Quirin 2015 Abstract MiPschool London 2015
|Targeting Opa1 to increase mitochondria-dependent apoptosis of cancer cells.|
Event: MiPschool London 2015
In addition to providing the majority of cellular ATP, mitochondria mediate intrinsic apoptosis, which is characterized by the release of apoptogenic factors, including cytochrome c, from the mitochondria into the cytosol. The majority of cytochrome c is stored in cristae formed by the inner mitochondrial membrane, which are tightened at the cristae junctions by oligomers of the GTPase Opa1. During the process of apoptosis these cristae undergo a conformational change, termed cristae remodeling, characterized by the disruption of Opa1 oligomers, the opening of the cristae junctions and the subsequent release of cytochrome c [1,2]. We hypothesize that pharmacological inhibition of the GTPase activity of Opa1 can facilitate cytochrome c release by inducing the opening of the cristae, and consequently sensitizing cells to apoptotic stimuli. Here we show the steps towards the identification of a pharmacological inhibitor of the GTPase activity of Opa1.
For that, an enzymatically active recombinant form of Opa1 is purified to perform a high-throughput screen of a small compound library for their effect on Opa1 GTPase activity. We characterized the kinetic parameters Km and kcat of Opa1 to adjust an enzymatic-screening assay accordingly in which the hydrolysis of GTP by Opa1 will be measured . Concurrently, we are generating an in silico model of the GTPase domain of Opa1 which allows us to analyze the intermolecular interactions of potential inhibitors with Opa1 to optimize lead molecules.
This progress shall lead to the identification of a pharmacological drug for a novel target, the GTPase Opa1.
Regulation: Cyt c
1-Biol Dept, Padova Univ, Italy
2-DTI, Venetian Institute of Molecular Medicine, Padova, Italy
3-Pharmacology Dept, Padova Univ, Italy. - [email protected]
- Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA, Korsmeyer SJ (2002) A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Developm Cell 2:55–67.
- Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T, Bartoli D, Polishuck RS, Danial NN, De Strooper B, Scorrano L (2006) OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion. Cell 126:177–89.
- Cogan EB, Birrell GB, Griffith OH (1999) A robotics-based automated assay for inorganic and organic phosphates. Analyt Biochem 271:29–35.