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Characterising the functional domains of the candidate ALS protein, cyclin F
thesisposted on 2022-03-28, 11:40 authored by Kimberley Duncan
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the rapid degeneration of motor neurons leading to muscle paralysis and death. The only known causes of ALS are gene mutations, several of which are intrinsic to protein degradation pathways. Dysfunctional protein degradation could lead to abnormal protein accumulation in neurons, a hallmark of ALS pathology. Our laboratory recently identified ALS-linked mutations in CCNF, which encodes cyclin F, an E3 ubiquitin ligase that mediates the destruction of aberrant proteins by the ubiquitin proteasome system (UPS). The overall aim of this project was to characterise the function of cyclin F domains and predict the effect of ALS-linked mutations on protein function. First, bioinformatic modelling was performed to visualise the proximity of cyclin F mutations to active sites and predict their effect on protein function. Next, a series of CCNF deletion clones were generated for expression in neuronal cell lines to assess the effect of deleting functional domains on sub-cellular localisation of cyclin F as well as toxicity. 3D modelling of the mutations within the cyclin box show that they may affect substrate binding, while deletion cloning studies show that mutations within PEST could affect protein stability and cause mis-localisation, leading to protein aggregation. This could lead to the enhanced cytotoxicity that was found in PEST cyclinF-transfected cells. These deletion constructs can be utilised in future in vitro studies to further examine the effects of individual domains on protein binding, stability and UPS function, to elucidate the potentail mechanisms that cause neuronal death.