RUI: Investigating ATP's Influence on Ferritin Structure, Function, and Cellular Iron Homeostasis

Iron is an element where you “can’t live without it but you can’t live with too much of it”. Too little iron can lead to a host of health issues including anemia, fatigue, and a weaker immune system, whereas too much iron can cause cell death and has been implicated in various neurodegenerative diseases. Ferritin is a ubiquitous and multi-subunit iron storage protein that plays key roles in minimizing iron toxicity and controlling cellular iron balance. The goal of this research project is to understand how cells regulate this essential nutrient by examining the interactions between ferritin, iron, and key cellular molecules like ATP and other nucleotides. Results from this project could have wide-reaching benefits including a deeper understanding of cellular iron storage, mobilization and re-distribution, and the implication of these processes on iron imbalance and iron-related diseases. Undergraduate students working on this project will have the opportunity to engage in multidisciplinary research encompassing bioanalytical, biophysical, machine learning, and molecular and structural biology methods, equipping them with valuable skills and knowledge for future and successful scientific careers. This research project has three main objectives. First, using a variety of thermodynamic and kinetic techniques (e.g. fluorescence quenching, isothermal titration calorimetry, and UV-vis light absorbance spectroscopy), the project seeks to study how ATP interacts with ferritin’s nanocage structure and influences the rates of iron uptake, oxidation, and storage. The experiments will be performed in aqueous buffered solutions and in bacterial, yeast, and mammalian cell lysates, which closely mimic the cytosolic environment of three different organisms. Using ferritin encapsulated fluorescent probe and fluorescence emission, dynamic light scattering, circular dichroism, synchronous fluorescence spectroscopy, differential scanning calorimetry, cryo-electron microscopy, and deep learning methods, the second objective will investigate the effect of ATP on ferritin integrity and stability and explore how ATP affects ferritin structural and conformational changes (e.g. ATP hydrotropic effects). Lastly, the effect of ATP on the kinetics of iron mobilization from ferritin will be investigated by two main routes, via direct chelation and via iron reductive mobilization mechanisms. The results of these studies will provide a detailed understanding of the effect of cytosolic components, such as ATP and other cytosolic nucleotides (or metabolites) on ferritin structural changes, iron mineralization and mobilization reactions, and ultimately the role that ATP and ferritin play in cellular iron homeostasis. 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.