Parkinson’s Disease (PD) is the second most frequent neurodegenerative disease, affecting 2% of the over-65 population. The aetiology of PD is unknown; however, it is a multifactorial disease in which both environmental and genetic factors play a role. Familial forms of PD, characterised by autosomal dominant or recessive inheritance, account for only 5-15% of cases but represent a crucial opportunity to shed light on the molecular mechanisms that underlie the disease pathogenesis. This thesis focuses on the two proteins encoded by the human genes PINK1 and UQCRC1. In 2004, nonsense and missense mutations in the PINK1 gene were associated with the development of autosomal recessive early onset PD. In a 2020 study, UQCRC1 gene mutations were linked to late-onset autosomal-dominant parkinsonism and polyneuropathy. In this project, (co)transfection, Western Blot, co-immunoprecipitation technique, and immunofluorescence were used to test the hypothesis of a putative interaction between the two proteins PINK1 and UQCRC1, both of which are localised in the mitochondria. Two human cancer cell lines, HeLa and HEK293T cells, were utilised to overexpress both proteins that were required for the execution of co-immunoprecipitation. Immunofluorescence was used to demonstrate the proteins’ co-localisation in these cell lines. The results showed in this work constitute preliminary data in support of a direct protein-protein interaction between PINK1 and UQCRC1, laying the ground for a new investigative line of research to better understand the implications of both mitochondrial proteins in the pathways of PD development. Further studies based on different techniques will be necessary to definitely confirm the hypothesis.
La malattia di Parkinson, che colpisce il 2% degli over 65, è la seconda malattia neurodegenerativa più frequente. L'eziologia della malattia di Parkinson è sconosciuta, tuttavia, si tratta di una patologia multifattoriale in cui sia fattori ambientali che genetici svolgono un ruolo fondamentale nello sviluppo. Le forme familiari sono caratterizzate da ereditarietà autosomica dominante o recessiva e rappresentano il 5-15% dei casi, ma costituiscono un'opportunità cruciale per far luce sui meccanismi molecolari che sono alla base della patogenesi della malattia. Questa tesi si concentra sulle due proteine codificate dai geni umani PINK1 e UQCRC1. Nel 2004, le mutazioni nonsense e missense nel gene PINK1 sono state associate allo sviluppo di malattia di Parkinson ad esordio precoce con ereditarietà autosomica recessiva. In uno studio del 2020, le mutazioni del gene UQCRC1 sono state collegate a forme di parkinsonismo e polineuropatia con ereditarietà autosomica dominante. In questo progetto, (co)trasfezione, Western Blot, co-immunoprecipitazione, e immunofluorescenza sono stati utilizzati per testare l'ipotesi di una presunta interazione tra le due proteine PINK1 e UQCRC1, entrambe localizzate nei mitocondri. Due linee cellulari tumorali umane, cellule HeLa e HEK293T, sono state utilizzate per over-esprimere entrambe le proteine che erano richieste per l'esecuzione della co-immunoprecipitazione. L'immunofluorescenza è stata usata per dimostrare la co-localizzazione delle proteine in queste linee cellulari. I risultati mostrati in questo lavoro costituiscono dati preliminari a sostegno di un'interazione diretta proteina-proteina tra PINK1 e UQCRC1, ponendo le basi per una nuova linea di ricerca per comprendere meglio i ruoli di entrambe le proteine mitocondriali nei pathways di sviluppo della malattia di Parkinson. Ulteriori studi basati su diverse tecniche saranno necessari per confermare definitivamente l'ipotesi.
Characterisation of a novel interaction between human PINK1 and UQCRC1, two mitochondrial proteins involved in Parkinson’s Disease
PEREZ MORRISSEY, ELENA MARIE
2021/2022
Abstract
Parkinson’s Disease (PD) is the second most frequent neurodegenerative disease, affecting 2% of the over-65 population. The aetiology of PD is unknown; however, it is a multifactorial disease in which both environmental and genetic factors play a role. Familial forms of PD, characterised by autosomal dominant or recessive inheritance, account for only 5-15% of cases but represent a crucial opportunity to shed light on the molecular mechanisms that underlie the disease pathogenesis. This thesis focuses on the two proteins encoded by the human genes PINK1 and UQCRC1. In 2004, nonsense and missense mutations in the PINK1 gene were associated with the development of autosomal recessive early onset PD. In a 2020 study, UQCRC1 gene mutations were linked to late-onset autosomal-dominant parkinsonism and polyneuropathy. In this project, (co)transfection, Western Blot, co-immunoprecipitation technique, and immunofluorescence were used to test the hypothesis of a putative interaction between the two proteins PINK1 and UQCRC1, both of which are localised in the mitochondria. Two human cancer cell lines, HeLa and HEK293T cells, were utilised to overexpress both proteins that were required for the execution of co-immunoprecipitation. Immunofluorescence was used to demonstrate the proteins’ co-localisation in these cell lines. The results showed in this work constitute preliminary data in support of a direct protein-protein interaction between PINK1 and UQCRC1, laying the ground for a new investigative line of research to better understand the implications of both mitochondrial proteins in the pathways of PD development. Further studies based on different techniques will be necessary to definitely confirm the hypothesis.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15513