PROJECT SUMMARY/ABSTRACT Metal ions are required nutrients for cellular function, but can also be toxic if misregulated. Our immune system leverages this dichotomy by deploying mechanisms both to withhold nutrient metals from pathogens as well as overwhelm them with toxic levels, particularly of Cu and Zn. Metal imbalances at the cellular level have also been implicated in neurodegenerative diseases, and are being investigated as possible anticancer strategies. But what exactly are the targets and mechanisms of cellular metal stress? The research proposed here explores this question of cellular metal stress by seeking to identify protein targets of aberrant metal interactions by measuring global changes in protein stability across the proteome. The proposed work builds on preliminary and recently published results from this collaborative team showing the utility of a pulse proteolysis mass spectrometry method developed in co-PI Fitzgerald’s laboratory to identify protein targets of Cu in E. coli, establishing these proteomic methodologies for the study of metal-protein interactions. The overall objective of the current application is to identify proteins that are functionally affected when cells experience stress induced by exposure to excess levels of Zn and Cu. This objective will be met by using a powerful combination of mass spectrometry-based proteomic methods to address four specific aims: 1) Determine global profiles of protein stability changes as a function of cellular metal overload and metal deficiency across bacterial, fungal, and human cancer cells; 2) Establish a mechanistic basis linking differential stability of Aim 1 proteins to function; 3) Identify the relative sensitivity of proteins across the proteome to misfolding induced by Cu and Zn binding; and 4) Establish a biophysical basis for understanding the relative sensitivity of proteins to metal-induced misfolding. Understanding how protein stability is impacted across the proteome upon exposure to normal or aberrant levels of Cu and Zn has important implications for understanding metal-induced toxicity and mechanisms cells use to maintain metal homeostasis in the face of metal-associated stress. By studying proteomes from microbial, fungal, and human cancer cells, the outcomes of these studies will advance our understanding of how these organisms respond at the proteome level to changing metal environments imposed by the host immune system. These studies will inform and impact the development of pharmacological agents against microbial pathogens and cancer cells, and align with the applicant’s long-term goals to develop chemical tools to manipulate biological metal ion location, speciation, and reactivity for potential therapeutic benefit.