Regulatory Roles of Zinc Fluxes in Metalloprotein Occupancy and Cell Cycle Progression

Zinc fluxes have recently been discovered to be key regulatory events in a variety of human physiologies. For instance, significant zinc mobilization events play essential roles in cortical neuron function, proper immune response, and insulin secretion. Despite the profound physiologies regulated by this transition metal, the mechanistic roles of zinc, the trafficking pathways and specific metalloprotein receptors in these signaling events are not well understood. Zinc is ubiquitous, and every cell in the human body requires high concentrations of this essential element. It has traditionally been viewed as a static cofactor involved in protein structure and enzyme catalysis. More recently, however, a number of studies have provided support for the idea that zinc binding sites in regulatory proteins respond to transient fluctuations in zinc availability and are switched on and off in way that regulates key cellular events. To interpret and eventually intervene in neurological disorders and metabolic diseases caused by disruption of such pathways, the biomedical community needs a basic mechanistic understanding of these zinc-dependent switching events. We will test the hypothesis that regulatory zinc fluxes exert instructive control of the mammalian cell cycle through specific receptor mediated processes. This hypothesis is based on multiple lines of evidence, including: (1) data showing zinc distribtion at various time points in the cell cycle for single cells; (2) live cell imaging that demonstrates the movement of waves of zinc; and (3) physiochemical methods for staining and fixing cells after growth in culture systems that show colocalization of zinc with specific factors. We will use mammalian gametes, i.e. the sperm and egg, as a model cellular system to understand how an essential zinc signaling pathway works. One objective is to identify molecular mediators and targets of regulatory zinc fluxes by examining the quantitative changes in the localization of zinc in single cells at specific points in the cell cycle. We will then correlate these spatio-temporal characteristics of metal availability with changes in protein chemistry of zinc receptors that are targets of the signaling events. At the project’s conclusion, we will have accomplished three innovative goals of significance to the biomedical community. First, we will provide fundamental new insights into how inorganic signaling events work at a molecular level. Second, we will develop methods to probe the metalloproteome and identify how key proteins involved in embryogenesis change zinc occupancy immediately following fertilization. Third, we will provide fundamental new insights into inorganic signaling events in sperm capacitation and acrosome reactions. Refinement and validation of new chemical probes and physical methods tools developed in these studies will substantially enhance our knowledge of a fundamental pathway that regulates cellular decision making processes. Taken together these results will allow for a robust definition of the zinc fluxes, both zinc sparks and cytosolic zinc waves, and their molecular roles in regulating the cell cycle.