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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 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 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 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 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.Item Open Access The novel proteasomal substrate Far10 contributes to control of mitotic exit in yeast(2005) Karnam, Harish Kumar; Hilt, Wolfgang (Priv. Doz. Dr. )Ubiquitin-Proteasome System (UPS) mediated proteolysis of an array of cellular proteins plays an important role in many basic physiological processes. Among these are control of cell cycle and division, differentiation and development, response to stress, transcriptional regulation, circadian rhythms, regulation of the immune and inflammatory responses, and biogenesis of organelles. Some of the well-known substrates of this system are cell cycle regulators such as cyclins, cyclin dependent kinase inhibitors, and proteins involved in sister chromatid separation, tumor suppressors, as well as transcriptional activators and their inhibitors [Glickman, 2002; Hilt, 2004; Wolf, D.H, 2004]. Due to these facts, identification and characterization of new substrates of the ubiquitin-proteasome system is important to reveal its cellular functions. For this purpose a high expression lethality [HEL] screen had been developed [Ledig, 1996; Velten, 1996, Velten, 2000]. This screen was based on the hypothesis that overexpression of a protein whose degradation by the ubiquitin-proteasome system is required for viability or growth, will cause a strong growth defect in cells where proteasome function is impaired, as for instance in pre1-1 pre4-1 mutants. An unknown protein originally designated as Hel48 now commonly termed as Far10 was identified, [Velten, 2000; Kemp and Sprague, Jr., 2003]. In this work cycloheximide chase experiments were undertaken to prove that Far10 is a novel substrate of the proteasome. Far10 expressed from its endogenous promoter on the chromosome either as N-terminally 19Myc tagged or as C-terminally 3Ha-tagged version was rapidly degraded in wild type cells and stabilized in pre1-1 pre4-1 proteosome mutants. Based on the ability of HA-tagged Far10 to cause lethality it was concluded that the tagged version of this protein is functional. Therefore the degradation rates seen with different tagged versions are supposed to be as wild type. The identical behavior of N-terminally and C-terminally tagged Far10 strongly support this idea. Regulatory proteolysis is an important mechanism for major cell cycle transitions such as the initiation of DNA replication, separation of sister chromatids and exit from mitosis [Jan-Michael Peters, 1998; Hilt, 2004]. APC, an ubiquitin-protein ligase, consisting of 12 known subunits in Saccharomyces cerevisiae is essential for ubiquitin-dependent proteolysis during mitosis [Harper et al., 2002; Jan-Michael Peters, 2002]. It requires two substrate specific co-activators: Cdc20 and Cdh1/Hct1. Substrates of APCCdc20 complex include non-cyclins such as Pds1 [Cohen-Fix et al., 1996; Michaelis et al., 1997; Ciosk et al., 1998; Nasmyth, 1999] and cyclins such as Clb2 and Clb5 [Bäumer et al., 2000; Wäsch, 2002; Irniger, 2002; Cross, 2003]. APCCdh1 complex initiates degradation of the mitotic cyclin Clb2 in telophase and also mediates proteolysis of other proteins such as the spindle-associated protein Ase1, Cdc20 and the polo-like kinase Cdc5 [Schwab et al., 1997; Visintin et al., 1997; Shirayama et al, 1998]. Thus, Cdc20 and Cdh1 ensure that different target proteins of the APC are degraded in a proper temporal order during mitosis. The participation of the anaphase-promoting complex and its co-activators in the degradation of Far10 was demonstrated by the observation of synthetic dosage effects in cdc23-1, cdc20-1 and hct1-?1 mutants. Cycloheximide decay analysis of 19Myc tagged Far10 in cdc23-1 APC mutants as well as cdc20-1 and proteasome mutants uncovered a clear proteolytic stabilization of N(myc)19Far10. On the contrary, a deletion of HCT1 had no effect on the degradation of Far10. These results confirm that Far10 is a genuine substrate of the APC and requires the specificity factor Cdc20 for its degradation. In addition to this, analysis of in-vivo ubiquitination experiments of Far10(HA)3 in wild type (WCG4) and pre1-1 pre4-1 proteasome mutants revealed that the polyubiquitinated forms of Far10(HA)3 accumulate in the pre1-1 pre4-1 proteasome mutants. Substrates of APC and Cdc20 in particular identified till date have a nine amino acid conserved motif called the destruction [D] box which has a consensus sequence: RXXLXXVXN/D/E. Far10 being a substrate of APCCdc20 has a nine amino-acid sequence similar to the D box motif, 340RRKLSGKYE348 residing in the C-terminal region. To check the relevance of this motif in the degradation of Far10, site directed mutagenesis of 1) first two arginines (340, 341) to alanine and leucine and 2) leucine (343) to alanine was carried out. Overexpression of these two different mutant versions of Far10 in the wild type yeast strains did not result in toxicity. Moreover, cycloheximide chase analyses of N(Myc)19Far10(L343A) expressed from the endogenous promoter on the chromosome showed that this mutant protein was not stabilized in wild type yeast strains. These data suggest that this sequence in Far10 may not confirm to a classical D-box and that the degradation signals might be located else where in the protein. It could also be possible that mutations in this D-box have to be collective in order for the desired effect(s) to be seen. Database analysis of FAR10 revealed an N-terminal FHA (fork head associated) domain and a C-terminal transmembrane domain. Cell fractionation experiments as well as immunofluorescence studies proved that Far10 localizes to the nuclear envelope [Velten, 2000]. To investigate the function of the C-terminal transmembrane domain, a deletion construct containing Far10 lacking the transmembrane domain, far10?TM was generated. In contrast to wild type Far10 this mutant protein was unable to cause synthetic dosage effects in pre1-1 pre4-1, cdc23-1 and cdc20-1 mutants. Immunofluorescence studies of Far10?TM(HA)2 revealed that this mutant protein was indeed mislocalized. These results provide evidence that the ability of Far10 to induce lethality depends on its correct localization to the nuclear membrane [Murray, 2001]. An investigation into the synthetic interactions of FAR10 with cdc20-1 mutant revealed that cdc20-1 far10? double mutants displayed a synthetic growth defect at 25°C. On the other hand far10? cdc23-1 double mutants showed no obvious growth effects when compared to cdc23-1 single mutants. The data imply that APC is fully active at 25°C in far10? cdc23-1 mutants. On the contrary cdc20-1 far10? double mutants at the same temperature may show a defective APC activity. These results propose that when APC-Cdc20 activity is disturbed, presence of Far10 is required. The mitotic exit network [MEN] in budding yeast is a complex signaling cascade consisting of Tem1 (a GTPase); Cdc15, Dbf2 and Cdc5 (protein kinases); Cdc14 (a protein phosphatase); Mob1 (a Dbf2 associated factor); Bub2-Bfa1/Byr4 (a two component GTPase-activating Protein; GAP); Lte1 (a guanine nucleotide exchange factor; GEF) and a scaffold protein, Nud1 [Amon, 2001]. Tem1 is a positive regulator of MEN. The ultimate effector of MEN is Cdc14 and it is held inactive in the nucleolus by its inhibitor Cfi1/Net1 during G1, S, G2 and early M phase. MEN is activated when the spindle pole body reaches daughter cell where Lte1 (GEF) exchanges a GDP for GTP on Tem1. Thus activated, Tem1-GTP binds to and activates Cdc15, which in turn activates Mob1-Dbf2 complex. Dbf2 facilitates release of Cdc14 from the nucleolus. The freed Cdc14 functions to shut down mitotic Cdk activity by promoting expression of the Cdk inhibitor Sic1 and stimulation of degradation of the essential mitotic cyclins. To outline the relation(s) of FAR10 with regulatory modules of the mitotic exit network a genetic method was executed. For this purpose, effects of FAR10 overexpression and inactivation were studied in MEN mutants. Overexpression of FAR10 in a string of MEN mutants was found to cause toxicity in cdc14-3, dbf2-2, and tem1-3 mutants, with cdc15-2 and cdc5-1 mutants displaying a mild effect and lte1? mutant showing no effect at all. These results prove that when MEN activation is defective cells become sensitive to Far10 overexpression. In contrast when MEN is hyperactive as in the case of bub2? mutants, the effect of overexpression of FAR10 is suppressed. FAR10 is not an essential gene and its deletion causes no obvious growth defects when compared to wild type (W303) strain. Though far10? cdc14-3 double mutants showed no detectable growth effects at either 25°C or 30°C when compared to cdc14-3 single mutants, deletion of FAR10 in dbf2-2 mutants had a moderate suppression effect at 25°C. In the case of far10? tem1-3 double mutants this suppression effect was enhanced at 32°C revealing that FAR10 may be an inhibitor of mitotic exit. Overexpression of FAR10 causes synthetic dosage effects in mutants that are defective in Clb-CDK inactivation such as hct1-?1 and sic1-?1. These results suggest that a defective Clb-CDK inactivation either to due impaired degradation or absence of inhibition by Sic1 makes cells susceptible to Far10 overexpression. In addition, hct1-?1 far10? and sic1-?1 far10? double mutants showed synthetic growth defect at 30°C, which was markedly enhanced at 37°C. The data prove that presence of Far10 is required under these conditions. The ultimate function of the mitotic exit network in budding yeast is inactivation of the mitotic Clb2-CDK activity, which is followed by cytokinesis resulting in the formation of two daughter cells [Visintin et al., 1998]. In relevance of these findings it was rationalized that an enhancement in Clb-CDK inactivation through ectopic overexpression of Sic1 might alleviate the toxic effects associated with FAR10 overexpression. Henceforth, SIC1(HA)1X was co-overexpressed with FAR10 in cdc23-1, cdc14-3, dbf2-2 and tem1-3 mutants. Results in this case show that co-overexpression of SIC1(HA)1X along with FAR10 did not restore wild type growth rates. An analogous result was obtained when Sic1 was overexpressed in MEN mutants that harbored a deletion of FAR10. The data here propose an ill-defined role for Far10 as an inhibitor of mitotic exit. Additionally, these results also provide evidence that FAR10 may act in parallel to MEN and/or co-operate in triggering exit from mitosis.Item Open Access In vitro- und in vivo-Untersuchungen zur Bedeutung des intestinalen Arzneimittelmetabolismus und -transportes beim Menschen(2003) Gläser, Hartmut; Wolf, Dieter H. (Prof. Dr.)Mit dem Nachweis von arzneimittelmetabolisierenden Enzymen (AME) und Arzneimitteltransportern im Dünndarm des Menschen wurde die Bedeutung des intestinalen Metabolismus für die Bioverfügbarkeit oral applizierter Arzneimittel zunehmend erkannt. Die mangelnde Verfügbarkeit von humanen Enterozyten stellt bei Untersuchungen zum intestinalen Arzneimittelmetabolismus und -transport ein wesentliches Problem dar. Zur Expression und ex vivo Funktion von Cytochrom P450 Enzyme sowie deren Modifikation durch Rifampicin in humanen Enterozyten existieren keine systematischen Daten. Auch sind die Kenntnisse über die Expression und in vivo Funktion von intestinalen Arzneimitteltransportern und deren Beeinflussung durch Rifampicin sehr gering. Mit einem multiluminalen Perfusionskatheter (MLPC) zur Gewinnung von humanen Enterozyten und einer Gewebesammlung von Dünndarm- und Leberproben von jeweils demselben Patienten wurde versucht, die o.g. Probleme zu bearbeiten. Die Charakterisierung der mit dem MLPC gewonnenen Zellen zeigte, dass vitale, abgeschilferte Enterozyten gewonnen, und sowohl zur Untersuchung der Expression von AMEs und Transporter als auch zur ex vivo Analyse des intestinalen Metabolismus verwendet werden können. Unter Einsatz des MLPCs wurde in einer Studie die Induktion von CYP2C8, 2C9 und 3A4 durch Rifampicin in den Enterozyten nachgewiesen. Mit Hilfe des MLPCs konnte auch die Funktion und Expression des Arzneimitteltransporters P-Glykoprotein (Pgp) untersucht werden. Für die funktionelle in vivo Analyse wurde Chinidin bei gleichzeitiger i.v. Gabe des Pgp-Substrates Digoxin luminal über den MLPC verabreicht. Damit konnte die Bedeutung des intestinal exprimierten Pgp sowohl für die systemische Elimination von Digoxin als auch für die Resorption von Chinidin nachgewiesen werden. Zudem konnte die Zunahme der Elimination von Digoxin bzw. Abnahme der Chinidinresorption nach Gabe von Rifampicin gezeigt werden. Die Expressionsanalyse von MRP2 in Dünndarm und Leber zeigte keinen Unterschied im Ausmaß der Expression in den beiden Geweben. Die Tatsache, dass im Dünndarm - nicht aber in der Leber - eine Korrelation zwischen MRP2 mRNA und Protein nachgewiesen wurde, gibt einen Hinweis auf unterschiedliche Regulationsmechanismen von MRP2 in den beiden Geweben.Item Open Access Protein-protein and protein-small-molecule inhibitor interactions in the measles virus replication complex(2013) Krumm, Stefanie A.; Wolf, Dieter (Prof. Dr.)The disease measles is caused by the highly contagious measles virus (MeV). MeV belongs to the paramyxovirus family together with respiratory syncytial virus, human parainfluenza viruses and metapneumovirus. Paramyxoviruses are responsible for major pediatric morbidity and mortality. Despite the availability of an effective MeV vaccine, measles case numbers increased alarmingly in the past few years especially in Europe. The return of endemic measles in the European population can directly be linked to the decrease in acceptance/use of the measles-mumps-rubella (MMR) vaccine. The Measles Initiative has set a goal to eliminate measles by 2015. The addition of an effective antiviral to quickly treat sporadic outbreaks and the surrounding communities would greatly aid in the measles eradication efforts. Fundamental understanding of the viral replication mechanism at the molecular level will be critical for the successful development of antivirals. Therefore the following dissertation examined the protein-protein interactions in the measles virus polymerase complex to understand the events taking place at the molecular level. Additionally, it engaged in protein-small-molecule interactions to identify small-molecule inhibitors of viral replication and their targets. The first part of the thesis focused on molecular interactions in the viral replication complex. The viral replication complex is an attractive target for antiviral therapy since it possesses unique features and is expressed and functions in a sub cellular compartment distinct from host cell polymerases. The polymerase complex consists of the phosphoprotein (P) and the polymerase (L) protein. The P-L complex only interacts with nucleoprotein (N) encapsidated RNA (RNP) for transcription and replication. MeV N contains a core domain involved in RNA encapsidation and a 125-residue carboxy (C)-terminal tail (Ntail) considered to mediate P-L binding to RNP for polymerization. Ntail of MeV is largely unstructured, but a terminal microdomain is implicated in P binding. C-terminal tail truncated N mutant proteins progressively eliminating this microdomain and upstream tail sections demonstrated that the interaction of the Ntail microdomain with a C-terminal domain in P is not required for polymerase recruitment and initial binding of L to its template. Additional investigations showed that disrupting the domain organization by insertion of an epitope tag in the Ntail did not affect polymerase activity, but rather affected particle assembly. Cell free virions contained reduced levels of envelope proteins which did not affect cell-to-cell fusion kinetics. However, the N-mutant virus was observed to have a kinetic delay of viral mRNA and genome production. Studies to identify and characterize small-molecule antiviral compounds and their targets were conducted in the second part of this thesis. Non-nucleoside small-molecules are suitable antiviral therapeutics. There are two main approaches in identifying antivirals. First, compounds that target the virus, for example the RNA replication machinery, can be assayed for. Alternatively, compounds that target a host factor that the virus requires can also be a viable strategy. Cellular factors may also be necessary for the entire family of viruses and therefore compounds aiming for host factors may be more likely to be broadly active inhibitors. A potent pathogen-directed small-molecule compound class had been identified in a high-throughput screen. Hit-to-lead chemistry yielded a highly potent and water soluble compound ERDRP-0519. It targets the L subunit of the morbillivirus polymerase complex directly, since resistance-mediating mutations were exclusively located in the L protein. Unparalleled efficacy of this orally available small-molecule inhibitor was demonstrated and pioneered a path towards an effective morbillivirus therapy that can support measles eradication efforts. Therapeutic targeting of host cell factors required for virus replication rather than of pathogen components opens new perspectives to counteract virus infections. JMN3-003 is a potent broadly active inhibitor of viral RdRp activity with a host factor mediated profile. It inhibited a wide range of different viral targets. Its antiviral activity was host cell species dependent and induced a temporary cell cycle arrest. While the compound inhibited viral mRNA and genome production, it left host cell mRNA and protein production unaffected. Taken together, this PhD studies changed the prevailing paradigm in polymerase recruitment and provided strong proof of concept for the potential of the development of pathogen- and host-directed antiviral therapy. These studies demonstrated how basic molecular research of protein-protein interactions critical for virus replication can complement a translational approach to identify, characterize, and improve novel antiviral candidates.Item Open Access Biochemical characterisation of TET DNA hydroxylases(2017) Ravichandran, Mirunalini; Jurkowski, Tomasz (Jun.-Prof. Dr.)Methylation of DNA in CpG dinucleotide plays an important role in mammalian development. The recent discovery of TET enzymes showed that DNA demethylation can occur through stepwise oxidation of 5-methylcytosine (5mC) to 5-hyrdoxymethylcytosine (5hmC), 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5-caC) followed by the removal of the higher oxidised bases by Thymine DNA glycosylase (TDG) and base excision repair mechanism. Genetic studies revealed that the TET enzymes are involved in numerous biological processes such as transcriptional regulation, hematopoietic stem cell differentiation, embryonic, primodial germ cells (PGCs) development and are commonly misregulated in cancer. While the biological functions of TET enzymes have been studied extensively, very little is known about their biochemical properties. In this body of work, the biochemistry of TET enzymes was investigated in detail with the focus on their catalytic and kinetic behaviour, which would allow us to understand the molecular mechanisms of TET enzymes. First of all, an in vitro system including a novel plate assay to quantify the oxidation products catalysed by TET enzymes, was established. As a proof of principle, several analogues of α-Ketoglutarate, the intermediates of citric acid cycle (oncometabolites) were tested. Moreover, the effect of divalent metal ions was tested both in vitro and in vivo and it was demonstrated that the addition of nickel ions to mammalian cells decreased the level of 5hmC through inhibition of TET enzymes by displacing the Fe2+ from the catalytic centre. Furthermore, using detailed biochemical studies, it was demonstrated that ascorbic acid (AscA) modulates the activity of TET enzymes through efficient recycling of Fe2+, which challenges the existing view of AscA as a bound cofactor of TET enzymes. Finally, using biochemical approach followed by next generation sequencing and bioinformatics analysis, the catalytic behaviour of TET enzymes on single molecule level was elucidated. Using linear double stranded DNA containing multiple 5hmC-substrates in different flanking sequence context, it was shown that mammalian TET enzymes oxidize 5hmC substrates in both CG and non-CG context. Importantly, both mammalian TET enzymes and Naegleria gruberi Tet1 like dioxygenase (nTet) showed a strong and distinct flanking sequence preference. In addition, it was shown that TET enzymes (both mammalian and nTet) might catalyse the substrates on DNA in distributive manner.