UCLA

Proper mitochondrial function contributes to cell’s health and integrity. Perturbations in mitochondrial homeostasis have been linked to diseases, specifically neurodegenerative diseases. Understanding the players of mitochondrial homeostasis will be beneficial for identification of potential therapeutic pathways. The mitochondrion is equipped with proteases that together serve as a quality control system to maintain homeostasis. While knockout using yeast genetics and RNAi approaches are powerful, new tools that are more rapid and specific are needed to elucidate the role of mitochondrial proteases. In this study, I utilize a high-throughput screening approach to discover and utilize small molecule modulators to characterize presequence-degrading protease (PreP).

PreP degrades N-terminal mitochondrial-targeting sequences and other small bioactive peptides, including amyloid-beta peptide, implicating it in Alzheimer’s Diseases. Nonetheless, not much is known about this protease. An in vitro fluorescence donor-quencher based assay was adapted for high-throughput screening of PreP modulators. From a collection of over 100,000 drug-like small molecules, we discovered numerous modulators of PreP.

MitoBloCK-60 was identified as a specific inhibitor of PreP. This piperazine-derivative does not uncouple mitochondria and affect viability of cells. Crystal structure of small molecule-bound protein at 2.3 Å revealed key bindings and specificity determinants. MitoBloCK-60 binds within the catalytic chamber of PreP, specifically at the substrate recognition exosite, inducing a conformational change in PreP that limits substrate binding. MitoBloCK-60 treatment in cultured cells suggests the role of PreP in mitochondrial quality control pathways. By being able to modulate PreP activity rapidly and selectively, we will provide insight into the involvement of PreP in mitochondria in general and how dysfunction of PreP could lead to diseases.