Understanding the role of C. elegans histidine-rich protein condensates in metal homeostasis

Biometals such as zinc and copper are fundamental to physiology, as roughly 40% of cell proteins require metal to function. Despite broad importance, the regulation of biometal concentration is narrowly understood. The nematode worm C. elegans is a powerful and economical model for discovery of fundamental physiology and cell biology principles broadly applicable to other organisms including humans. When exposed to heavy metals or zinc-displacing toxins, C. elegans activates two protective proteins named NUMR-1/2 and MYMR-3 that are rich in a metal-binding amino acid (histidine). This project will define regulation and cellular functions of NUMR-1/2 and MYMR-3 and explore their chemical and metal-binding properties. Given that principles of biometal regulation are fundamental and histidine-rich proteins are found in most organisms, these studies should provide insights with broad relevance to future studies on normal cell and organ function, with implications for understanding the effects of biometal-induced dysfunction in fungal infection, virulence, cancer, immune responses, neurodegeneration, and medicinal chemistry. Furthermore, understanding interactions between NUMR-1/2 and MYMR-3 proteins and metals has potential biotechnology applications in heavy metal exposure therapy, environmental sensing and bioremediation, and agricultural bioengineering. This project will also train undergraduate, high school, and graduate students in cellular, molecular, and analytical chemical skills via laboratory courses, internships, and research mentoring. NUMR-1/2 and MYMR-3 are predicted to be intrinsically disordered proteins (IDPs) with histidine-rich domains similar to metal-binding sensors and chaperones. NUMR-1/2 regulation and function are linked to RNA splicing and the protein is localized to nuclear ‘biomolecular condensates’ in response to biometal dyshomeostasis. IDPs phase-separate and drive formation of biomolecular condensates, which are dynamic membraneless organelles that organize signaling, sequestration, and RNA metabolism. IDPs and condensates function during immune responses and heat or oxidative stress, but roles in biometal homeostasis and inducible regulation are largely unexplored. The central hypothesis of this project is that NUMR-1/2 and MYMR-3 are biometal-responsive IDPs that interact with metals to promote formation and/or homeostasis of biomolecular condensates. Biometal homeostasis is disrupted by metals from geological events, natural disasters, and industrial exposures, as well as natural metal-displacing toxins and medicines. Objectives are to: A) define in vivo NUMR-1/2 and MYMR-3 localization/dynamics, function, and regulation, and; B) define conformations and metal binding of NUMR-1/2 and MYMR-3 peptide fragments. This project combines expertise of a C. elegans stress physiology lab and a protein chemistry lab to integrate in vivo protein localization, dynamics, regulation, and physiological function with in vitro biophysical and structural characterization of protein domains and their interactions with metals. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.