UC San Diego

NEET proteins belong to a class of iron–sulfur proteins that harbor a unique labile [2Fe–2S] cluster and play a major role in preventing mitochondrial dysfunction. Loss of function in mitochondria, the key organelle responsible for cellular energy production, can result in many human pathologies including diabetes, neurodegeneration, and cancer. Despite the recent advances in molecular medicine, cancer remains a significant challenge as it is the second leading cause of death in developed countries. Therefore, additional therapies are needed to increase therapeutic options to cure diseases related to NEET proteins. Since the exciting discovery of the novel mitochondrial target, MiNT, for the thiazolidinedione (TZD) insulin sensitizing drug pioglitazone, the NEET family has emerged as an important class of human proteins that are implicated in multiple pathologies. There are only a few compounds that have thus far been discovered or designed as NEET ligands that control NEET [2Fe–2S] stability. The Jennings laboratory and collaborators have shown that NEET proteins act as central sensors/transducers of iron/ reactive oxygen species (ROS) metabolism were deregulation results in cell death. Specifically, recent studies have shown that small molecules can bind to NEETs destabilizing the [2Fe-2S] cluster, leading to release of iron, induction of ROS and cell death. This effect is selective for cancer cells that upregulate NEETs. These findings demonstrate, for the first time, the significance of NEETs as drug targets for a chemotherapeutic treatment of cancer. This thesis focuses on the role of NEET proteins, specifically MiNT(CISD3), in different diseases using drug binding to examine protein stability. To further detail the unique structural difference of MiNT, as it is a monomeric protein containing two Fe-S clusters, we use a combination of biochemical and biological assays to characterize the effects of ligand binding. Specifically, we use ultraviolet visible (UV-Vis) spectroscopy and circular dichroism (CD) to monitor cluster stability over time. Together, these assays use temperature and secondary structure analysis to determine stability effects because of ligand interactions. Given the broadened understanding of the MiNT function and the initial results from binding interactions highlighted here, it might be possible to begin the development of therapeutic agents that may be useful in mitigating several different diseases including neurodegeneration, breast cancer, diabetes, and inflammation.