Decoding Mitochondrial AAA Protease Specificity

The removal of misfolded, damaged, and regulatory proteins by AAA proteases is essential for maintaining cellular integrity and health across all kingdoms of life. Disruptions in this process can lead to cellular dysfunction and the accumulation of cytotoxic protein aggregates. Mitochondrial protein quality control is particularly crucial, as proteins within the inner membrane and matrix are highly susceptible to oxidative damage. To prevent inappropriate protein degradation, substrate selection by AAA proteases is tightly regulated. It is thought that these enzymes primarily recognize unstructured sequences at the C- or N-termini of target proteins, using ATP hydrolysis to unfold substrates and translocate them into the proteolytic chamber of a peptidase for degradation. Adaptor proteins add an additional regulatory layer by either promoting or inhibiting the degradation of specific substrates. However, the precise mechanisms by which these enzymes maintain substrate specificity, interact with adaptors, and target substrates under different cellular conditions remain poorly understood. This proposal seeks to elucidate the mechanisms of substrate recognition and adaptor mediated degradation by soluble human ClpXP and membrane-bound m-AAA and i-AAA proteases, along with their accessory adaptors. Mutations in these proteases have been linked to various neurological disorders, and human ClpXP (hClpXP) has emerged as a promising therapeutic target in cancer treatment. Despite the high sequence similarity between bacterial and hClpXP, the mechanisms of substrate recognition and adaptor interaction in the human complex remain unclear. The mechanism by which membrane-bound AAA proteases recognize and degrade both soluble and membrane protein substrates is notably murky. To address these gaps, we will employ a multidisciplinary approach, integrating structural biology, biochemical analysis, genetics, and mass spectrometry to understand the mechanisms of substrate specificity and processing by these enzymes. Pulse-labeling mass spectrometry and genetic approaches will be used to identify substrates for each AAA protease and quantify degradation rates in vivo. This will be followed by biochemical reconstitution of a subset of these substrates in degradation assays to identify degron sequences and understand how adaptors influence substrate recognition and degradation by AAA proteases. Finally, we will determine the cryo-EM structures of these complexes, along with their cognate adaptors, in substrate-free and during substrate recognition, recruitment, and unfolding. Given that many aspects of these degradation mechanisms are still not well understood, the MIRA award will allow the Pl to allocate more time and resources to bridging this knowledge gap while also focusing on training and mentoring a diverse group of scientists. This research will offer critical insights into substrate specificity and adaptor-mediated proteolysis, carrying significant implications for both fundamental science and therapeutic advancements.