@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}
}