Development of Oma1 inhibitors for the analysis of mitochondrial fusion pathways, apoptosis and mitochondrial related diseases.
Final Report Abstract
Defects in mitochondrial function are responsible for a wide range of diseases including cancer, cardiovascular disease, and degenerative neural and muscular disorders. Particularly, mitochondrial dysfunction has been linked to defects in apoptosis and mitochondrial fusion pathways. More specifically, the mitochondrial proteolytic system plays a key role in processing proteins involved in mitochondrial fusion pathways and apoptosis. However, how the mitochondrial proteolytic system regulates mitochondrial homeostasis and dynamics is not clearly understood. Studies have shown that Oma1, a mitochondrial inner membrane zinc-metalloprotease, is responsible for the cleavage of Opa1, a member of the dynamin-related GTPases and involved in the controlling of mitochondrial fusion, when mitochondrial membrane potential is lost, or the mitochondrial ATP level is low. The cleavage of Opa1 leads to impaired mitochondrial fusion and consequently to apoptosis. Therefore the development of tools that modulate the function of the mitochondrial protease Oma1 would provide a platform to subsequently dissect the role of the mitochondrial proteolytic system to mitochondrial-based diseases. The goal of my postdoctoral studies at UCLA was to develop small molecule effectors that modulate the proteolytic activity of Oma1. For the development of Oma1 inhibitors, two strategies are being employed. The first strategy was the purification of recombinant human Oma1 for in vitro high throughput screens. Oma1 could be successfully purified from E. coli, resulting in a yield of 100 µg/l cell culture. However, purified Oma1 did not show proteolytic activity with a subset of substrates, including a peptide designed around the putative S1 cleavage site of Opa1. Therefore, additional approaches to optimize purification and activity are needed to move forward in a robust high throughput screen. The second strategy is the development of an in vivo yeast screen around the degradation of a thermosensitive Oxa1 mutant. The yeast strains needed were successfully generated. As expected the yeast strain carrying the thermosensitive Oxa1 mutation was not able to grow on non-fermentable carbon sources at the restrictive temperature of 37ºC, because the mutation prevents the assembly of respiratory chain complexes. The subsequent knockout of Oma1 partially restored the growth at 37ºC. Therefore this strategy of inhibiting Oma1 with small molecule effectors could be exploited for an in vivo screen although further optimizations are needed. In conclusion, this fellowship year was used to prepare reagents and develop methodology for high throughput screens to identify Oma1 inhibitors. Additional experiments are required to further optimize these approaches, but having both in vitro and in vivo screens in place will result in overlapping approaches to develop small molecule modulators.
