Structural and functional analyses of the HECT E3 ligase HACE1
Dissertation
Datum der mündl. Prüfung:2024-03-26
Erschienen:2024-04-19
Betreuer:Dr. Sonja Lorenz
Gutachter:Dr. Sonja Lorenz
Gutachter:Prof. Dr. Peter Rehling
Dateien
Name:Dissertation_Düring.pdf
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Description:Dissertation
Diese Datei ist bis 25.03.2025 gesperrt.
Zusammenfassung
Englisch
Many crucial cellular processes rely on post-translational modifications. In particular, the covalent attachment of ubiquitin molecules, termed ubiquitylation, onto substrates, typically via lysine residues, is most commonly associated with a signal for the proteasomal degradation of the respective modified proteins, among many other roles. Ubiquitylation is mediated by a cascade of enzyme classes: few different ubiquitin-activating enzymes (E1) transfer ubiquitin onto several different ubiquitin-conjugating enzymes (E2), before the final transfer to specific substrates is mediated by many different ubiquitin ligases (E3). HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1) belongs to the HECT-subclass of E3 ligases and as such employs a catalytic cysteine to accept thioester-linked ubiquitin from E2 enzymes and directly catalyze the isopeptide bond-formation between ubiquitin and substrates. Initially characterized as a tumor suppressor absent in a Wilm’s tumor (kidney cancer) patient, loss of HACE1 function is associated with a broad spectrum of cancers as well as neurodevelopmental and -degenerative diseases. This dissertation describes the structural and functional elucidation of HACE1. It demonstrates that recombinantly expressed and purified HACE1 forms a stable homodimer, as shown by size exclusion chromatography, mass photometry, and small-angle X-ray scattering. The physiological relevance of this self-association was furthermore supported in ectopic and near-endogenous cellular co-immunoprecipitation experiments as well as native PAGE analyses of endogenous protein in murine brain homogenates. Dimerization is mediated by an N-terminal helix contacting the C-terminal HECT domain in trans, as shown by a cryo-EM structure of the full-length dimeric HACE1 complex as well as hydrogen-deuterium exchange mass spectrometry. The N-terminal helix thereby obstructs the canonical E2-binding site of the HECT domain, explaining why the HACE1 dimer represents an inactive, autoinhibited state deficient in accepting thioester-linked ubiquitin from E2 enzymes. Disruption of dimerization by mutating or truncating the helix lifts autoinhibition and induces strong ubiquitylation of the HACE1 model substrate RAC1, both in vitro and in cellular assays. A similar effect was achieved with certain phosphomimetic substitutions, indicating a potential mode of regulation via kinases. This dissertation provides biochemical and structural insights into the regulation of a HECT E3 ligase. The monomeric HACE1 variants developed here could be used to identify and characterize oligomerization-dependent interactors and regulators of HACE1 and better understand pathologies.
Keywords: Ubiquitin; E3 ligase; HECT; HACE1; RAC1; Autoinhibition; Cryo-EM