Investigating the role of human mitochondrial matrix AAA+ proteases in proteostasis and disease

Submission note: This thesis is submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry and Genetics, School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Victoria.

Mitochondria play crucial roles in energy production and the regulation of metabolic pathways and as such the correct maintenance of mitochondrial integrity is essential to the overall health of a cell. An important aspect of mitochondrial integrity is maintenance of protein homeostasis (proteostasis) within the organelle, and key to this is the regulated removal of damage or unwanted proteins in a timely fashion. This process is carried out by ATP-dependent proteases such as LONM, CLPXP and m-AAA protease and mutations in these components are associated with diverse human diseases. To date, however, little is known about the physiological role of these machines or the substrates they recognise. Similarly, the mechanism of action of substrate binding and specificity of these proteases also remains poorly defined. In this study, the two matrix localised ATP-dependent proteases, LONM and CLPXP were examined. In the first part of this study the recognition motif of two known, but poorly defined, “native” substrates of LONM (StAR and SDH5) was analysed. This analysis identified a general binding motif for LONM, in which “native” protein substrates contain two elements for recognition by LONM. Mutation or deletion of these elements rendered the protein stable in vitro. Significantly, these findings suggest that preventing the premature clearance of some proteins may serve as a useful therapeutic option to treat disease states associated with a proteostasis imbalance. In the second part of this study, the molecular basis of Perrault syndrome was examined through generating disease-specific point mutations in CLPP (T145P, C147S and Y229D). Interestingly, although all three mutants were successfully imported into mitochondria, they each exhibited a range of defects. While C147S exhibited relatively mild defects, Y229D exhibited a loss of interaction with the partner peptidase CLPX. In contrast T145P exhibited broad defects in oligomerisation, interaction with the partner unfoldase CLPX and surprisingly exhibited a hyper-stimulated peptidase activity. Collectively these data provide a molecular basis for the underlying cause of Perrault syndrome.