Approaches to study Zn(II) deficiency and transport in biology

Abstract

Divalent zinc, Zn(II), is an abundant and essential metal ion for human health. Across diverse biological settings, it stabilizes the structure of proteins, serves as a catalytic cofactor in enzymes with disparate functions, and mediates important signaling events. The ability of cells to apply Zn(II) in all these roles is contingent upon their ability to ensure adequate, but not excessive, Zn(II) levels. This control process, or homeostasis, is maintained by at least 24 transporters, including 14 ZIPs that increase the transition metal ion concentration of the cytosol and 10 ZnTs that decrease the transition metal ion concentration of the cytosol. Zn(II) homeostasis can be challenged either by excessive or inadequate nutritional Zn(II) or by interference of other metal ions with Zn(II) uptake transporters. Neither the molecular consequences of Zn(II) deficiency nor the molecular basis of ZIP-mediated selective metal uptake is well defined.

To address both these issues, I developed and applied new methodologies to study transition metal homeostasis. First, I report the preparation and use of "A12-resin", comprising the Zn(II)-binding protein S100A12 conjugated to agarose, that is capable of selective depletion of Zn(II) from diverse biological media. I deplete cell culture media of Zn(II) by using this resin and characterize the effects of Zn(II) insufficiency on the metabolism, transcriptome, and metallome of HEK293 cells. Second, I further apply Zn(II)-depleted cell culture media in a Zn(II) uptake assay. I show that repletion of Zn(II) depleted media with ⁷⁰Zn(II), a naturally low-abundance, stable isotope of Zn(II), enables sensitive, inductively coupled plasma-mass spectrometry-based measurements of Zn(II) uptake. Finally, I apply this assay to characterize the metal ion selectivity of human LIV-1 subfamily Zn(II) transporters.

I show that the kinetic parameters associated with ZIP4, ZIPS, ZIP8, and ZIP10 transport of Mn(II), Cd(II), and Zn(II) are distinct, and that metal ion selectivity is conferred by the transmembrane domains of the proteins rather than by the extracellular N-terminal domains. Taken together, the work presented in this thesis enables and motivates future work to interrogate transition metal homeostasis in human cells.