03 Fakultät Chemie
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Item Open Access Regulation of the catalytic activity and specificity of DNA nucleotide methyltransferase 1(2014) Bashtrykov, Pavel; Jeltsch, Albert (Prof. Dr.)DNA nucleotide methyltransferase 1 (Dnmt1) is mainly responsible for the maintenance of DNA methylation in mammals and plays a crucial role in the epigenetic control of gene expression. Dnmt1 recognizes and methylates hemimethylated CpG sites formed during DNA replication. In the present work, the mechanistic details of the substrate recognition by the catalytic domain of Dnmt1, the possible role of the CXXC and RFTS domains of Dnmt1 in the regulation of specificity and activity of Dnmt1, and the influence of the Ubiquitin-like PHD and RING finger domain-containing 1 (Uhrf1) protein on the enzymatic properties of Dnmt1 was investigated. Using modified substrates, the functional roles of individual contacts of the Dnmt1 catalytic domain with the CpG site of the DNA substrate were analysed. The data show that the interaction with the 5-methylcytosine:guanine pair is required for the catalytic activity of Dnmt1, whereas the contacts to the non-target strand guanine are not important, since its replacement with adenine increased the activity of Dnmt1. It was proposed that the CXXC domain binding to unmethylated CpG sites increases the specificity of Dnmt1 for hemimethylated DNA. Our data showed that the CXXC domain does not influence the enzyme’s specificity in the full-length Dnmt1. In contrast, mutagenesis in the catalytic domain introducing an M1235S exchange resulted in a significant reduction in specificity. Therefore, the readout for the hemimethylated DNA occurs within its catalytic domain. It was observed in a crystal structure that the RFTS domain of Dnmt1 inhibits the activity of the enzyme by binding to the catalytic domain and blocking the entry of the DNA. By amino acid substitution in the RFTS domain its positioning within the catalytic domain was destabilized and a corresponding increase in the catalytic rate was observed, which supports this concept and suggests a possible mechanism to allosterically regulate the activity of Dnmt1 in cells. Uhrf1 has been shown to target Dnmt1 to replicated DNA, which is essential for DNA methylation. Here it is demonstrated that Uhrf1 as well as its isolated SRA domain increase the activity and specificity of Dnmt1 in an allosteric mechanism. The stimulatory effect was independent of the SRA domain’s ability to bind hemimethylated DNA. The RFTS domain of Dnmt1 is required for the stimulation, since its deletion or blocking of its interaction with the SRA domain, significantly reduced the ability of Uhrf1 to increase the activity and specificity of Dnmt1. Uhrf1, therefore, plays multiple roles that support DNA methylation including targeting of Dnmt1, its stimulation and an increase of its specificity.Item Open Access Biochemical characterisation of tRNA-Asp methyltransferase Dnmt2 and its physiological significance(2014) Shanmugam, Raghuvaran; Jeltsch, Albert (Prof. Dr.)Methylation of tRNA plays important roles in the stabilisation of tRNAs and accurate protein synthesis in cells. In eukaryotes various tRNA methyltransferases exist, among them DNMT2 which methylates tRNAAsp at position C38 in the anticodon loop. It is also called tRNA-aspartate methyltransferase 1 (Trdmt1) and the enzyme is highly conserved among eukaryotes. In this work, I investigated the mechanism of DNMT2 interaction with tRNAAsp, characterised the function of the only prokaryotic Dnmt2 homolog found in G. sulfurreducens and studied the physiological importance of the C38 methylation of tRNAAsp in mammalian cells. The molecular details of the interaction of DNMT2 and tRNAAsp are unknown due to lack of the co-crystal structure. Here, I characterised the important residues in DNMT2 required for the tRNA binding and catalysis. By site-directed mutagenesis of 20 conserved lysine and arginine residues in DNMT2, I show that 8 of them have a strong effect on the catalytic activity of the enzyme. They map to one side of the enzyme where the catalytic pocket of DNMT2 is located. The binding of most of the mutant enzymes to tRNA was unaffected suggesting a role of these residues in transition state stabilisation. Manual docking of tRNAAsp into the surface cleft decorated by the 8 residues suggested that DNMT2 interacts mainly with the anticodon stem/loop of tRNAAsp. In my second project, I characterised the function of Dnmt2 homolog found in G. sulfurreducens (GsDnmt2). Here, I show that GsDnmt2 methylates tRNAGlu more efficiently than tRNAAsp. I also report the molecular basis for the swapped substrate specificity of GsDnmt2 and show that the variable loops of G.sulfurreducens tRNAAsp and tRNAGlu of eukaryotes contain a -GG- dinucleotide which is not preferred by Dnmt2. Exchange of the variable loop of mouse tRNAAsp to that tRNAGlu led to dramatic decrease in the activity of human DNMT2. This identifies the variable loop of tRNA as a specificity determinant in the recognition by Dnmt2. In my final project, I investigated the physiological importance of the tRNAAsp C38 methylation in aminoacylation and cellular protein synthesis. Here, I report that C38 methylation enhances the rate of aspartylation on tRNAAsp by 4-5 folds. Concomitant with this, a decrease in the charging levels of tRNAAsp was observed in Dnmt2 knockout MEF cells, which also showed a reduced efficiency in the synthesis of proteins containing poly-Asp sequences. A gene ontology searches for proteins with poly-Asp sequences showed that a significant number of these proteins are associated with transcriptional regulation and gene expression functions. With this I propose that the mild phenotype observed with the Dnmt2 KO cells under stress condition could be correlated to a disregulation of protein synthesis.Item Open Access Cytosolic protein quality control of the orphan protein Fas2, a novel physiological substrate of the E3 ligase Ubr1(2013) Scazzari, Mario; Wolf, Dieter H. (Prof. Dr.)Cellular protein quality control (PQC) monitors the proper folding of polypeptides, assembly of protein subunits into protein complexes as well as the delivery of terminally misfolded proteins to degradation. The components of PQC known best at the moment are molecular chaperones and the ubiquitin proteasome system. In contrast to the well-described protein quality control system of the endoplasmic reticulum (ERAD), less is known about how misfolded proteins in the cytosol are recognized and degraded. The cytosolic fatty acid synthase complex (FAS) of Saccharomyces cerevisiae, which is composed of six Fas1- and six Fas2-subunits, is rather stable to proteolysis in vivo. In the absence of the Fas1 subunit (FAS1 deletion strain) the remaining Fas2 subunit becomes an orphan protein which is proteolytically unstable and is targeted to the 26S proteasome for degradation (Egner et al, 1993). In my work, I used the orphan Fas2 protein as object of investigation in order to identify new cellular components that are involved in the recognition and degradation of a natural unassembled protein subunit in S. cerevisiae. In addition, it was elucidated how these newly identified factors act in the quality control process of a naturally occurring orphan protein. Due to previous reports (Heck et al, 2010; Prasad et al, 2010) showing that some cytosolic misfolded proteins are imported into the nucleus for proteasomal degradation the cellular localization of orphan Fas2 was determined. Using laser-scanning microscopy it could be shown that C-terminally EGFP-tagged (enhanced green fluorescent protein) orphan Fas2 is localized to the cytosol, thus representing a potential substrate for the cytosolic quality control system (CytoQC). Furthermore, glycerol step density gradient centrifugation experiments revealed that the majority of the orphan Fas2 proteins are organized in high molecular assembly intermediates, which consist mostly of Fas2 homohexamers. By using the thermosensitive ssa1-45 mutant carrying in addition the gene deletions of SSA2, SSA3 and SSA4, it could be shown that the proteasomal degradation of the orphan protein is dependent on the Hsp70 chaperone Ssa1. It is likely that Ssa1 is required for keeping orphan Fas2 soluble. All members of the Hsp90-, Hsp100-, and Hsp110-chaperone family as well as the small heat shock proteins Hsp26 and Hsp42 were shown to have no effect on the degradation of orphan Fas2. Selected members of the Hsp40 chaperone family, including Apj1, Xdj1 and even Ydj1 also did not show a significant influence on the Ssa1-dependent elimination of the substrate. To prove whether other components of the UPS than the proteasome are required for degradation of orphan Fas2 different E2- and E3 gene deletion mutants were analyzed. It was found that the elimination of orphan Fas2 is strongly delayed in a strain carrying a UBC2 UBC4 double deletion. As single deletions of UBC2 and UBC4 have no significant effect on the turnover of the substrate, it can be assumed that these E2 enzymes have complementing functions in the degradation process of orphan Fas2. In a search for the responsible E3 ubiquitin ligase(s) required for orphan Fas2 degradation the E3 RING ligase Ubr1 was identified. Deletion of UBR1 leads to a strongly delayed degradation of orphan Fas2. The expression of an Ubr1 RING mutant (C1220S) or of an Ubr1 type-1 N-end rule mutant (D176) from a high-copy plasmid in the UBR1 deletion strain cannot complement the strongly delayed degradation of orphan Fas2. In contrast, the stabilization of the orphan protein in the UBR1 deletion strain is reversed, when the same strain harbours a high-copy plasmid expressing wild type Ubr1 or an Ubr1 type-2 N-end rule mutant (P406S) or even a cytosolically-located version of the nuclear E3 RING ligase San1 due to deletion of the nuclear localization sequence. Interaction studies revealed that the E3 RING ligase Ubr1 is physically associated with orphan Fas2. In addition, it was found that Ubr1 mutants harbouring either a defect RING domain or a defect in one of the N-end rule substrate-binding sites (type-1 or type-2) were still able to physically interact with orphan Fas2. Further studies showed that the physical association of orphan Fas2 and Ubr1 remains stable in the conditional ssa1-45 mutant carrying in addition deletions of SSA2 to SSA4. This indicates that already E3-bound orphan Fas2 may not require a functional peptide-binding domain of Ssa1 to maintain the physical contact to Ubr1. Finally, the AAA-ATPase Cdc48 was identified to be necessary for the elimination process of orphan Fas2. Cdc48 may function in the dissociation process of orphan Fas2 assembly intermediates, which mainly consist of Fas2-homohexamers.Item Open Access Proteinqualitätskontrolle im endoplasmatischen Retikulum : Identifizierung und Charakterisierung von Komponenten des Abbaus missgefalteter sekretorischer Proteine(2003) Hitt, Reiner; Wolf, Dieter H. (Prof. Dr.)Das endoplasmatische Retikulum (ER), der Faltungsort von Proteinen des sekretorischen Systems, besitzt ein komplexes Netzwerk faltungsunterstützender und kontrollierender Proteine. Sekretorische Proteine, die ihre endgültige räumliche Konformation jedoch nicht erlangen, werden dem Ubiquitin-Proteasom-System zum Abbau zugeführt. Dieser Prozess wird im Allgemeinen mit ER-Degradation oder ER-assoziierte Degradation (ERAD) bezeichnet. In der vorliegenden Arbeit wurden drei zur endoplasmatischen Proteinqualitätskontrolle gehörende Themengebiete bearbeitet: Als erstes wurde die Klonierung des bisher unbekannten DER7 Gens mit Hilfe der isolierten ERAD Mutante der7-1 durchgeführt. Danach erfolgte eine weitergehende Charakterisierung des Der1 Proteins und der Vergleich mit den Der1-Homologen Ydr411c (S. cerevisiae) und R151.6 (C. elegans). Zuletzt wurde der Einfluss einiger zytosolischer Chaperone auf den Abbau des ERAD Modellsubstrats CPY* untersucht. Die bisher nicht charakterisierte ERAD Mutante der7-1 bewirkt einen verlangsamten Abbau der ERAD Substratproteine CPY* und PrA*. Zusätzlich ist die Beweglichkeit dieser Proteine im elektrischen Feld verringert. Es konnte gezeigt werden, dass dies auf eine defekte Prozessierung der N-Glykoside im endoplasmatischen Retikulum zurückzuführen ist. Durch Kreuzen des der7-1 Allels mit Nullmutanten von Glukosidase I und II, durch Komplementation mit plasmidkodierter Glukosidase I und durch meiotische Kartierung wurde gefunden, dass DER7 mit CWH41 identisch ist. CWH41 kodiert für das Enzym Glukosidase I, welches bei der Prozessierung der Oligosaccharide im endoplasmatischen Retikulum einen terminalen alpha1,2-Glukoserest entfernt. Korrekt getrimmte Oligosaccharide scheinen daher für eine effiziente Proteindegradation notwendig zu sein. Das bestätigt sich durch den, für Doppelmutanten des ERAD und der „unfolded protein response“ (UPR) typischen, temperaturabhängigen Wachstumsdefekt von der7-1 deltaire1 Mutanten. Das Protein Der1 wurde schon in früheren Arbeiten als eine Komponente der ERAD-Maschinerie identifiziert. Bisher wurde jedoch eine Beteiligung nur am Abbau einiger löslicher missgefalteter Proteine nachgewiesen. Durch den Vergleich des Der1 abhängigen Abbaus von löslicher CPY* und membranständigem CTG* (CPY*-Transmembrandomäne-GFP) konnte nun gezeigt werden, dass die Funktion von Der1 unabhängig von der Missfaltung ist und sich wahrscheinlich auf die Degradation löslicher Proteine beschränkt. Eine topologische Charakterisierung von Der1 zeigte, dass der N- und der C-Terminus im Zytosol lokalisiert sind und Der1 insgesamt vier Transmembrandomänen besitzt. Es wurde außerdem gefunden, dass Der1 sehr empfindlich auf Veränderungen am Protein reagiert. Einzig kleinere Modifikationen des C-Terminus, wie das Einfügen eines einzelnen HA-Epitops oder einer N-Glykosylierungs-Konsensussequenz, erhalten die Funktionalität des Proteins. Um potentielle Interaktionspartner von Der1 zu finden, wurde nach High-Copy-Suppressoren der Temperatursensitivität von der1-2 deltaire1 Doppelmutanten gesucht. Es wurden 19 Genbankplasmide erhalten, die im Genbankinsert weder DER1 noch IRE1 enthielten. Ihre Wirkungsweise ist jedoch möglicherweise indirekt, da der Abbau von CPY* durch ihre Expression in der1-2 Mutanten nicht wiederhergestellt wurde. Die fortschreitende Genomsequenzierung ermöglichte inzwischen die Identifizierung einer „Der1-ähnlichen“ Familie mit mehr als 15 Vertretern aus den unterschiedlichsten Organismen. Zwei dieser homologen Proteine sind Ydr411c aus S. cerevisiae und R151.6 aus C. elegans. Das Der1 homologe Protein Ydr411c wurde wegen seiner Zugehörigkeit zur „Der1-ähnlichen“ Familie Dfm1 (Der1-like family member) benannt. Ebenso wie Der1, besitzt Dfm1 einen zytosolischen C-Terminus und ist im endoplasmatischen Retikulum lokalisiert. Im Gegensatz zur deltader1 deltaire1 Doppelmutation, führt die deltadfm1 deltaire1 Doppeldeletion jedoch nicht zu temperatursensitiven Hefestämmen. Des Weiteren konnte nur ein äußerst schwacher Einfluss von Dfm1 auf den Abbau des ERAD Substrats CPY* festgestellt werden. Das C. elegans Protein R151.6 hingegen, scheint dieselbe Funktion wie Der1 der Hefe auszuüben. Seine heterologe Expression in deltader1 deltaire1 Doppelmutanten hob die konditionale Letalität dieses Stamms auf. Das deutet darauf hin, dass die Funktion von Der1 auch in höheren Organismen konserviert ist. Als weiteres stellte sich die Frage ob die zytosolischen Chaperone der Hsp90 und Hsp100/Clp am Abbau löslicher ERAD Substrate beteiligt sind. Die in dieser Arbeit getesteten deltahsc82, deltahsp82, deltasti1, deltasba1 und deltahsp104 Nullmutanten zeigten jedoch keinen Einfluss auf die Abbaugeschwindigkeit von CPY*. Ob zytosolische Chaperone generell nur beim Abbau von Membransubstraten eine Rolle spielen, müssen weitere Untersuchungen zeigen.Item Open Access Endoplasmic reticulum associated protein degradation (ERAD): the function of Dfm1 and other novel components of the pathway(2011) Stolz, Alexandra; Wolf, Dieter H. (Prof. Dr.)Proteins, featured with a multitude of enzymatic activities as well as structural and other physiological functions are the main operators in the cell. Proteins are synthesized in the cytosol by ribosomes, which use m-RNA as a template to translate DNA based structural information into an amino acid sequence. During translation many errors occur resulting in so-called defective ribosomal products. In addition, stresses as heat, heavy metal ions and oxygen lead to the formation of partially unfolded and misfolded proteins. In human accumulation of these proteins results in severe diseases as are Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and many others. Therefore quality control systems exist, which recognize unfolded or misfolded proteins and support their folding process. If a protein is unable to reach its native conformation or to refold, the quality control system marks it as terminally misfolded and hands it over to the degradation machinery of the cell. In case of proteins of the secretory pathway this process is called endoplasmic reticulum quality control and associated protein degradation (ERQD). ERQD includes the recognition of the misfolded protein species, the trimming of glycan trees to signal misfolding, retrograde transport out of the ER lumen into the cytosol, ubiquitylation of the misfolded protein and degradation by the proteasome. The following thesis was engaged in the identification of new components of ERQD and tried to get insights into some mechanistic functions of the involved proteins. The proteins Dfm1, Mnl2 and Ubr1 were found as new components of the endoplasmic reticulum associated protein degradation (ERAD) machinery. Mnl2 was identified as a putative α-1,2-mannosidase. It was shown to be involved in the degradation of the misfolded glycoprotein CPY*. Most probably Mnl2 trims down the glycan trees of ERAD substrates, which are subsequently recognized by the lectin Yos9. Yos9 accelerates the degradation of terminally misfolded glycoproteins which expose these glycan structures. However, Yos9 does not seem to act only on glycosylated proteins but also seems to affect the degradation kinetics of unglycosylated ERAD substrates. In contrast to misfolded glycoproteins Yos9 delays degradation in case of the unglycosylated ERAD substrate CPY*0000. Most likely Yos9 has a chaperone like function in addition to its lectin function and provides more time for refolding of the misfolded protein. This function is, however, independent of its MRH domain that recognizes glycans. The other new ERAD component, the polytopic ER membrane localized Dfm1 protein, was found to form distinct complexes with the ligases Hrd1/Der3 and Doa10 as well as with the AAA type ATPase Cdc48. Degradation of different ERAD substrates containing a transmembrane domain was tested for Dfm1 involvement. The degradation and ubiquitylation of the ERAD-C substrate Ste6* was shown to depend on Dfm1. In addition, Dfm1 seems to be involved in a new degradation pathway, which acts independently of the ubiquitin ligases Hrd1/Der3 and Doa10. In the absence of these canonical ER ligases the cytosolic ubiquitin ligase Ubr1 seems to be recruited to maintain degradation of at least some ERAD substrates by the proteasome. Extraction of the misfolded protein species no longer depends on Cdc48 in all cases, but the driving force of other machines, most probably chaperones of the Ssa family of Hsp70 chaperones, were found to be sufficient to keep extraction and degradation of the substrates going.Item Open Access The unfolded protein response in fission yeast : Ire1 modulates stability of select mRNAs to maintain protein homeostasis(2013) Kimmig, Philipp; Wolf, Dieter (Prof. Dr.)Virtually all proteins that eukaryotic cells display on their surface or that are secreted into the extracellular spare are first folded and assembled in the membrane-surrounded organelle endoplasmic reticulum (ER). Only properly folded and assembled proteins depart from the ER to the cell surface. If the ER does not have enough capacity to fold, a condition termed “ER stress”, a signal pathway called the “Unfolded Protein Response” (UPR), is switched on to increase the protein folding capacity, to expand the surface area and volume of the compartment. All eukaryotic cells, from unicellular yeasts to mammalian cells, contain a highly conserved protein-folding sensor Ire1. In all species analyzed to date, Ire1, an ER membrane-resident kinase/endoribonuclease, is known to activate the UPR through an unconventional messenger RNA (mRNA) splicing mechanism to induce translation of a potent transcription factor Hac1 (in yeast) or XBP1 (in metazoans). This unique splicing event provides the switch that drives a comprehensive gene expression program in which the production of ER components is increased to boost the protein folding capacity of the compartment. In this thesis an organism is identified, the yeast Schizosaccharomyces pombe, in which the UPR does not involve mRNA splicing or the initiation of a gene expression program to increase the folding capacity of the ER. Rather Schizosaccharomyces pombe lacking, a Hac1/XBP1 ortholog, utilizes Ire1 RNase activity to an entirely different end. We found that activation of Ire1 in S. pombe leads to the decay of a specific class of mRNAs that all encode proteins entering the ER. Interestingly, the set of down-regulated mRNA targets are particularly enriched for those encoding proteins involved in sterol metabolism, suggesting a potential qualitative change of the physiology of the cell. The deletion of the cytosolic mRNA degradation pathway shows an accumulation of RNA cleavage fragments of the down-regulated mRNA targets upon ER stress, an event by means that the Ire1 endonuclease directly cleaves these mRNAs. Intriguingly, the down-regulated mRNAs contain a short three-nucleotide base UG/C consensus at the Ire1 cut sites where cleavage occurs after G. Thus, rather than increasing the protein folding capacity of the ER when faced with an increased protein folding load, S. pombe cells correct the imbalance by decreasing the load via mRNA cleavage. Besides decreasing the ER load, a single mRNA—the mRNA that encodes the molecular chaperone BiP, which is one of the major protein-folding components in the ER—uniquely escapes this decay. Rather then being degraded, Ire1 truncates Bip1 mRNA in its 3’ UTR, which—counter-intuitively stabilizes the non-polyadenylated 5’ fragment and results in increased Bip1 translation. Decreasing the protein load by selective mRNA degradation and the single up-regulation of the major chaperone in the ER illustrate how a universally conserved machinery has been invented to maintain ER homeostasis in fission yeast.Item Open Access The active subunits of the 20S Proteasome in Saccharomyces cerevisiae : mutational analysis of their specificities and a C-terminal extention(2008) Estiveira, Rui José Cabrita; Heinemeyer, Wolfgang (PD Dr.)The proteasome is a large multi-subunit complex ubiquitous in eukaryotes and archaebacteria. It contains proteolytic subunits that function simultaneously to digest protein substrates into oligopeptides. In eukaryotic cells, it is involved in the removal of abnormal, misfolded or incorrectly assembled proteins, but additionally it has regulatory functions. For example it is responsible for the degradation of cyclins in cell-cycle control and for the destruction of transcription factors or metabolic enzymes in metabolic adaptation. Finally, the proteasome is also involved in MHC (major histocompatibility complex) class I mediated cellular immune response. These cellular functions are linked to an ubiquitin- and ATPrequiring protein degradation pathway involving the 26S proteasome whose proteolytic core is formed by the 20S proteasome. The 20S proteasome has a cylindrical shape and is composed of four rings, each formed by seven α- or seven β-subunits and stacked in the order αββα. In eukaryotic cells, the 20S proteasome is composed of two copies of 14 different subunits, 7 distinct α-type and 7 distinct β-type subunits. Only three of the β-type subunits are proteolytically active and have N-terminal threonine residues acting as nucleophiles. They differ in their major specificities: β5/Pre2, β2/Pup1 and β1/Pre3 are classified as having "chymotrypsin-like", "trypsin-like" and "peptidylglutamyl peptide hydrolysing" (PGPH or caspase-like) activities, respectively. This classification is based on the preferred amino acid residues found at the site of hydrolysis in peptide or protein substrates. These three active β-type subunits have a fixed location in the proteasome, with the two β5/Pre2 copies separated from the clustered β2/Pup1 and β1/Pre3 subunits. In yeast a hierarchy of individual subunit activities for proteasomal function was established: β5/Pre2>> β2/Pup1 > β1/Pre3. Part of this work aimed at clarifying whether this hierarchy is solely dependent on the specificities or whether topological conditions lead to the dominance of the β5/Pre2 activity over the others, which could involve inter-subunit communication mediated by interjacent inactive β-subunits. Stepwise site-directed mutagenesis of key residues forming the substrate binding pockets was used to swap specificities between the yeast β5/Pre2 and β1/Pre3 active sites. Consequences of these mutations were then analysed in regard to maturation of the modified subunits, their specificities towards peptide substrates diagnostic for chymotrypsin-like and PGPH activity and changes in their rank in the hierarchy of functional importance. By mutating the key residue methionine 45 into an arginine, the β5/Pre2 was able to mature and showed some PGPH activity. Combinations with mutant strains, having the other active subunits inactivated, revealed that Pre2 lost its functional dominance. When other key residues were additionally replaced by those present in β1/Pre3 (A20T, V31T, I35T), instead of an further increase in PGPH activity, the β5/Pre2 subunit showed an overall decrease in activity. An unexpected exception was the pre2-M45R-I35T mutant with a strong increase in chymotrypsin-like activity. Maturation of the Pre2 subunit occurred normally like in wild-type in all combinations of mutations tested. When the residue arginine 45 was mutated into a methionine in Pre3, this subunit lost any detectable peptidase activity. Attempts to stabilise the methionine 45 by introducing strategic point mutations at residue 52 were unsuccessful. Additional alterations in the substrate binding site of β1/Pre3 (T20A, T31V, T35I) completely abolished the autolytic maturation and thus any gain of activity. Strains lacking both the Pre3 propeptide and Nα-acetyltransferase were used to confirm that the mutations result in activity loss, even when the autolytic removal of the propeptide was not required. In a second project, the role of the long C-terminal extension of the yeast β2/Pup1 subunit was examined. This 37 amino acid structure embraces the β-ring neighbor subunit β3/Pup3 and reaches the next subunit β4/Pre1. It also contacts β7/Pre6 of the opposite β-ring. Mutations of residues that could loosen contact to the surface of β3/Pup3 (Y204A, R208G and T211A) where without effect. Complete deletion of this extension or truncation of 25 residues was lethal and deletion of the last 20 amino acids caused a strong cell growth defect. When replacing the last 20 amino acids of this C-terminal extension by a FLAG tag, the growth phenotype was lost. The lethal mutations were over-expressed in wild type strains, but the mutated β2 subunits did not incorporate into proteasomes. This indicates that removal of the distal half from the β2/Pup1 C-terminal extension impedes the integration of this subunit during early assembly stages of the 20S proteasome.Item Open Access Characterization of novel proteins involved in catabolite degradation of fructose-1,6-bisphosphatase in saccharomyces cerevisiae(2006) Pfirrmann, Thorsten; Wolf, Dieter (Prof. Dr.)Glycolysis and gluconeogenesis are reciprocally controlled central metabolic pathways in cells. Catabolite degradation of fructose-1,6-bisphosphatase (FBPase) is a key regulatory step, when Saccharomyces cerevisiae cells switch from anabolic gluconeogenesis to catabolic glycolysis. Addition of glucose to cells growing on a non-fermentable carbon source causes FBPase phosphorylation resulting in a decrease of enzymatic activity. This is followed by a proteolytic breakdown of the enzyme via the ubiquitin-proteasome system with a half-life of 20-30 min. In a genome wide screen nine so called gid mutants (glucose induced degradation deficient) defective in proteasome-dependent catabolite degradation of FBPase were identified. Analysis of Gid2 revealed that this protein is a part of a soluble, cytosolic protein complex with a molecular mass of at least 600kDa (Regelmann et al., 2003). The work of this thesis focuses on the analysis of the novel Gid proteins and their possible role in a higher molecular mass protein complex. To be able to detect Gid proteins immunologically, functional chromosomally HA eptitope tagged versions of Gid5, Gid6, Gid7, Gid8 and Gid9 proteins were generated. Using step glycerol gradient centrifugation it could be shown that Gid5/Vid28, Gid7, Gid8 and Gid9 are also components of a higher molecular mass complex of about 600kDa. Gid1/Vid30, Gid/Ubc8 and Gid4/Vid24 exhibit a sedimentation profile of lower molecular mass slightly overlapping with 600kDa aminopeptidase I. Gid6/Ubp14 is only present in its monomeric form. Use of Gid7 as a bait protein in a co-immunoprecipitation experiment, led to the identification of Gid1/Vid30, Gid2, Gid4/Vid24, Gid5/Vid28, Gid7, Gid8 and Gid9 as interacting components. The protein Gid6/Ubp14 is not part of this protein complex. The direct interaction of Gid4 with Gid5 could be shown via the two hybrid method. Expression profiles on ethanol or glucose of Gid1, Gid2, Gid5, Gid6, Gid7, Gid8 and Gid9 were similar. Gid4/Vid24 was not expressed on ethanol but appears when cells are treated with glucose. As found for Gid3/Ubc8 (Schüle et al., 2000), Gid4/Vid24 seems to disappear during incubation on glucose in a time-dependent fashion. Fructose-1,6-bisphosphatase was found to interact with Gid1 and Gid7 protein. As shown for GID2 (Regelmann et al., 2003) deletion of GID1 and GID7 leads to a block in fructose-1,6-bisphosphatase polyubiquitination. This shows that Gid proteins are directly involved in the ubiquitination process which preceeds proteasome degradation. Two discovered short RING domains in Gid2 and Gid9 (ShRING domains) as well as the discovery of 5 WD40 domains within Gid7 suggest a role of the Gid complex as a novel E3 ubiquitin ligase. The targeted mutation of conserved cysteine residues within the shRING domain of Gid2 could support this theory. Biochemical and molecular methods were used to identify the localization of Gid1, Gid6, Gid7 and Gid8. Interestingly all four Gid proteins were found to be localized in the nucleus. The direct interaction of FBPase with these Gid proteins raised the question of whether FBPase itself had a function in the nucleus of the cell or not. To investigate this question GFP-fusions with FBPase were constructed and localisation studies were performed. An increasing signal of FBPase within the nucleus after onset of catabolite degradation gave proof of the existence of this enzyme in the nucleus as well. Several mutants known to have a defect in nuclear import were tested for the catabolite degradation of FBPase. The protein kinaseA pathway was shown to be the signal transduction pathway triggering FBPase degradation. This led to the discovery of novel putative phosphorylation sites within FBPase by bioinformatics.Item Open Access Untersuchungen zur Expression, Funktion und Regulation der ABC-Transporter P-Glykoprotein und Multidrug Resistance Protein 3 beim Menschen(2003) Hitzl, Monika; Wolf Dieter H. (Prof. Dr.)Das Ausmaß der Wirkung eines Arzneimittels hängt von seiner Konzentration am Wirkort ab. Zu niedrige Konzentrationen des Wirkstoffes haben keinen erwünschten therapeutischen Effekt beim Patienten. Im Gegensatz dazu bewirken zu hohe Arzneimittelkonzentrationen häufig toxische Nebenwirkungen. Arzneimitteltransporterproteine sind neben Arzneimittel-metabolisierenden Enzymen für die Pharmakakonzentration am Wirkort verantwortlich. Das Verständnis der Ursachen interindividueller Unterschiede in der Expression solcher Transporterproteine kann somit dazu beitragen, interindividuelle Unterschiede in der Arzneimittelwirkung zu verstehen. In dieser Arbeit wurden zwei ATP-binding cassette Transporterproteine, P-Glykoprotein und Multidrug Resistance Protein 3 untersucht. Es konnte gezeigt werden, dass ein Polymorphismus in Exon 26 des humanen MDR1-Gens (C3435T) mit einer reduzierten P-Glykoproteinexpression und -funktion assoziiert ist. Träger dieses Polymorphismus (3435 TT) zeigten eine niedrigere P-Glykoproteinexpression in Leukozyten als Probanden mit dem CC-Genotyp. Zur Ermittlung der P-Glykoproteinfunktion wurde der Efflux des P-Glykoproteinsubstrates Rhodamin123 aus Leukozyten mit der höchsten P-Glykoproteinexpression (CD56+ NK-Zellen) gemessen. Die P-Glykoproteinfunktion in CD56+ NK-Zellen von Individuen mit dem Genotyp 3435 TT in Exon 26 des MDR1-Gens war signifikant niedriger als bei Individuen mit dem CC-Genotyp. Die Abhängigkeit der P-Glykoproteinexpression vom MDR1-Genotyp wurde auch in Plazenta- und Kolongewebe untersucht. Es konnte gezeigt werden, dass die P-Glykoproteinexpression in humanem Plazentagewebe durch den Polymorphismus C3435T in ähnlicher Weise wie in den CD56+ NK-Zellen beeinflusst wurde. In Fällen, in denen Mutter und Kind den gleichen Genotyp hatten, führte die Mutation in Exon 26 des MDR1-Gens (3435 TT) zu einer 40 %igen Reduktion der P-Glykoproteinexpression in Plazentagewebe. Diese Befunde bedeuten, dass Arzneimittel, welche P-Glykoproteinsubstrate sind (z. B. HIV-Proteaseinhibitoren), niedrigere Konzentrationen in den Leukozyten von Individuen mit dem Genotyp 3435 CC erreichen und somit möglicherweise eine geringere Wirksamkeit haben (z. B. bei HIV). Eine genetisch-bedingte, niedrigere P-Glykoproteinexpression in der Plazenta dürfte die Anreicherung von Xenobiotika im Feten verstärken und könnte somit ein erhöhtes Teratogenitätsrisiko darstellen. Im zweiten Teil dieser Arbeit wurde die Expression und Regulation von Multidrug Resistance Protein 3 untersucht. Eine Analyse von 62 humanen Lebern zeigte eine erhebliche Variabilität der Multidrug Resistance Proteins 3 Expression. Der Protonenpumpenhemmer Omeprazol wurde als Faktor identifiziert, der zu einer signifikanten Erhöhung der Multidrug Resistance Proteins 3 Expression im Lebergewebe von Patienten führt. Durch in vitro Experimente konnte die Induktion des Multidrug Resistance Proteins 3 durch Omeprazol bestätigt werden. Weitere Experimente zeigten, dass der Aryl Hydrokarbon Rezeptor am Mechanismus der Induktion von Multidrug Resistance Protein 3 durch Omeprazol beteiligt ist. Es konnte außerdem nachgewiesen werden, dass Gallensäuren die Expression von Multidrug Resistance Protein 3 in CaCo-2 Zellen induzieren. Da die Expression von ABC-Transporterproteinen häufig in Tumorgewebe induziert ist und somit zur Multidrug Resistance bei der Chemotherapie beiträgt, wurde die MRP3-Expression in Kolon-Tumorgewebe untersucht. Es konnte jedoch bei diesem Tumor eine niedrigere MRP3-Expression im Vergleich zu gesundem Kolongewebe nachgewiesen werden. Mit dieser Arbeit wurden Faktoren identifiziert, die zu einer variablen Transporterprotein-expression beitragen. Das bessere Verstehen dieser Einflussfaktoren könnte somit einen Beitrag zu einer individualisierten, sichereren Arzneimitteltherapie leisten.Item Open Access Die HECT-Ligase Hul5, eine neue Komponente der ER-assoziierten Proteindegradation(2007) Kohlmann, Sonja; Wolf, Dieter H. (Prof. Dr.)Die meisten sekretorischen Proteine der eukaryontischen Zellen erreichen durch das endoplasmatische Retikulum (ER) den sekretorischen Signalweg. Sie gelangen vom Zytoplasma durch einen Kanal in der ER-Membran in das ER, wo sie ihre native Konformation erhalten. Das ER enthält ein strenges Qualitätskontrollsystem, welches fehlgefaltete Proteine erkennt, im ER zurückhält und letztendlich der ER-assoziierten Degradation (ERAD) zuführt. Die ER-Qualitätskontrolle und die ER-assoziierte Degradation sind eng miteinander verknüpft, und werden unter der Bezeichnung ER-Qualitätskontrolle und assoziierte Degradation (ERQD) zusammengefasst. Die ERQD ist von der Hefe bis hin zum Menschen ein hoch konservierter Prozess. Aus diesem Grund wird die Hefe Saccharomyces cerevisiae als Modellorganismus zur Erforschung solcher sogenannter „housekeeping“ Prozesse genutzt. In dieser Arbeit wurden durch einen genomweiten Screen neue Komponenten der ER-assoziierten Degradation identifiziert. Für diesen Screen wurden die EUROSCARF Hefe-Deletionsbank und das Screeningsubstrat Sec61-2L verwendet. Die Deletionsbank besteht aus etwa 5000 diploiden S. cerevisiae Stämmen mit je einer homozygoten Einfachdeletion. Bei dem Screeningsubstrat Sec61-2L handelt es sich um das nicht glykosylierte, mutierte Translokonprotein Sec61-2 und einer C-terminalen zytosolischen Fusion mit der 3-Isopropylmalat-Dehydrogenase (Leu2). Das für Sec61-2L kodierende Plasmid wurde in die leu2-auxotrophen Deletionsstämme transformiert und der Wachstumsphänotyp bei 38° auf Leucin-defizientem Medium getestet. Aufgrund der Punktmutation faltet sich Sec61-2 bei 38°C in einer Art und Weise, dass es der ER-Degradation unterliegt. Nur wenn Sec61-2L stabil vorliegt, also in Deletionsstämmen mit einem Defekt in der Erkennung oder der Degradation des fehlgefalteten Sec61-2L, ist ein Wachstum auf Leucin-defizientem Medium möglich. Auf diese Weise konnten über 40 bisher unbekannte, potentielle Komponenten der ER-Qualitätskontrolle und ER-assoziierten Degradation identifiziert werden. Unter anderem wurde durch diesen genomischen Screen die E4-Ligase Hul5 als Komponente des ERAD für dieses nicht glykosylierte Substrat gefunden. Des Weiteren war bereits durch einen entsprechenden Screen mit dem Substrat CTL* bekannt, dass Hul5 auch am Abbau dieses glykosylierten ERAD-Substrats beteiligt ist. In der vorliegenden Arbeit wurde nachgewiesen, dass für den vollständigen Abbau der ERAD-Substrate Sec61-2Lmyc und CTL*myc die katalytische Funktion der E4-Ligase Hul5 benötigt wird. Außerdem wurde gezeigt, dass der Abbau der Substrate Sec61-2Lmyc und CTL*myc im Wildtypstamm sowie in der HUL5 Deletionsmutante am N-Terminus einsetzt und über definierte Zwischenprodukte verläuft. Im Wildtypstamm kann auf diese Weise der Abbau der Substrate vollständig verlaufen. Im Gegensatz dazu erfolgt in der HUL5 Deletionsmutante ein Abbruch der Degradation am Proteasom, was zu einer Akkumulation der C-terminalen Abbaufragmente truncSec61-2Lmyc und truncCTL*myc führt. Des Weiteren wurde gezeigt, dass für den Abbau des N-terminalen Anteils von CTL*myc keine Extraktion des Proteins aus der ER-Membran notwendig ist. Demzufolge muß der N-Terminus von CTL*myc durch die ER-Membran in das Zytosol der Zelle ragen, wo die Ubiquitinierung und die Degradation des Substrats einsetzen. Außerdem wurde gefunden, dass auch das Proteasom an der Extraktion von CTL*myc aus dem ER beteiligt ist. Es ist bekannt, dass die E4-Ligase Hul5 gemeinsam mit dem deubiquitinierenden Enzym Ubp6 und dem Ubiquitin-konjugierenden Enzym Ubc4 den Abbau anderer proteasomaler Substrate regulieren kann. In dieser Arbeit wurde gezeigt, dass die Degradation des ERAD-Substrats CTL*myc durch Hul5, jedoch nicht durch Ubp6 und Ubc4 beeinflusst wird. Dieses Ergebnis gibt Hinweise auf weitere mit Hul5 agierende deubiquitinierende und Ubiquitin-konjugierende Enzyme.