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Identification and characterization of a Drosophila proteasome regulatory network
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.
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.

Place, publisher, year, edition, pages
2005. Vol. 25, no 11, 4662-4675 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-22621DOI: 10.1128/MCB.25.11.4662-4675.2005OAI: oai:DiVA.org:su-22621DiVA: diva2:189175
Note
Part of urn:nbn:se:su:diva-101Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2017-12-13Bibliographically 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.
Keyword
Proteasome, PA28gamma, Transcriptional regulation, Drosophila, 26S
National Category
Biochemistry and Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2004-04-07 Created: 2004-04-07 Last updated: 2009-12-22Bibliographically approved
2. Studies of metazoan proteasome function and regulation
Open this publication in new window or tab >>Studies of metazoan proteasome function and regulation
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological processes depend upon the structural and functional quality of the molecules that comprise living organisms. The integrity of molecules such as DNA, RNA, proteins, carbohydrates and lipids is crucial and the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Within a cell, each protein must be present at a specific concentration during certain specific conditions. To maintain cellular homeostasis and the ability to respond to the environment the proteome is in a dynamic state of synthesis and degradation. In eukaryotic cells the ubiquitin-proteasome pathway is the principal mechanism for regulated protein turnover in both the cytoplasm and the nucleus. The 20S proteasome is a cylindrical multi-subunit protease. Proteasomes play an essential role in the targeted and timely ordered degradation of key regulatory proteins and their inhibitors. The 26S proteasome is a 2.500 kDa complex composed of the 20S proteasome sandwiched between two 19S regulators. This is the enzymatic complex responsible for ATP-dependent ubiquitin mediated protein degradation. A polyubiquitin chain attached to a protein serves as a general recognition signal for destruction via the 26S proteasome. It is known that the 19S regulator confers ubiquitin recognition and substrate unfolding to the 20S proteasome, however, the specific functions for many of the different subunits within the 19S complex are not known. We have used RNA interference to study the S13/Rpn11 and S5a/Rpn10 subunits of Drosophila melanogatser proteasomes. We have produced stable cell lines with the human S13 gene under inducible promoters that was used to rescue the knockdown phenotype after RNA interference. The rescue was successful in demonstrating that the human protein is a functional homologue to the Drosophila protein. We call the technique RNAi+c (RNA interference + complementation). This procedure enabled us to also test different mutants of the human S13 protein for their ability to function in the proteasome. Using RNA interference to a Drosophila proteasome subunit in combination with complementation with a corresponding human protein we have been able to study residues important for the deubiquitinating activity of this subunit (Paper I). Interestingly, upon a decrease of either S13 or S5a we see an induction in the levels of active 20S proteasomes. Increase in the levels of the non-targeted 19S subunit can be detected when RNAi treatment is carried out on either S13 or S5a. We have used RNA interference and proteasomal inhibition together with whole genome microarray analysis to reveal a co-regulated network of proteasome genes. This network likely contributes to an overall regulatory system that maintains proper proteasome levels in the cell. Initial studies of the mechanism of transcriptional co-regulation of proteins involved in the 26S proteasome pathway were also performed (Paper II). Finally, the biological function of the proteasome regulator PA28g/REGg is not known. We have studied this regulator in Drosophila using RNA interference and promoter mapping (Paper III).

Place, publisher, year, edition, pages
Stockholm: Institutionen för molekylärbiologi och funktionsgenomik, 2005. 72 p.
Keyword
proteasome ubiquitin RNAi microarray
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-496 (URN)91-7155-062-3 (ISBN)
Public defence
2005-05-26, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 14-18, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2005-05-04 Created: 2005-05-04 Last updated: 2010-06-30Bibliographically approved

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