Objective: To explore the role of ubiquitin signalling defects in Parkinson’s Disease.

Background: Defects in ubiquitin signalling have been implicated in the pathogenesis of neurodegenerative diseases including Parkinson’s Disease (PD)¹. This is best demonstrated by the E3 ubiquitin ligase Parkin (PRKN), which regulates mitochondrial autophagy², being the most common pathogenic mutation in autosomal recessive PD. However, an unbiased exploration of the overall ubiquitin landscape (ubiquitome) in PD is lacking and may provide new insights into the disease.

Method: The ubiquitomes of 15 early-onset PD patient-derived human dermal fibroblasts (HDFs) were compared against 6 healthy controls. The PD cohort consisted of 6 PRKN mutants, 4 GBA1 mutants and 5 idiopathic (no identified pathogenic PD mutation). Ubiquitinated proteins or ubiquitin-bound proteins were enriched from cell lysates using tandem ubiquitin binding elements (TUBEs). These ubiquitin-enriched fractions were subjected to mass spectrometry analysis. Mass spectrometry analysis of the whole cell proteome was examined in parallel to the ubiquitome.

Results: Overall, approximately 7000 ubiquitinated proteins were identified in total through mass spectrometry analysis after TUBEs pulldown. Comparisons of each genetic PD cohort vs healthy controls revealed distinct differences in significantly (p<0.05) differentially expressed ubiquitinated proteins (116 in GBA1 v control, 49 in PRKN v control, 16 in idiopathic v control). Examination of the proteome identified approximately 8000 proteins. There were higher numbers of differential expressed proteins in the same PD cohort comparisons (276 in GBA1 v control, 115 in PRKN v control, 102 in idiopathic v control). Comparing all PD vs controls resulted in fewer significantly differentially expressed proteins (9 in the ubiquitome and 10 in the proteome).

Conclusion: We have optimised a workflow to characterise the ubiquitome in patient derived HDFs. Using this approach, we identified/observed significant differences in the ubiquitome and proteome in 3 early-onset PD cohorts vs healthy controls. These differences are being explored to provide new mechanistic insights into disease pathogenesis or as potential biomarkers in PD.

References: 1. Schmidt MF, Gan ZY, Komander D, Dewson G. Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities. Cell Death Differ. 2021 Feb;28(2):570-590. doi: 10.1038/s41418-020-00706-7. Epub 2021 Jan 7. PMID: 33414510; PMCID: PMC7862249.
2. Kamienieva I, Duszyński J, Szczepanowska J. Multitasking guardian of mitochondrial quality: Parkin function and Parkinson’s disease. Transl Neurodegener. 2021 Jan 20;10(1):5. doi: 10.1186/s40035-020-00229-8. PMID: 33468256; PMCID: PMC7816312.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/exploring-the-ubiquitome-in-parkinsons-disease/