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The Path to Destruction: Understanding the mechanism and regulation of proteasomal degradation
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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 [en]
Proteasome, ubiquitin, p97, Cnc-C, transcription factors, Nrf1, Nrf2
National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-63762ISBN: 978-91-7447-384-1 (print)OAI: oai:DiVA.org:su-63762DiVA: diva2:452267
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
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. 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
3. Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells
Open this publication in new window or tab >>Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells
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2010 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 38, no 1, 17-28 p.Article in journal (Refereed) Published
Abstract [en]

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here, we report that nuclear factor erythroid-derived 2-related factor 1 (Nrf1), a transcription factor of the cap ""n"" collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1(-/-) MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101, and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

National Category
Biological Sciences
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-52207 (URN)10.1016/j.molcel.2010.02.029 (DOI)000276627800004 ()
Note
authorCount :6Available from: 2011-01-14 Created: 2011-01-13 Last updated: 2017-12-11Bibliographically approved

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