Abstract

Mitochondrial ATP synthase is at the core of energy conservation in mammalian cells. It is organized in catalytic F1 and intramembrane FO subcomplexes connected by a central stalk (CS) and a peripheral stalk (PS). Besides ATP production and cristae formation, ATP synthase is involved in generating the permeability transition pore (PTP), a channel mediating fast Ca2+ release (short open times) and cell death (long open times). We will investigate the mechanisms underlying the transition of ATP synthase to the PTP and further define its roles in pathophysiology.

The hypothesis about PTP formation is that replacement of Mg2+ with Ca2+ at the F1 catalytic site causes a conformational change transmitted through oligomycin-sensitivity conferral protein (OSCP) to the PS, then to the Fo “bundle region” connecting subunits b, g and e, and finally relayed to the c-ring through a pulling action of the C-terminus of subunit e, which connects with the outer lipid plug of the c-ring. PTP opening is favored by binding of CyPD to OSCP, which would increase the rigidity of the PS. We will attack the problem as follows (Fig.1).

1. We have generated mice bearing a conditional T213S mutation in subunit beta of ATP synthase (Atp5bT213S), which decreases the affinity for Ca2+ but not Mg2+ desensitizing the PTP to the inducing effects of Ca2+. Expression of the T213S mutant is triggered by crossing with CRE-expressing mice followed by tamoxifen treatment. The PTP will be characterized in isolated mitochondria (initially from liver) before and after activation of the transgene to analyze the phenotypic effects of the mutation. In parallel, a full phenotypic characterization of the Atp5bT213S mice will be performed. Mutant cell lines will also be generated and characterized for growth rates, replication time, incidence of apoptosis, mitochondrial phenotype, respiratory activity, propensity to PTP opening by Ca2+, electrophysiological features of the PTP.
2. We have generated cells where subunit e has been replaced by a truncated version lacking 10 Aa at the C-terminus, which allows assembly of a functional ATP synthase able to form dimers; and we are generating Drosophila larvae and adults after knockdown of subunits e and g to gain more insights about the function of dimerization subunits in a living animal. These studies will be matched by molecular dynamics investigations of the possible events underlying channel formation.
3. We have found that the N-terminal tail of CyPD is highly mobile and its deletion markedly increases CyPD binding to OSCP in in vitro experiments. We will define how this domain, located just before the isomerase domain, regulates PTP opening by modification of CyPD addressing and/or by influencing its global post-translation modifications.

Partenariato

• Università di Padova
• Università degli studi di Udine

Importo del progetto

Importo totale del progetto        Euro 164.201,00
Importo del progetto Uniud        Euro 87.654,60
Finanziamento Uniud                Euro 76.546,00

Durata

• Dal 04.02.2025
• Al 03.02.2027