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Investigating the activation and regulation of the proteasome: an essential proteolytic complex
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The proteasome is a major non-lysosomal proteolytic complex present in eukaryotic cells and has a central role in regulating many protein levels. The complex has been shown to participate in various intracellular pathways including cell cycle regulation or quality control of newly synthesized proteins and many other key pathways. This amazing range of substrates would not be possible without the help of regulators that are able to bind to the 20S proteasome and modulate its activity. Among those, the PA700 or 19S regulator and the PA28 family are the best characterized in higher eukaryotes. The 19S regulatory particle is involved in the recognition of ubiquitinated proteins, targeted for degradation by the proteasome. The PA28 (also termed 11S REG) family is composed of two members the PA28αβ and PA28γ. The function of PA28αβ is related to the adaptive immune response with a proposed contribution in MHC class I peptide presentation whereas the biological role PA28γ remains unknown. The main objectives of the laboratory, and subsequently of this thesis are to use Drosophila melanogaster model system and its advantages to better understand the precise contribution of these different activators in the regulation of the proteasome. Using genomic resources, a unique Drosophila PA28 member was identified, characterized and was shown to be a proteasome regulator with all the properties of PA28γ. Through site-directed mutagenesis we identified a functional nuclear localization signal in the homolog-specific insert region. Study of the promoter region revealed that transcription of Drosophila PA28γ (dPA28γ) gene is under control of DREF, a transcription factor involved in the regulation of genes related to DNA synthesis and cell proliferation. To confirm that dPA28γ has a role in cell cycle progression, the effect of removing dPA28γ from S2 cells was tested using RNA interference. Drosophila cells depleted of dPA28γ showed partial arrest in G1/S cell cycle transition confirming a conserved function between Drosophila and mammalian forms of PA28γ. Finally, characterization of the Dictyostelium regulator, an evolutionarily distant member of the PA28γ, was carried out using fluorogenic degradation assays. We are currently knocking-out the gene in order to determine the biological function of the activator. A second part of my work consisted in the generation of a Drosophila assay used to identify in vivo substrates of the 19S regulator, an assay system that has been originally engineered by Dantuma and coworkers in human cell lines. This was achieved by cloning of GFP behind a series of modified ubiquitins that create substrates degraded through different pathways involving the proteasome pathways. The last project of my thesis concerns the characterization of the mechanism for upregulation of proteasomal gene mRNA after MG132 (proteasome inhibitor) treatment. So far, we found that the 5´-UTR of the genes is responsible for this induction. We are now looking for the precise motif involved in this regulation.

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik , 2004. , 56 p.
Keyword [en]
Proteasome, PA28gamma, Transcriptional regulation, Drosophila, 26S
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-101ISBN: 91-7265-837-1 (print)OAI: oai:DiVA.org:su-101DiVA: diva2:189176
Public defence
2004-04-30, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2009-12-22Bibliographically approved
List of papers
1. Identification and characterization of a Drosophila nuclear proteasome activator: a homolog of human 11S REggamma (PA28gamma)
Open this publication in new window or tab >>Identification and characterization of a Drosophila nuclear proteasome activator: a homolog of human 11S REggamma (PA28gamma)
2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 2, 1383-1390 p.Article in journal (Refereed) Published
Abstract [en]

We report the cloning and characterization of aDrosophila proteasome 11 S REGγ (PA28) homolog. The 28-kDa protein shows 47% identity to the human REGγ and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the DrosophilaREG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. TheDrosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REGγ proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that γ class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the α and β REG classes.

Identifiers
urn:nbn:se:su:diva-22617 (URN)10.1074/jbc.M007379200 (DOI)
Note
Part of urn:nbn:se:su:diva-101Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2017-12-13Bibliographically approved
2. Drosophila Proteasome Regulator REGγ: Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression
Open this publication in new window or tab >>Drosophila Proteasome Regulator REGγ: Transcriptional Activation by DNA Replication-related Factor DREF and Evidence for a Role in Cell Cycle Progression
2003 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 327, no 5, 1001-1012 p.Article in journal (Refereed) Published
Abstract [en]

The proteasome regulator REG (PA28γ) is a conserved complex present in metazoan nuclei and is able to stimulate the trypsin-like activity of the proteasome in a non-ATP dependent manner. However, the in vivo function for REGγ in metazoan cells is currently unknown. To understand the role of Drosophila REGγ we have attempted to identify the type of promoter elements regulating its transcription. Mapping the site of the transcription initiation revealed a TATA-less promoter, and a sequence search identified elements found typically in Drosophila genes involved in cell cycle progression and DNA replication. In order to test the relevance of the motifs, REGγ transcriptional assays were carried out with mutations in the proposed promoter. Our results indicate that a single Drosophila replication-related element sequence, DRE, is essential for REGγ transcription. To confirm that REGγ has a role in cell cycle progression, the effect of removing REGγ from S2 cells was tested using RNA interference. Drosophila cells depleted of REGγ showed partial arrests in G1/S cell cycle transition. Immuno-staining of Drosophila embryos revealed that REGγ is typically localized to the nucleus during embryogenesis with increased levels present in invaginating cells during gastrulation. The REGγ was found dispersed throughout the cell volume within mitotic domains undergoing cell division. Finally, database searches suggest that the DRE system may regulate key members of the proteasome system in Drosophila.

Keyword
REGγ; PA28γ; proteasome; DRE; DREF
Identifiers
urn:nbn:se:su:diva-22618 (URN)10.1016/S0022-2836(03)00188-8 (DOI)
Note
Part of urn:nbn:se:su:diva-101. Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2017-12-13Bibliographically approved
3. Use of RNA interference and Complementation to study the function of the Drosophila and Human 26S proteasome Subunit S13
Open this publication in new window or tab >>Use of RNA interference and Complementation to study the function of the Drosophila and Human 26S proteasome Subunit S13
2003 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 23, no 15, 5320-5330 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23824 (URN)
Note
Part of urn:nbn:se:su:diva-101. Available from: 2005-05-04 Created: 2005-05-04 Last updated: 2017-12-13Bibliographically approved
4. Identification and characterization of a Metazoan Proteasome Regulator 11S REG (PA28) in Dictyostelium Discoideum
Open this publication in new window or tab >>Identification and characterization of a Metazoan Proteasome Regulator 11S REG (PA28) in Dictyostelium Discoideum
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-22620 (URN)
Note
Part of urn:nbn:se:su:diva-101Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2010-01-13Bibliographically approved
5. Identification and characterization of a Drosophila proteasome regulatory network
Open this publication in new window or tab >>Identification and characterization of a Drosophila proteasome regulatory network
2005 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 25, no 11, 4662-4675 p.Article in journal (Refereed) Published
Abstract [en]

Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5' untranslated regions (UTRs). Transfer of the 5' UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-22621 (URN)10.1128/MCB.25.11.4662-4675.2005 (DOI)
Note
Part of urn:nbn:se:su:diva-101Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2017-12-13Bibliographically approved

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