inhibitors
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(English)Manuscript (preprint) (Other academic)
Keyword
p97, proteasome, proteasome inhibition, high-throughput screening
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-43698 (URN)
Available from: 2010-10-26 Created: 2010-10-26 Last updated: 2011-10-28Bibliographically approved
3. Basic Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome
Open this publication in new window or tab >>Basic Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome
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2011 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 31, no 4, 897-909 p.Article in journal (Refereed) Published
Abstract [en]

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-63770 (URN)10.1128/MCB.00799-10 (DOI)000286596600025 ()
Available from: 2011-10-28 Created: 2011-10-28 Last updated: 2017-12-08Bibliographically approved

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