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Basic Leucine Zipper Protein Cnc-C is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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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.

Place, publisher, year, edition, pages
2011. Vol. 31, no 4, 897-909 p.
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-63770DOI: 10.1128/MCB.00799-10ISI: 000286596600025OAI: oai:DiVA.org:su-63770DiVA: diva2:452364
Available from: 2011-10-28 Created: 2011-10-28 Last updated: 2017-12-08Bibliographically 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)
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
2. The Path to Destruction: Understanding the mechanism and regulation of proteasomal degradation
Open this publication in new window or tab >>The Path to Destruction: Understanding the mechanism and regulation of proteasomal degradation
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A majority of intracellular proteins are degraded by the ubiquitin proteasome system (UPS). In this thesis, both the mechanism of the degradation and the regulation of UPS have been investigated. The importance of the p97 ATPase for proteasomal degradation of cytosolic substrates was examined. It was shown that tightly folded model substrates were dependent on p97 for their degradation. In addition to this, it was shown that an extended flexible peptide sequence on the substrate allowed degradation to occur directly by the proteasome. We propose that p97 works as an unfoldase on substrates that lack initiation regions. These results were originally achieved with experiments using Drosophila melanogaster S2 cell culture. Corresponding experiments were carried out in human cell lines. We observed that the human proteasome also needed assistance from the human p97 protein complex when model substrates lacked unfolded tagged regions. To identify the transcription factor(s) that regulate the expression of proteasomal genes, a large scale RNAi screen was performed. A library consisting of dsRNA to all known and predicted transcription factors in Drosophila was used. Drosophila S2 cells expressing the cytosolic UbG76V-GFP substrate were used in the screen. Since thisfusion protein isdependent on the UPS for its degradation,failure in UPS can easily be detected viafluorescent stabilization.When dsRNA targeted the bZIP transcription factor Cnc-C,it lead to a reduction of the proteasome subunit protein levels as well as decreased mRNA levels. Phylogenetic analysis together with sequence alignments were used to learn how Cnc-C is related to the bZIP CNC genes in other metazoans and in particular mammalian cells. In mammalian cells, NF-E2, Nrf1, Nrf2 and Nrf3 are present and we propose that Cnc-C is related to a common ancestor transcription factor for all these four genes. This contradicts earlier studies proposing that Cnc-C is a homolog of the mammalian Nrf2 protein.In the last study, theproteasome recovery pathway was examined tounderstand which bZIPtranscription factor in human cells is responsible for the expression of proteasome genes after proteasome inhibition.Different cancer cell lines were used to examine theexpression level of proteasome genes after treating the cells with proteasome inhibitors when either the bZIP protein Nrf1 or Nrf2 wereknocked down. It was shown that Nrf1-/- cellslacked the ability toupregulate proteasome genes after proteasomeinhibition. In contrast, Nrf2-/- cells still had the capacity to restore proteasome levels. This lead to the conclusion thatNrf1 is responsible for the proteasome recovery pathway in mammalian cells.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2011. 65 p.
Keyword
Proteasome, ubiquitin, p97, Cnc-C, transcription factors, Nrf1, Nrf2
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-63762 (URN)978-91-7447-384-1 (ISBN)
Public defence
2011-12-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2011-11-10 Created: 2011-10-28 Last updated: 2013-12-06Bibliographically approved

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