@phdthesis{MaierverhHartmann2024,
  author    = {Maier [verh. Hartmann], Carina Ramona},
  title     = {Regulation of the Mevalonate Pathway by the Deubiquitinase USP28 in Squamous Cancer},
  doi       = {10.25972/OPUS-34874},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-348740},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The reprogramming of metabolic pathways is a hallmark of cancer: Tumour cells are dependent on the supply with metabolites and building blocks to fulfil their increased need as highly proliferating cells. Especially de novo synthesis pathways are upregulated when the cells of the growing tumours are not able to satisfy the required metabolic levels by uptake from the environment. De novo synthesis pathways are often under the control of master transcription factors which regulate the gene expression of enzymes involved in the synthesis process. The master regulators for de novo fatty acid synthesis and cholesterogenesis are sterol regulatory element-binding proteins (SREBPs). While SREBP1 preferably controls the expression of enzymes involved in fatty acid synthesis, SREBP2 regulates the transcription of the enzymes of the mevalonate pathway and downstream processes namely cholesterol, isoprenoids and building blocks for ubiquinone synthesis. SREBP activity is tightly regulated at different levels: The post-translational modification by ubiquitination decreases the stability of active SREBPs. The attachment of K48-linked ubiquitin chains marks the transcription factors for the proteasomal degradation. In tumour cells, high levels of active SREBPs are essential for the upregulation of the respective metabolic pathways. The increased stability and activity of SREBPs were investigated in this thesis. SREBPs are ubiquitinated by the E3 ligase Fbw7 which leads to the subsequential proteolysis of the transcription factors. The work conducted in this thesis identified the counteracting deubiquitination enzyme USP28 which removes the ubiquitin chains from SREBPs and prevents their proteasomal degradation. It further revealed that the stabilization of SREBP2 by USP28 plays an important role in the context of squamous cancers. Increased USP28 levels are associated with a poor survival in patients with squamous tumour subtypes. It was shown that reduced USP28 levels in cell lines and in vivo result in a decrease of SREBP2 activity and downregulation of the mevalonate pathway. This manipulation led to reduced proliferation and tumour growth. A direct comparison of adenocarcinomas and squamous cell carcinomas in lung cancer patients revealed an upregulation of USP28 as well as SREBP2 and its target genes. Targeting the USP28-SREBP2 regulatory axis in squamous cell lines by inhibitors also reduced cell viability and proliferation. In conclusion, this study reports evidence for the importance of the mevalonate pathway regulated by the USP28-SREBP2 axis in tumour initiation and progression of squamous cancer. The combinatorial inhibitor treatment of USP28 and HMGCR, the rate limiting enzyme of the mevalonate pathway, by statins opens the possibility for a targeted therapeutic treatment of squamous cancer patients.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Xu2022,
  author    = {Xu, Wenshan},
  title     = {Regulation of the DNA Damage Response by the Ubiquitin System},
  doi       = {10.25972/OPUS-16006},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160064},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {DNA damage occurs frequently during normal cellular progresses or by environmental factors. To preserve the genome integrity, DNA damage response (DDR) has evolved to repair DNA and the non-properly repaired DNA induces human diseases like immune deficiency and cancer. Since a large number of proteins involved in DDR are enzymes of ubiquitin system, it is critical to investigate how the ubiquitin system regulates cellular response to DNA damage. Hereby, we reveal a novel mechanism for DDR regulation via activation of SCF ubiquitin ligase upon DNA damage. As an essential step for DNA damage-induced inhibition of DNA replication, Cdc25A degradation by the E3 ligase β-TrCP upon DNA damage requires the deubiquitinase Usp28. Usp28 deubiquitinates β-TrCP in response to DNA damage, thereby promotes its dimerization, which is required for its activity in substrate ubiquitination and degradation. Particularly, ubiquitination at a specific lysine on β-TrCP suppresses dimerization. The key mediator protein of DDR, 53BP1, forms oligomers and associates with β-TrCP to inhibit its activity in unstressed cells. Upon DNA damage, 53BP1 is degraded in the nucleoplasm, which requires oligomerization and is promoted by Usp28 in a β-TrCP-dependent manner. Consequently, 53BP1 destruction releases and activates β-TrCP during DNA damage response. Moreover, 53BP1 deletion and DNA damage promote β-TrCP dimerization and recruitment to chromatin sites that locate in the vicinity of putative replication origins. Subsequently, the chromatin-associated Cdc25A is degraded by β-TrCP at the origins. The stimulation of β-TrCP binding to the origins upon DNA damage is accompanied by unloading of Cdc45, a crucial component of pre-initiation complexes for replication. Loading of Cdc45 to origins is a key Cdk2-dependent step for DNA replication initiation, indicating that localized Cdc25A degradation by β-TrCP at origins inactivates Cdk2, thereby inhibits the initiation of DNA replication. Collectively, this study suggests a novel mechanism for the regulation of DNA replication upon DNA damage, which involves 53BP1- and Usp28-dependent activation of the SCF(β-TrCP) ligase in Cdc25A degradation.},
  subject      = {DNS-Sch{\"a}digung},
  language  = {en}
}
@phdthesis{LiessneeEller2021,
  author    = {Liess [n{\´e}e Eller], Anna Katharina Luise},
  title     = {Understanding the regulation of the ubiquitin-conjugating enzyme UBE2S},
  doi       = {10.25972/OPUS-20419},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The ubiquitination of proteins serves as molecular signal to control an enormous number of physiological processes and its dysregulation is connected to human diseases like cancer. The versatility of this signal stems from the diverse ways by which ubiquitin can be attached to its targets. Thus, specificity and tight regulation of the ubiquitination are pivotal requirements of ubiquitin signaling. Ubiquitin-conjugating enzymes (E2s) act at the heart of the ubiquitination cascade, transferring ubiquitin from a ubiquitin-activating enzyme (E1) to a ubiquitin ligase (E3) or substrate. When cooperating with a RING-type E3, ubiquitin-conjugating enzymes can determine linkage specificity in ubiquitin chain formation. Our understanding of the regulation of E2 activities is still limited at a structural level. The work described here identifies two regulation mechanisms in UBE2S, a cognate E2 of the human RING-type E3 anaphase-promoting complex/cyclosome (APC/C). UBE2S elongates ubiquitin chains on APC/C substrates in a Lys11 linkage-specific manner, thereby targeting these substrates for degradation and driving mitotic progression. In addition, UBE2S was found to have a role in DNA repair by enhancing non-homologous end-joining (NHEJ) and causing transcriptional arrest at DNA damage sites in homologous recombination (HR). Furthermore, UBE2S overexpression is a characteristic feature of many cancer types and is connected to poor prognosis and diminished response to therapy. The first regulatory mechanism uncovered in this thesis involves the intramolecular auto-ubiquitination of a particular lysine residue (Lys+5) close to the active site cysteine, presumably through conformational flexibility of the active site region. The Lys+5-linked ubiquitin molecule adopts a donor-like, 'closed' orientation towards UBE2S, thereby conferring auto-inhibition. Notably, Lys+5 is a major physiological ubiquitination site in ~25\% of the human E2 enzymes, thus providing regulatory opportunities beyond UBE2S. Besides the active, monomeric state and the auto-inhibited state caused by auto-ubiquitination, I discovered that UBE2S can adopt a dimeric state. The latter also provides an auto-inhibited state, in which ubiquitin transfer is blocked via the obstruction of donor binding. UBE2S dimerization is promoted by its unique C-terminal extension, suppresses auto-ubiquitination and thereby the proteasomal degradation of UBE2S. Taken together, the data provided in this thesis illustrate the intricate ways by which UBE2S activity is fine-tuned and the notion that structurally diverse mechanisms have evolved to restrict the first step in the catalytic cycle of E2 enzymes.},
  subject      = {E2},
  language  = {en}
}
@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{Peter2014,
  author    = {Peter, Stefanie},
  title     = {Hemmung der Myc-Funktion durch niedermolekulare Inhibitoren der E3-Ubiquitin-Ligase Huwe1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-104449},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Die Deregulation des Transkriptionsfaktors Myc ist ein zentraler Mechanismus in der kolorektalen Karzinogenese. Die Myc-Deletion in Tumormodellen hemmt das Wachstum von Kolonkarzinomen, somit stellt die Inaktivierung von Myc einen Ansatzpunkt in der Behandlung von kolorektalen Tumoren dar. Die direkte Inhibition von Myc ist schwierig, da Myc keine katalytische Aktivit{\"a}t besitzt und stattdessen f{\"u}r die Myc-Funktion n{\"o}tige Protein-Protein- oder Protein-DNA-Interaktionen angegriffen werden m{\"u}ssen. Die E3-Ubiquitin-Ligase Huwe1 interagiert sowohl mit Myc als auch mit dem Myc-interagierenden Protein Miz1 und ist im Kolonkarzinom {\"u}berexprimiert. Huwe1 ubiquitiniert Myc und induziert dar{\"u}ber dessen Transaktivierungsfunktion. Die Inaktivierung von Huwe1 ist somit eine vielversprechende M{\"o}glichkeit f{\"u}r die Inhibition der Myc-Funktion und die Therapie des Kolonkarzinoms. In dieser Arbeit wird mittels shRNA-vermittelter Depletion von Huwe1 in Zellkulturexperimenten gezeigt, dass Huwe1 f{\"u}r die Proliferation von Kolonkarzinomzelllinien und f{\"u}r die Transaktivierung von Myc-Zielgenen ben{\"o}tigt wird. Mit zwei von Boehringer Ingelheim identifizierten niedermolekularen Huwe1-Inhibitoren (BI8622 und BI8626) ist es m{\"o}glich, die Huwe1-Funktion spezifisch in Zellen zu blockieren. Die Huwe1-Inhibitoren induzieren einen Proliferationsarrest in kolorektalen Karzinomzelllinien, wohingegen die Substanzen auf embryonale Stammzellen keine Auswirkungen haben. Die Inaktivierung von Huwe1 f{\"u}hrt zu einer Akkumulation von Miz1 an Promotoren Myc-aktivierter Zielgene und dar{\"u}ber zu einer vermehrten Bildung repressiver Myc/Miz1-Komplexe, was mit einer Deacetylierung von Histon H3 und einer transkriptionellen Repression Myc-gebundener Gene assoziiert ist. Miz1 akkumuliert nach Huwe1-Inhibition ebenso an direkten Miz1-Zielgenen, deren Expression bleibt aber unbeeinflusst. Diese Daten weisen darauf hin, dass eine kontinuierliche Degradierung von Miz1 durch Huwe1 zur Transaktivierung von Myc-Zielgenen in Kolonkarzinomzellen n{\"o}tig ist. Damit wurde ein neuer Mechanismus identifiziert, {\"u}ber den Huwe1 die Myc-Transaktivierung reguliert und der eine tumorzellspezifische Repression der Myc-Funktion mit Hilfe von Huwe1-Inhibitoren erm{\"o}glicht.},
  subject      = {Myc},
  language  = {de}
}