Structural insights into zinc homeostasis

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Project Summary / Abstract The Zrt-, Irt-like Protein (ZIP, or Solute Carrier 39 (SLC39)) family of membrane proteins mediate zinc influx1. Zinc homeostasis is a critical regulator of a variety of physiological processes, including immune function, insulin secretion, and cell communication, and plays key roles in protein structure and catalysis2. Because both excess and deficient zinc levels are cytotoxic, zinc homeostasis is tightly controlled in mammalian cells via an abundant network of membrane proteins that act as zinc importers (ZIP / SLC39 family) or zinc exporters (ZnT / SLC30 family). Zinc homeostasis proteins are potential pharmacological targets in neurodegenerative disorders and cancer, but progress is hampered by a lack of structural and mechanistic information. No full length mammalian structures of ZIP family members have been solved and no in vitro functional assays exist. ZIP9 (SLC39A9) represents a novel treatment avenue for prostate cancer3. The prostate has the highest concentration of zinc in the body, which promotes apoptosis and helps maintain low levels of cellular proliferation. In prostate cancer, zinc levels decrease, leading to tumor proliferation4. Prostate zinc levels cannot be rescued with dietary supplements5,6. Recently, ZIP9 was identified as a membrane Androgen Receptor (mAR) which mediates testosterone-induced apoptosis7. ZIP9 is upregulated in prostate cancer, but to realize its potential as a pharmacological target a full structural and functional characterization is required. My objective is to elucidate the structure and mechanism of ZIP9. Aim 1 is to develop a proteoliposome-based functional assay using stopped-flow fluorescence spectroscopy and site-directed mutagenesis to characterize the mechanism of ZIP9. Aim 2 is to determine the structural basis of zinc influx by ZIP9 using Microcrystal Electron Diffraction (MicroED). Aim 3 is to examine ZIP9's mAR characteristics by determining the molecular and structural basis of its interactions with testosterone and G proteins using biochemistry and high resolution single particle cryogenic electron microscopy (cryoEM). This research will unravel the mechanism of ZIP-mediated Zn2+ influx and clarify ZIP9's putative role as a membrane Androgen Receptor, broadening our understanding of zinc homeostasis and paving the way for the use of ZIPs as a pharmacological target in prostate cancer and beyond.