University of Cambridge
The discovery of a role of FBXO7/PARK15 in intracellular trafficking
Abstract
dc:description.abstractParkinson’s Disease (PD) is the second most common neurodegenerative disease globally, and yet our understanding of the underlying mechanisms causing the disease is poor, making development of novel therapies challenging. Intracellular trafficking pathways, including Kinesin and Dynein motor transport and the secretory pathway, are emerging as dysregulated pathways in PD contributing to Alpha-synuclein accumulation and neuronal dysfunction. These mechanisms can be studied using genetic cell- or animal models expressing PARK-gene mutations. PARK15/FBXO7 encodes the F-box protein Fbxo7, which has a wide range of functions both as the substrate recruiting subunit of a Skp1-Cullin1-F-box (SCF) E3 Ubiquitin ligase complex and independent of SCF-binding. Known functions of Fbxo7 include regulation of mitophagy, the cell cycle and the proteasome. Loss of Fbxo7 has been associated with reduced proteasome transport to synapses via the proteasome regulator PI31, but a direct link between Fbxo7 and intracellular trafficking remains to be uncovered. We identified over 30 trafficking proteins in a substrate screen for SCFFbxo7 ubiquitination, including the Dynein regulators Lis1 and Nde1. I validated Lis1, Nde1 and the Nde1 paralog protein Ndel1 as interacting proteins of Fbxo7 by co-immunoprecipitation experiments. The results presented in this thesis show that steady state levels of Lis1 are not affected by Fbxo7, while it may have a stabilising effect on Nde1. Furthermore, in vivo and in vitro ubiquitination assays established that Lis1 is a substrate of SCFFbxo7 ubiquitination. I discovered the secretory pathway to be strongly affected by Fbxo7 KD in multiple different cell types and showed secretion defects using both Retention Using Selective Hooks (RUSH) assays and Immunoglobulin G (IgG) secretion assays. Cells with reduced Fbxo7 displayed abnormal Golgi morphology and accumulation of secretion cargo in the Golgi. By complementing Fbxo7 KD cells with transgenic Fbxo7, I rescued the secretion defect and showed that this required the ubiquitin ligase activity of Fbxo7. In addition to Dynein regulation, Lis1 is a regulator of the phospholipase complex PAFAH1B, which functions in Golgi tubule formation required for efficient secretory trafficking. Based on results from experiments using Lis1 ubiquitination mutants and LC-MS analysis, I present a model where SCFFbxo7-mediated ubiquitination of Lis1 works as a switch between its functions as a Dynein regulator and a PAFAH1B regulator. In conclusion, a novel role for Fbxo7 has been discovered in secretory trafficking and this is associated with ubiquitination of Lis1. This highlights the secretory pathway and ubiquitination of trafficking proteins as promising targets for therapeutic targeting in PD-treatment.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Bjone, Hanna
- Advisor dc:contributor.advisor
-
- Laman, Heike
Subjects
dc:subject × 8Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.125220
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/395850