ABSTRACT The primary goal of this MIRA R35 application is to understand the molecular basis and mechanisms by which protein AMPylation regulates proteostasis in normal physiology and in the context of protein aggregation- associated diseases. Protein AMPylation is conferred by dedicated AMPylases, which are present in a single copy in most metazoans. These enzymes are increasingly recognized to control proteostasis, modulating how and when amyloidogenic proteins – such as amyloid-β (Aβ), α-synuclein (α-Syn) and poly-glutamine repeat proteins – aggregate. However, our knowledge of AMPylase regulation, target preference and their roles in physiological as well as in disease states remains elusive. Closing these gaps in knowledge is key for gaining fundamentally important insights into how post-translational protein AMPylation controls proteostasis, and will potentially enable the development of therapeutic interventions that capitalize on this process. The goals of this proposal are to 1) Dissect the mechanistic and physiological impact of FIC-1/FICD-mediated protein AMPylation on proteostasis, and 2) elucidate the mechanistic basis of protein AMPylation in the cytoplasm and nucleus. We will use a multi-faceted, translational and multi-organism approach to address the genetics and biochemistry associated with these central questions in protein aggregation and proteostasis regulation. Our research program is utilizing four complementary model systems: the nematode C. elegans, purified proteins for in vitro biochemistry, human cell lines and in vivo mouse models, each offering its unique set of advantages to examine protein AMPylation in the context of health and disease. We will leverage our unique in vivo and in vitro assays and tools, while continuing to design novel approaches, to define the enzymes, targets, signaling cascades and biological processes involved in AMPylation-mediated proteostasis regulation in unpreceded details. In the next five years, our goal is to fully develop current projects and to devise new tools and techniques to continuously produce genetic and mechanistic data with translational potential. A mechanistic understanding of these processes will advance our knowledge of protein folding and proteostasis pathways which will represent key steps towards the discovery of novel interventions to prevent or treat protein aggregation-associated disorders.