Stefano Conti-Nibali

Ciclo: XXXIV

Data inizio: 31/10/2018

Curriculum: Biomolecolari

Borsa: UniCT

Titolo tesi: Electrophysiological analysis of human VDAC proteins incorporated into the Nanodiscs and investigation of VDAC isoform 3 cysteine residues in mitochondrial functionality and ROS buffering

Abstract: Mitochondria are crucial organelles for eukaryotic cells since they support the huge energy demand required to maintain cellular homeostasis. Communication between cytosol and mitochondria is allowed by mitochondrial porins, also known as Voltage-Dependent Anion selective Channels (VDACs), localized in the outer mitochondrial membrane of all eukaryotes. VDACs allow metabolite exchanges across the organelle and participate in numerous cellular pathways due to the interaction with cytosolic enzymes and apoptotic factors. There are three different isoforms in mammals: VDAC1, VDAC2, and VDAC3. Although sharing a high sequence homology, the three isoforms present different functions and characteristics.

The first part of this Ph.D. thesis focuses on the electrophysiological analysis of VDAC proteins produced using a cell-free expression system associated with nanodiscs (NDs), which offer a native- like environment. This new expression method represents a powerful alternative to avoid protein refolding procedures. In this work, the in vitro protein synthesis system was used for the first time to express the three human VDAC isoforms. After their reconstruction into Planar Lipid Bilayer, the electrophysiological features were compared with those obtained by canonical expression protocol. Furthermore, we analyzed the impact of both reducing agents in the buffer and cysteine removal on the biophysical characteristics of human VDAC3. Our results showed that this new system did not change the electrophysiological features of VDACs, and additionally validated the extreme importance of cysteine residues in the channel functionality of VDAC3.

The second part of the thesis concerns the investigation of human VDAC3 contribution in oxidative stress response. Among the three isoforms in mammals, VDAC3 has long been the research topic of De Pinto’s group as it represents the least abundant and the most enigmatic one. It features untypical biophysical properties that have been attributed to the specific oxidative status of its cysteine residues. Moreover, clues within the literature have suggested VDAC3 as a putative redox sensor, stressing its involvement in mitochondrial reactive oxygen species homeostasis. A direct proof, however, is missing. This work aimed to evaluate the role of VDAC3 on mitochondrial functionality using the near-haploid human HAP1 cell line devoid of VDAC3. This work revealed that VDAC isoform 3 prevents mitochondrial impairment induced by oxidative stress. For the first time, we have also proved that cysteine residues of VDAC3 are responsible for the ability of the protein to counteract mitochondrial ROS overload.

Tutor: De Pinto

Data Conseguimento Titolo: 29/03/2021

Linkedin: Indicate il link

Email: stefano92co@hotmail.it

Periodi all'estero- Sede e data: Si

Esperienze post-Dottorato ed attuale occupazione: Research Fellow dal 01/03/2022 presso Università di Pavia, Dipartimento di Medicina Molecolare