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Studies of metazoan proteasome function and regulation
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
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 [en]
proteasome ubiquitin RNAi microarray
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-496ISBN: 91-7155-062-3 (print)OAI: oai:DiVA.org:su-496DiVA: diva2:194664
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
List of papers
1. 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, 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: 2009-12-22Bibliographically approved
2. 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: 2010-06-30Bibliographically approved
3. 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: 2010-06-21Bibliographically approved

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