Molecular and structural studies of proteins required for mitochondrial DNA maintenance

Abstract

Mitochondria are membrane-bound organelles that produce the majority of ATP used to drive metabolic processes in eukaryotic cells. A unique feature of mitochondria is the existence of a separate mitochondrial DNA (mtDNA), which codes for thirteen proteins needed for oxidative phosphorylation (OXPHOS). The double-stranded mtDNA is copied by a dedicated enzymatic machinery, which includes mitochondrial DNA polymerase γ (POLγ) and a set of accessory proteins. Mitochondria also harbor transcription and translation machineries, which are distinct from those in the nucleus. Mutations that affect factors required for mtDNA maintenance or expression can cause mitochondrial disease.

In the present thesis, we have characterised a subset of proteins which play an important role in replication and transcription of mtDNA, both from a structural and functional point of view. We have studied mitochondrial helicase TWINKLE and analysed the molecular consequences of disease-causing mutations in this protein. By means of electron microscopy, we have correlated structural changes caused by these mutations, with functional consequences on the protein activity. Our findings demonstrate that mutations impair TWINKLE’s ability to undergo large-scale conformational changes required for proper function.

We have also studied one of the most prevalent mitochondrial disease-causing mutations affecting POLγ, A467T. Using a combination of biochemical characterisation and in vivo studies using a murine model, we demonstrate that the mutation impairs interactions between the catalytic and accessory subunits of POLγ. As a result, POLγ dissociates and the catalytic subunit is degraded by LONP1, a protein involved in maintaining mitochondrial matrix protein homeostasis.

Details of LONP1 function is further characterised in the two other studies. In these works, we use cryo electron microscopy (cryo-EM) to determine the structure of full-length, human mitochondrial LONP1. In combination with a range of biochemical experiments we use this structural information to establish the mechanisms by which LONP1 can degrade mitochondrial proteins. We also analyse the functional consequences of two LONP1 mutations causing human disease. In vitro analysis demonstrated that the mutations impaired LONP1’s ability to bind and degrade substrates in an ATP-dependent manner.

In summary, our work has provided new structural and functional information about POLγ, TWINKLE, and LONP1 – three key factors required for proper mitochondrial function. Our work has also characterised disease-causing mutations affecting these proteins and established the molecular consequences of these alterations.