@phdthesis{Dogan2009,
  author    = {Dogan, Taner},
  title     = {Role of IAPs in modulating C-RAF stability and cell motility},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39827},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {The data of this thesis show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signaling pathway and cell migration},
  subject      = {IAP},
  language  = {en}
}
@phdthesis{Jaenicke2015,
  author    = {J{\"a}nicke, Laura Annika},
  title     = {Regulation of MYC Activity by the Ubiquitin-Proteasome System},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123339},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The oncogenic MYC protein is a transcriptional regulator of multiple cellular processes and is aberrantly activated in a wide range of human cancers. MYC is an unstable protein rapidly degraded by the ubiquitin-proteasome system. Ubiquitination can both positively and negatively affect MYC function, but its direct contribution to MYC-mediated transactivation remained unresolved. To investigate how ubiquitination regulates MYC activity, a non-ubiquitinatable MYC mutant was characterized, in which all lysines are replaced by arginines (K-less MYC). The absence of ubiquitin-acceptor sites in K-less MYC resulted in a more stable protein, but did not affect cellular localization, chromatin-association or the ability to interact with known MYC interaction partners. Unlike the wild type protein, K-less MYC was unable to promote proliferation in immortalized mammary epithelial cells. RNA- and ChIP-Sequencing analyses revealed that, although K-less MYC was present at MYC-regulated promoters, it was a weaker transcriptional regulator. The use of K-less MYC, a proteasomal inhibitor and reconstitution of individual lysine residues showed that proteasomal turnover of MYC is required for MYC target gene induction. ChIP-Sequencing of RNA polymerase II (RNAPII) revealed that MYC ubiquitination is dispensable for RNAPII recruitment and transcriptional initiation but is specifically required to promote transcriptional elongation. Turnover of MYC is required to stimulate histone acetylation at MYC-regulated promoters, which depends on a highly conserved region in MYC (MYC box II), thereby enabling the recruitment of BRD4 and P-TEFb and the release of elongating RNAPII from target promoters. Inhibition of MYC turnover enabled the identification of an intermediate in MYC-mediated transactivation, the association of MYC with the PAF complex, a positive elongation factor, suggesting that MYC acts as an assembly factor transferring elongation factors onto RNAPII. The interaction between MYC and the PAF complex occurs via a second highly conserved region in MYC's amino terminus, MYC box I. Collectively, the data of this work show that turnover of MYC coordinates histone acetylation with recruitment and transfer of elongation factors on RNAPII involving the cooperation of MYC box I and MYC box II.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{Mueller2010,
  author    = {M{\"u}ller, Judith},
  title     = {Die Rolle der HectH9/Mcl1-Interaktion in der Myc-induzierten Apoptose und Auswirkungen der Myc V394D-Mutation auf die von c-Myc gesteuerten Tumorgenese in einem transgenen Mausmodell},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-55789},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {W{\"a}hrend der Entstehung von Tumoren k{\"o}nnen zwei Mechanismen auftreten, die beide von der Aktivit{\"a}t der Onkogene abh{\"a}ngig sind und die Tumorgenese einschr{\"a}nken. F{\"u}r das Onkogen Myc ist gezeigt, dass es sowohl Apoptose als auch unter bestimmten Umst{\"a}nden Seneszenz ausl{\"o}sen kann und damit sein eigenes onkogenes Potential limitiert. Im Rahmen dieser Arbeit konnte ich mich mit diesen Tumor-suppressiven Mechanismen in zwei unabh{\"a}ngigen Teilprojekten besch{\"a}ftigen. Eine erh{\"o}hte Expression von Myc steigert die Proliferation der Zellen, induziert aber gleichzeitig Doppelstrangbr{\"u}che an der DNA. Durch den dadurch entstandenen Schaden wird die DNA-Schadensantwort ausgel{\"o}st, die zum Beispiel zur Phosphorylierung von H2A.X durch die Kinasen Atm und Atr f{\"u}hrt. Ein weiteres putatives Zielprotein dieser Kinasen ist HectH9, das abh{\"a}ngig vom DNA-Schaden das mitochondriale Protein Mcl1 ubiquitiniert und es damit f{\"u}r den proteasomalen Abbau markiert. Im ungestressten Zustand interagiert das in der mitochondrialen Membran lokalisierte Protein Mcl1 mit proapoptotischen Proteinen und h{\"a}lt deren inerten Status aufrecht. Die Reduktion der Mcl1-Mengen ist essentiell, um die proapoptotischen Proteine zu aktivieren, dadurch die Freisetzung von Zytochrom C aus dem Mitochondrium zu veranlassen und damit den Prozess der Apoptose einleiten zu k{\"o}nnen. Anhand der in dieser Arbeit dokumentierten Daten bietet sich Mcl1 als potentielles Zielprotein f{\"u}r pharmazeutisch Strategien zur Therapie Myc-induzierter Tumore an. Im Idealfall erh{\"o}ht eine verst{\"a}rkte Reduktion seiner Proteinmengen die zellul{\"a}re Apoptose und verringert somit das Tumorwachstum. Im murinen T-Zell-Lymphom wird die Myc-abh{\"a}ngige Tumorgenese durch eine Mutation der Proteinsequenz von Myc verlangsamt. Diese Mutation unterbindet die Bindung von Myc zu Miz1 und verhindert dadurch die Repression von Zielgenen. Abh{\"a}ngig von der Interaktion von Myc zu Miz1 gelingt die Inhibition der Transkription des Zellzyklusinhibitors p15Ink4b. Die Interaktion von Myc und Miz1 ist essentiell um die TGFbeta-abh{\"a}ngige Seneszenz zu umgehen. Dar{\"u}ber hinaus ist Myc direkt an der Repression von TGFbeta beteiligt. Entgegen der bisher verwendeten Modelle konnte in dieser Arbeit gezeigt werden, dass Myc unabh{\"a}ngig von Miz1 zu den Promotoren der reprimierten Zielgene rekrutiert wird und die Bindung der beiden Proteine offensichtlich nur f{\"u}r die Transrepression essentiell ist.},
  subject      = {Myc},
  language  = {de}
}
@phdthesis{Orth2021,
  author    = {Orth, Barbara},
  title     = {Identification of an atypical peptide binding mode of the BTB domain of the transcription factor MIZ1 with a HUWE1-derived peptide},
  doi       = {10.25972/OPUS-25044},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250447},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Ubiquitination is a posttranslational modification with immense impact on a wide range of cellular processes, including proteasomal degradation, membrane dynamics, transcription, translation, cell cycle, apoptosis, DNA repair and immunity. These diverse functions stem from the various ubiquitin chain types, topologies, and attachment sites on substrate proteins. Substrate recruitment and modification on lysine, serine or threonine residues is catalyzed by ubiquitin ligases (E3s). An important E3 that decides about the fate of numerous substrates is the HECT-type ubiquitin ligase HUWE1. Depending on the substrate, HUWE1 is involved in different processes, such as cell proliferation and differentiation, DNA repair, and transcription. One of the transcription factors that is ubiquitinated by HUWE1 is the MYC interacting zinc finger protein 1 (MIZ1). MIZ1 is a BTB/POZ (Bric-{\`a}-brac, Tramtrack and Broad-Complex/Pox virus and zinc finger) zinc finger (ZF) protein that binds to DNA through its 13 C2H2-type zinc fingers and either activates or represses the transcription of target genes, including genes involved in cell cycle arrest, such as P21CIP1 (CDKN1A). The precise functions of MIZ1 depend on its interactions with the MYC-MAX heterodimer, but also its heterodimerization with other BTB-ZF proteins, such as BCL6 or NAC1. How MIZ1 interacts with HUWE1 has not been studied and, as a consequence, it has not been possible to rationally develop tools to manipulate this interaction with specificity in order to better understand the effects of the interaction on the transcriptional function of MIZ1 on target genes or processes downstream. One aspect of my research, therefore, aimed at characterizing the MIZ1-HUWE1 interaction at a structural level. I determined a crystal structure of the MIZ1-BTB-domain in complex with a peptide, referred to as ASC, derived from a C terminal region of HUWE1, previously named 'activation segment'. The binding mode observed in this crystal structure could be validated by binding and activity assays in vitro and by cell-based co-IP experiments in the context of N-terminally truncated HUWE1 constructs. I was not able to provide unambiguous evidence for the identified binding mode in the context of full-length HUWE1, indicating that MIZ1 recognition by HUWE1 requires yet unknown regions in the cell. While the structural details of the MIZ1-HUWE1 interaction remains to be elucidated in the context of the full-length proteins, the binding mode between MIZ1BTB and ASC revealed an interesting, atypical structural feature of the BTB domain of MIZ1 that, to my knowledge, has not been described for other BTB-ZF proteins: The B3 region in MIZ1BTB is conformationally malleable, which allows for a HUWE1-ASC-peptide-mediated β-sheet extension of the upper B1/B2-strands, resulting in a mixed, 3 stranded β-sheet. Such β-sheet extension does not appear to occur in other homo- or heterodimeric BTB-ZF proteins, including MIZ1-heterodimers, since these proteins typically possess a pre-formed B3-strand in at least one subunit. Instead, BCL6 co repressor-derived peptides (SMRT and BCOR) were found to extend the lower β-sheet in BCL6BTB by binding to an adjacent 'lateral groove'. This interaction follows a 1:1 stoichiometry, whereas the MIZ1BTB-ASC-complex shows a 2:1 stoichiometry. The crystal structure of the MIZ1BTB-ASC-complex I determined, along with comparative binding studies of ASC with monomeric, homodimeric, and heterodimeric MIZ1BTB variants, respectively, suggests that ASC selects for MIZ1BTB homodimers. The structural data I generated may serve as an entry point for the prediction of additional interaction partners of MIZ1 that also have the ability to extend the upper β-sheet of MIZ1BTB. If successful, such interaction partners and structures thereof might aid the design of peptidomimetics or small-molecule inhibitors of MIZ1 signaling. Proof-of-principle for such a structure-guided approach targeting BTB domains has been provided by small-molecule inhibitors of BCL6BTB co-repressors interactions. If a similar approach led to molecules that interfere with specific interactions of MIZ1, they would provide intriguing probes to study MIZ1 biology and may eventually allow for the development of MIZ1-directed cancer therapeutics.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Ries2020,
  author    = {Ries, Lena Kerstin},
  title     = {From recognition to reaction: Mechanistic analysis of the interactions of the HECT ligase E6AP with ubiquitin},
  doi       = {10.25972/OPUS-17960},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179609},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The ubiquitination of proteins controls a multitude of physiological processes. This versatility of ubiquitin as a molecular signal arises from the diverse ways by which it can be attached to target proteins. Different ubiquitination patterns are then translated into different downstream consequences. Due to the enormous complexity of possible ubiquitin modifications, the ubiquitination machinery must be highly specific and tightly controlled. Ubiquitination proceeds through an enzymatic cascade, the last step of which is catalyzed by the E3 enzyme family. E3 enzymes are the crucial regulators since they dictate the specificity of substrate selection and modification. Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity, regulation and specificity in this crucial ligase are incompletely understood. One aim of this study was to unravel the role of the a1'-helix N-terminal to the HECT domain that was found to be a key element mediating regulation and oligomerization in other HECT ligases. I found that most N-terminally extended HECT domain constructs were insoluble when expressed in E. coli, indicating that additional regions N-terminal to the tested fragments may be essential to protect this highly hydrophobic helix from causing aggregation. Another question addressed in this study was how E6AP builds ubiquitin chains. Using single-turnover experiments, I showed that ubiquitin-loaded E6AP is unable to transfer an additional ubiquitin molecule onto a stably linked ubiquitin-E6AP complex. This indicates that E6AP cannot assemble chains on its active site and may instead follow a sequential addition mechanism in which one ubiquitin molecule is transferred at a time to the target protein. Using NMR spectroscopy and extensive mutational analyses, the determinants of ubiquitin recognition by the C-lobe of E6AP were unraveled and assigned to particular steps in the catalytic cycle. A functionally critical interface was identified that is specifically required during thioester formation between the C-terminus of ubiquitin and the ligase active site. This interface resembles the one utilized by NEDD4-type enzymes, suggesting a conserved ubiquitin binding mode across HECT ligases, independent of their linkage specificities. Moreover, I identified critical surface patches on ubiquitin and in the N- and C-terminal portions of the catalytic domain of E6AP that are important for the subsequent step of isopeptide bond formation. I also uncovered key determinants of the Lys48-linkage specificity of E6AP, both in the E6AP HECT domain and ubiquitin itself. This includes the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site, Lys48. It is thus tempting to speculate that ubiquitin linkage formation by E6AP is substrate-assisted. Taken together, my results improve our mechanistic understanding of the structure-function relationship between E6AP and ubiquitin, thus providing a basis for ultimately manipulating the functions of this HECT ligase for therapeutic applications.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Schuelein2011,
  author    = {Sch{\"u}lein, Christina},
  title     = {Die Regulation von Fbw7 durch PI3K-abh{\"a}ngige Phosphorylierung und Charakterisierung eines konditionalen Usp28-Knockout-Mausmodells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70963},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Das Proto-Onkoprotein Myc ist an der Entstehung und Aufrechterhaltung einer Vielzahl humaner Tumore entscheidend beteiligt. In der vorliegenden Arbeit wurde Serin 227 in Fbw7 als Ziel f{\"u}r eine PI3K-abh{\"a}ngige Phosphorylierung identifiziert. Diese Phosphorylierung f{\"u}hrt zur Stabilisierung von Fbw7 und steigert die F{\"a}higkeit von Fbw7, Substratproteine zu ubiquitinieren und abzubauen. Um die Bedeutung von Usp28 in der Myc-induzierten Tumorentstehung und in der normalen Gewebehom{\"o}ostase zu untersuchen, wurde ein konditionales Knockout-Mausmodell f{\"u}r Usp28 charakterisiert. M{\"a}use mit einer Keimbahndeletion von Usp28 sind lebensf{\"a}hig, fertil und ph{\"a}notypisch unauff{\"a}llig. Weder in Organen der Usp28-negativen Tiere, noch in entsprechenden murinen embryonalen Fibroblasten kann eine Destabilisierung von Myc festgestellt werden. Allerdings zeigen Fibroblasten mit heterozygotem Usp28-Verlust einen Proliferationsdefekt und in Eμ-Myc-Lymphomen dieses Genotyps werden tendenziell niedrigere Myc-Proteinmengen gefunden. Das tumorfreie {\"U}berleben ist bei den Eμ-Myc; Usp28 +/- Tieren verl{\"a}ngert.},
  subject      = {Myc},
  language  = {de}
}
@phdthesis{Zovko2013,
  author    = {Zovko, Josip},
  title     = {Die E3-Ubiquitinligase HectD1 reguliert die Stabilit{\"a}t des antiapoptotischen Bcl-2-Familienmitglieds A1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87922},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Die Bcl-2-Familienmitglieder A1 und sein humanes Homolog Bfl-1 gew{\"a}hrleisten das {\"U}berleben der Zelle. Gleichzeitig tr{\"a}gt eine Dysregulation der Expression von A1/ Bfl-1 zur Krebsentstehung bei. Die Stabilit{\"a}t von A1/ Bfl-1 wird durch deren Ubiquitinylierung sowie die anschließende proteosomale Degradation gesteuert. Mit Hilfe eines Yeast-Two-Hybrid-Screens wurde die E3-Ubiquitinligase HectD1 als potentieller Interaktionspartner von A1/ Bfl-1 identifiziert. Die Interaktion von A1 und HectD1 des Yeast-Two-Hybrid-Screens konnte in S{\"a}ugerzellen best{\"a}tigt werden. Desweiteren konnte gezeigt werden, dass lediglich 87 Aminos{\"a}uren f{\"u}r eine Interaktion von HectD1 und A1 n{\"o}tig sind. Da membrangebundenes HectD1 zu einer Translokation von zytosolischem A1 an die Zellmembran f{\"u}hrt, kann man davon ausgehen, dass beide Proteine auch in vivo miteinander interagieren. Eine dominant negative HectD1-Mutante schließlich beeinflusst die Ubiqutinylierung von A1 und f{\"u}hrt somit zu dessen Stabilisierung. Diese Daten legen nahe, dass HectD1 ein wichtiger negativer Regulator von A1/ Bfl-1 ist und dass HectD1 f{\"u}r die Regulierung der A1/ Bfl-1-Proteinmenge in (Krebs)zellen sehr wichtig ist.},
  subject      = {Zelltod},
  language  = {de}
}