Iron is an essential nutrient that functions as a key co-factor for many cellular proteins involved in aerobic respiration, nucleotide metabolism, gene expression, and DNA repair. Given its central role in sustaining life, it is not surprising that cells have established a variety of elaborate regulatory mechanisms to control iron availability and usage. While characterizing components of the ubiquitination machinery that are deregulated in tumorigenesis, we identified a ubiquitin ligase subunit called FBXL5. In preliminary work, we demonstrated the FBXL5 is a novel regulator of iron metabolism that proteolytically controls the expression of important effectors of iron metabolism and cytoplasmic iron-sulfur cluster assembly. The major goal of this proposal is to examine the biological mechanisms by which FBXL5 regulates and is regulated by iron-related pathways. In specific aim 1, we will examine the molecular basis of the iron-regulated interaction of FBXL5 with IRP2, a key regulator of iron homeostasis. The experiments in specific aim 2 will revolve around the regulation of FBXL5 itself and elucidating the novel proteolytic pathway responsible for its proteasome-dependent degradation under conditions of low iron availability. Specific aim 3 will focus on the establishing the functional relevance of the interaction between FBXL5 and MMS19 and CIAO1, two putative components of the cytosolic iron assembly (CIA) pathway which is required for the assembly of Fe/S clusters in extramitochondrial proteins. Investigation of these three aims will provide a comprehensive view of how FBXL5 is able to influence multiple iron-associated processes. Ultimately, we hope that this work will offer insight into how the deregulation of FBXL5 and its downstream pathways may contribute to tumorigenesis while simultaneously highlighting potential new therapeutic strategies. PUBLIC HEALTH RELEVANCE: The attachment of the ubiquitin to proteins is an important signal for cellular communication and is believed to be disrupted in multiple diseases including cancer, neurodegenerative diseases such as Huntington's disease, and diabetes. This proposal is focused on understanding how cells use this tagging system to regulate iron levels and usage.