SUMMARY Transition metals are essential micronutrients required for cellular function across all kingdoms of life. Zinc (Zn) is the second most abundant metal in humans, functioning as a cofactor for both structural and enzymatic proteins for an estimated 10% of the proteome. Metalloproteins require metal binding to execute fundamental chemical reactions necessary for cellular physiology. As such, perturbations in Zn levels are detrimental to cellular homeostasis and promote diseases including neurodegeneration and cancer. In conditions of Zn limitation, it is predicted that specialized proteins called metallochaperones are necessary for the transfer of metals to critical metalloprotein clients. Predicted bacterial metallochaperones have been identified, including COG0523 proteins which have demonstrated roles in metal homeostasis. However, eukaryotic mechanisms that transfer Zn to metalloproteins remain completely unknown presenting a significant gap in knowledge. Comparative genomic analyses have identified COG0523 paralogs in vertebrates named Cob-W domain containing proteins (CBWD). Moreover, COG0523 expression is induced during conditions of Zn starvation in both bacteria and vertebrates, suggesting these proteins serve a conserved role in the maintenance of Zn homeostasis. While CBWD exists as a family of paralogs in humans, zebrafish possess a single cbwd gene with high sequence similarity to human CBWD1. Genetic and pharmacological tractability, high fecundity, and optical transparency make the zebrafish model well-suited for in vivo investigation of COG0523 function. Preliminary experiments from our lab indicate vertebrate Cbwd binds to and transfers Zn to metalloprotein targets, suggesting it functions as a Zn metallochaperone. We have identified candidate zebrafish Cbwd client Zn metalloproteins associated with protein processing and cellular proliferation, including the methionyl-aminopeptidase Metap1 and the Zn-finger transcription factor Zfhx3. Thus, we will test the hypothesis that Cbwd is a Zn metallochaperone that regulates cellular proliferation and organismal development via interactions with Metap1 and Zfhx3 using biochemical, genetic, and pharmacological approaches in zebrafish and cell culture. This work is expected to determine the impact of Cbwd mediated Zn transfer on client metalloprotein enzymatic function (Aim 1), test the role of Cbwd-Metap1 interaction on cellular protein homeostasis and proliferation (Aim 2), and define the Cbwd-dependent Zfhx3 signaling pathways important for organismal development (Aim 3). Completion of these aims has the potential to identify CBWD as the first eukaryotic Zn metallochaperone described in nature and will further define the role of Cbwd in cellular and organismal metal homeostasis. These findings will shed light on the in vivo role of ancient COG0523 proteins and will vertically advance our understanding of Zn homeostasis and contributions to the progression of human cancers and ...