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Identification and characterization of a Drosophila nuclear proteasome activator: a homolog of human 11S REggamma (PA28gamma)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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.

Place, publisher, year, edition, pages
2001. Vol. 276, no 2, 1383-1390 p.
URN: urn:nbn:se:su:diva-22617DOI: 10.1074/jbc.M007379200OAI: diva2:189171
Part of urn:nbn:se:su:diva-101Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2010-06-21Bibliographically approved
In thesis
1. Investigating the activation and regulation of the proteasome: an essential proteolytic complex
Open this publication in new window or tab >>Investigating the activation and regulation of the proteasome: an essential proteolytic complex
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.
Proteasome, PA28gamma, Transcriptional regulation, Drosophila, 26S
National Category
Biochemistry and Molecular Biology
urn:nbn:se:su:diva-101 (URN)91-7265-837-1 (ISBN)
Public defence
2004-04-30, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 13:00 (English)
Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2009-12-22Bibliographically approved

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