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Doctoral thesis
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Drivers of cell cycle progression: structures and functions of the E3 ubiquitin ligases TRIP12 and APC/C

ContributorsHoefler, Annaorcid
Imprimatur date2023-12-04
Defense date2023-11-13
Abstract

Ordered progression through the cell cycle is the most fundamental task in the life of a cell. To ensure it happens timely and correctly, the synthesis and degradation of regulatory proteins are orchestrated through the ubiquitination and ubiquitination degradation pathway to guarantee correct protein turn-over. This turn-over is regulated by E3 ubiquitin ligases, the substrate defining enzymes of this mechanism. In humans more than 600 have been identified, each specialised to target a specific set of protein substrates. The Anaphase-Promoting complex, also known as cyclosome (or APC/C), is an E3 ubiquitin ligase of megadalton size and a key component in the temporal turn-over of cyclins and cell cycle regulatory proteins. APC/C itself is regulated through several independent regulatory mechanisms, such as the inhibiting proteins and protein complexes EMI1 or EMI2, the mitotic checkpoint complex, phosphorylation and the mutually exclusive binding to CDH1 and CDC20 cofactors.

TRIP12 is a different E3 ubiquitin ligase regulating the P53 response pathway and is involved in the initiation of apoptosis and cell cycle arrest. To do this, TRIP12 ubiquitinates P14ARF, which is essential to stabilise P53 in the nucleus. P14ARF turn-over allows P53 degradation and guarantees progression through the cell cycle.

This PhD work focusses on the structural and biochemical characterisation of the E3 ubiquitin ligases TRIP12 and APC/C by single particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. The results of this study identify TRIP12 as a functional monomer, with a flexible hinge between the N-terminal Armadillo repeat domain and the HECT domain at its C-terminus. In contrast to previous publications, no in vitro ubiquitination of P14ARF was detectable in this study. However, it is hypothesised that P14ARF is shielded from TRIP12 ubiquitination by MDM2.

High-resolution cryo-EM structures of the APC/CCDH1:EMI1 and the APC/Capo complexes at resolutions of 2.9 Å and 3.2 Å were obtained. Improvements of cryo-EM maps allowed the modelling of previously unassigned protein sequences and the identification of a novel anchoring mechanism of the APC/C cofactor CDH1. The N-terminal helix of CDH1 was structurally resolved for the first time and shown to interact with the APC/C subunits APC1 and APC8. Based on this information a novel discrimination mechanism between CDH1 and CDC20 was discovered. Analysis of the cryo-EM map of the apo form of APC/C confirmed the binding of the autoinhibitory segment of APC1 to one of the CDH1 and CDC20 binding sites, providing the first experimental structural evidence of this inhibitory mechanism. In addition, a previously unknown Zn2+-binding module in the APC2 subunit was identified.

In an unrelated project, the leptin receptor extracellular domain (LR-ECD) was structurally and evolutionarily investigated. The leptin receptor belongs to the class I cytokine receptors and is involved in the regulation of appetite and food uptake. Biochemical experiments and low-resolution structural information obtained in this study show the appearance of monomeric proteins with a 1:1 stoichiometry, however, small quantities of higher order oligomeric states were confirmed. Newly published information in 2023 showed the determination of the structure of the LR-ECD by two independent groups and corroborated the evidence collected in this work.

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Citation (ISO format)
HOEFLER, Anna. Drivers of cell cycle progression: structures and functions of the E3 ubiquitin ligases TRIP12 and APC/C. 2023. doi: 10.13097/archive-ouverte/unige:176383
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Creation04/03/2024 7:09:47 PM
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