ESR 15 Project - Role of redox signalling on mtDNA expression and regulation
Work Package 5: Novel insights into maintenance of mitochondrial genome.
Partner: Jan Smeitink
Institution: Khondrion, Nijmegen, The Netherlands
Duration: 36 months
Objectives: Oxidative stress is a common patho-mechanism described in many mitochondrial (-related) disorders. Oxidative stress can occur via free radical-induced macromolecular damage (unpaired electrons) but also via non-radical (2-electrons) disruption of cellular redox circuits, which normally function in cell signalling and physiological regulation. The mitochondrial Electron Transport Chain (ETC) is one of the cellular sources of reactive oxygen species (ROS). Dysfunctions in the ETC functioning are often causative of higher mitochondrial ROS production, but also of a disturbance in the redox regulating systems (i.e. oxidation state of peroxyredoxin and thioredoxin systems, GSH/GSSG, NADH/NAD+ levels). Blood samples from mitochondrial patients are indeed characterized by a more oxidized mitochondrial environment as compare to healthy individuals. Although much research has focused on the oxidation and consequent mutation of the mitochondrial DNA (mtDNA) specially in relation to ageing the oxidation of mitochondrial proteins should also be considered a target for ROS. Protein oxidation results mainly in the introduction of carbonyl groups, which may alter both the structure and function of the protein. In particular, the mitochondrial DNA polymerase (Pol γ), because of its location within the mitochondria and its association with the mtDNA, is exposed to various ROS and ist function can be impaired upon oxidation. Considering the tight relationship among the proteins involved in mtDNA gene expression, it can be expected that oxidative modifications of one or more of these proteins might affect the whole mtDNA gene expression. Moreover a concomitant disturbance of the redox regulating circuits can affect the mitochondrial-nuclear crosstalk, fundamental for proper expression of the mtDNA. Mitochondrial patient cell lines (primary fibroblasts) with clear oxidative stress features (i. e. high ROS levels, impaired redox regulation) will be used to visualize and quantify (in-cell immunoblotting) key proteins participating in mtDNA gene expression (i.e. Pol γ, POLRMT, TFAM) in order to generate a protein profile of the specific mitochondrial patient cell lines. Additionally the oxidation state of the mitochondrial proteome will be assessed under physiological and/or oxidative stress-stimulating conditions. The large amount of data that will be generated will be analyzed with computational tools in order to: (i) Find a protein “signature” of the disease (amount, localization and oxidation state) -> possible diagnostic tools; (ii) Find new biomarkers; (iii) Identify new targets for possible treatments; (iv) Better understand the molecular mechanism of the disease. Khondrion library of bioactive compounds with antioxidant and redox modulating properties will be then tested on cell lines showing aberrant protein oxidation patterns for their ability to reverse oxidative damage.
Expected results: This project aims to characterize novel factors involved in the correct assembly of human mitochondrial ribosomal proteins to facilitate normal protein synthesis, as well as to determine the molecular mechanisms that govern the assembly process.