Regulation of the activity of ubiquitin ligase HACE1

master's thesis

• Title (croatian): Regulacija aktivnosti ubikvitinske ligaze HACE1
• Author: Julia Jelena Toplak
• Mentor: Sonja Lorenz (mentor)
• Mentor: Dunja Leljak-Levanić (komentor)
• Committee member: Dunja Leljak-Levanić (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Ivana Ivančić Baće (član povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-09-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

The HECT ubiquitin ligase HACE1 is involved in membrane dynamics regulation, mitophagy,
redox homeostasis, and cytoskeleton organization. Despite HACE1 being a tumor suppressor and
a significant factor in neural development, it remains biochemically poorly characterized, and its
regulatory mechanisms are not known. This master’s thesis explores the impact of phosphorylation
as a regulatory mechanism and its effect on HACE1 activity and dimerization. Phosphomimetic
variants were generated and characterized using functional assays, mass photometry, and analytical
size exclusion chromatography. The findings reveal that the phosphomimetic mutations within the
N-terminal helix disrupted dimerization and activated HACE1 activity, providing insights into how
the monomer could be stabilized in its active state. Furthermore, recombinantly purified kinases
PAK1 and TBK1 were investigated as potential regulators of HACE1 activity, and the sites targeted
by these kinases in HACE1 sites were identified through mass spectrometry.

Abstract (croatian)

Ubikvitinska HECT ligaza HACE1 sudjeluje u regulacije dinamike membrana, mitofagiji, redokshomeostazi i organizaciji citoskeleta. Unatoč tome što je ligaza HACE1 tumor-supresor i značajan
faktor u razvoju živčanog sustava, nije detaljno biokemijski karakterizirana, a regulatorni
mehanizmi nisu poznati. Ovaj diplomski rad istražuje utjecaj fosforilacije kao regulatornog
mehanizma na aktivnost i dimerizaciju HACE1. Pripremljene su fosfomimetičke varijante i
karakterizirane korištenjem funkcionalnih eseja, masene fotometrije i analitičke kromatografije
temeljene na isključivanju. Rezultati otkrivaju da fosfomimetičke varijante unutar N-terminalnog
heliksa narušavaju dimerizaciju i aktiviraju aktivnost ligaze HACE1, pružajući uvid u to kako se
monomer može stabilizirati u aktivnom stanju. Nadalje, rekombinantno pročišćene kinaze PAK1 i
TBK1 istražene su kao potencijalni regulatori. Mjesta u ligazi HACE1 koje te kinaze ciljaju
identificirana su putem masene spektrometrije.

• Keywords: ubiquitin ligase; HACE1; ubiquitylation; phosphorylation; HECT
• Keywords (croatian): ubikvitin ligaza; HACE1; ubikvitilacija; fosforilacija
• Language: english
• URN:NBN: urn:nbn:hr:217:358497
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-09-21
• Created on: 2023-10-25 10:22:08