PROJECT SUMMARY The proper folding of proteins is essential for all life, making aberrant protein folding severely problematic. The protein homeostasis (proteostasis) network is a tightly regulated system that ensures that proteins are folded and degraded as necessary. However, misfolded proteins can overwhelm proteostasis. Misfolding is associated with numerous devastating neurodegenerative, heart, and kidney disorders, as well as forms of cancer. Protein disaggregases can engage misfolded substrates and dissolve them, promoting their return to proper fold and function. Disaggregases may serve as the final defense against collapse of proteostasis. Our central hypothesis is that disaggregases play key roles in maintaining cellular health, but are vulnerable when the proteostasis network becomes overwhelmed in disease. Therefore, technologies that enhance disaggregase activity may be therapeutically useful. However, disaggregases are the least characterized branch of the proteostasis network. My research program focuses on advancing our understanding of how disaggregases counter misfolding, both in health and under stress. We seek to elucidate how cells maintain proteostasis, how proteostasis fails, and how protein disaggregases might ultimately be applied to prevent or reverse collapse of proteostasis. During the first period of this award, we focused on engineering tailored Hsp104 variants, better understanding mechanisms of misfolding, and have begun characterizing human disaggregases. During the next phase, we will continue and expand our efforts to better understand the roles disaggregases play in safeguarding against misfolding. We will focus on two primary themes: (1) we will develop finely-tuned Hsp104 variants and use these variants as probes to test the effects of disaggregation in different systems. These studies will also reveal insights into the mechanism of Hsp104, and we aim to better understand how Hsp104 recognizes and distinguishes among substrates. Further, because Hsp104 is a member of the AAA+ family of proteins, which serve crucial roles across biology, our findings can be broadly applied. (2) We will characterize and develop approaches to modulate human disaggregases. Here, we will focus on understanding the structure, mechanism, and substrate repertoire of human disaggregases. We also aim to develop approaches to modulate the activity of human disaggregases. Beyond their possible therapeutic utility, finely-tuned disaggregases can be used as probes to test the hypothesis that misfolded species are toxic, and that restoration of proteins to their native folds and functions can reverse disease phenotypes. Ultimately we aim to apply the findings from these studies to develop new strategies to treat protein-misfolding diseases. This is especially important because, despite intense efforts, there are no effective therapeutics available to treat numerous protein-misfolding disorders.