Defining mechansims of AAA+ disaggregases - Project Summary Our research program seeks to define, at a basic level, the mechanistic underpinnings of the AAA+ (ATPases associated with diverse activities) disaggregases, Hsp104, and its partial human homolog, Skd3 (human ClpB), which remain incompletely understood. In non-metazoan eukaryotes, Hsp104 couples ATP hydrolysis to the disaggregation of diverse proteins trapped in disordered aggregates, preamyloid oligomers, phase-separated condensates, prions, and amyloids. Hsp104 can even dissociate very stable structures, including α-synuclein (α-syn) oligomers and amyloids. However, Hsp104 activity is limited against α-syn and high Hsp104 concentrations are required for optimal effects. Thus, we have engineered potentiated Hsp104 variants, which dissolve fibrils formed by -syn as well as TDP-43 and FUS. Though potent disaggregases, these potentiated Hsp104 variants are prone to off-target toxicity. To address this design issue, we engineered new potentiated Hsp104 variants with finely tuned collaboration with Hsp70 or selectivity for α-syn, which exhibited minimal off- target effects. By engineering enhanced Hsp104 variants, we have learned a great deal about how the protein disaggregase works at a basic level. Curiously, Hsp104 does not have an exact metazoan ortholog. Remarkably, we have found that a partial homolog of Hsp104 found in human mitochondria, an AAA+ protein called Skd3 (human ClpB), has powerful protein disaggregase activity. Skd3 is critical for human health as mutations in Skd3 underlie severe mitochondrial disorders. Despite these important advances, our basic mechanistic understanding of Hsp104 and Skd3 remains limited. Here, we will focus on several challenging questions, which currently limit our basic understanding of how these disaggregases operate. For Hsp104, we do not understand: (1) how Hsp104 translocates substrates during disaggregation; (2) how to engineer Hsp104 variants with selectivity for any given substrate; and (3) how specific C-terminal mutations in the middle domain and nucleotide-binding domain 2 of Hsp104 potentiate activity. For Skd3, we do not understand: (1) whether Skd3 and isoforms have a broad amyloid-disaggregase activity or how Skd3 dissociates amyloid structures; (2) how Skd3 structure enables disaggregase activity; and (3) whether additional factors regulate Skd3 disaggregase activity in mitochondria. Here, we will address these key knowledge gaps and thereby secure an enhanced mechanistic and structural view of Hsp104 and Skd3, which will advance our basic understanding of protein disaggregases in maintaining proteostasis.
