DJ-1 is a small, highly conserved protein of 189 amino acids, which is ubiquitously expressed. In humans, DJ-1 is encoded by the PARK7 gene, which was first linked to early onset, autosomal recessive familial forms of Parkinson’s disease (PD) in 2003. It is still unclear how DJ-1 contributes to PD pathogenesis: the high number of different physiological functions attributed to DJ-1 represent an important drawback in the comprehension of how its mutations cause PD. DJ-1 can work as an oxidative or stress level sensor, but it can also directly participate in ROS neutralizations as scavenger. Furthermore, the protein has been shown to function as a molecular chaperone, protease, transcriptional regulator, RNA-binding protein, mitochondrial function regulator and autophagy regulator, and it is involved in regulation of catecholamine homeostasis. Recent studies developed in our lab explores DJ-1 modulation of cell organelles contacts, how its concentration affects the number and the form of organelles tethering, such as that between endoplasmic reticulum (ER) and mitochondria or lysosomes and mitochondria. To visualize organelle contacts sites the laboratory where I spent my thesis project has developed new recombinant tool, the SPLICS probes, based on the SplitGFP and Bimolecular Fluorescence Complementation (BiFC) technology. Their functioning takes advantage from the ability of two non-fluorescent fragments of the GFP to restore the fully fluorescent signal when they are close enough to reconstitute the full GFP by complementation. This property was exploited to visualize contact sites between the membrane of two different organelles upon the selective targeting the two GFP portions to the membrane of the organelles of interest by the addition of specific localization sequences. In my thesis project it has been specifically examined how DJ-1 overexpression in mammalian cells affects contact sites between mitochondria and ER or lysosomes.
DJ-1 è una piccola proteina di 189 amminoacidi altamente conservata ed espressa in modo ubiquitario. Nell’uomo DJ-1 è codificata dal gene PARK7, il quale era stato correlato nel 2003 a forme autosomiali recessive del morbo di Parkinson familiare. Ancora non è chiaro il contributo che DJ-1 ha nell’insorgenza del Parkinson poiché l’elevato numero di funzioni fisiologiche diverse attribuite a questa proteina rende complicata la comprensione del legame tra le sue mutazioni e la patologia. DJ-1 funziona da sensore dello stress ossidativo ma può anche partecipare direttamente nella neutralizzazione di ROS come scavenger. Inoltre, la proteina può svolgere funzioni di chaperonina, proteasi, regolatore trascrizionale, proteina legante RNA, regolatore delle funzioni mitocondriali e regolatore autofagico, oltre a poter essere coinvolta nella regolazione dell’omeostasi delle catecolamine. Studi recenti condotti nel nostro laboratorio hanno esaminato l’effetto che DJ-1 può avere sulla modulazione dei contatti tra organelli cellulari e come la sua concentrazione può influire sul numero e la forma di questi siti di contatto, come quelli tra reticolo endoplasmatico e mitocondri o tra lisosomi e mitocondri. Per visualizzare i contatti, il laboratorio che ho frequentato per il mio progetto di tesi ha sviluppato un nuovo strumento ricombinante, le sonde SPLICS, basate sulla tecnologia della complementazione della fluorescenza bimolecolare (BiFC). Il loro funzionamento sfrutta l’abilità di due frammenti di GFP non fluorescenti di ricostituire la GFP completa quando si trovano sufficientemente vicini, ripristinando così il segnale fluorescente. Questa proprietà è stata utilizzata per visualizzare i siti di contatto tra le membrane di due diversi organelli attraverso il targeting selettivo delle due porzioni non fluorescenti con delle sequenze specifiche di localizzazione. Nel mio progetto di tesi viene indagato come l’overespressione di DJ-1 in cellule di mammifero influenza i siti di contatto tra mitocondri e reticolo endoplasmatico o lisosomi.
Exploring mitochondrial contact sites in DJ-1 overexpressing cells
PIOVAN, ANDREA
2021/2022
Abstract
DJ-1 is a small, highly conserved protein of 189 amino acids, which is ubiquitously expressed. In humans, DJ-1 is encoded by the PARK7 gene, which was first linked to early onset, autosomal recessive familial forms of Parkinson’s disease (PD) in 2003. It is still unclear how DJ-1 contributes to PD pathogenesis: the high number of different physiological functions attributed to DJ-1 represent an important drawback in the comprehension of how its mutations cause PD. DJ-1 can work as an oxidative or stress level sensor, but it can also directly participate in ROS neutralizations as scavenger. Furthermore, the protein has been shown to function as a molecular chaperone, protease, transcriptional regulator, RNA-binding protein, mitochondrial function regulator and autophagy regulator, and it is involved in regulation of catecholamine homeostasis. Recent studies developed in our lab explores DJ-1 modulation of cell organelles contacts, how its concentration affects the number and the form of organelles tethering, such as that between endoplasmic reticulum (ER) and mitochondria or lysosomes and mitochondria. To visualize organelle contacts sites the laboratory where I spent my thesis project has developed new recombinant tool, the SPLICS probes, based on the SplitGFP and Bimolecular Fluorescence Complementation (BiFC) technology. Their functioning takes advantage from the ability of two non-fluorescent fragments of the GFP to restore the fully fluorescent signal when they are close enough to reconstitute the full GFP by complementation. This property was exploited to visualize contact sites between the membrane of two different organelles upon the selective targeting the two GFP portions to the membrane of the organelles of interest by the addition of specific localization sequences. In my thesis project it has been specifically examined how DJ-1 overexpression in mammalian cells affects contact sites between mitochondria and ER or lysosomes.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/34754