Regulatory roles of the micronutrient zinc in phosphorylations

Zinc is an essential micronutrient and indispensable to the growth and development of organisms. Though historically less explored than iron, zinc has become the centre of recent attention because of its involvement in about 3000 human proteins. Zinc biochemistry is now a major frontier in the biosciences, poised for ample opportunities and breakthrough discoveries. There is hardly any cellular process that does not depend on zinc in some way. Zinc has unparalleled significance in protein structure, enzymatic catalysis, and cellular regulation. The planned experiments address regulatory functions of zinc(II) as inter- and intracellular messengers. Zinc inhibits enzymes at unprecedented, minute concentrations - a new paradigm for the biological functions of metal ions.
We have identified protein tyrosine phosphatases (PTPs), proteins critically involved in controlling cellular functions, as targets of zinc(II) ions. We suspect a new way of cellular control in which zinc(II) ions regulate these thoroughly investigated enzymes. We plan to investigate whether zinc(II) ions are general inhibitors of PTPs or control only a subset of them. We will achieved this goal by a combination of structural methods to identify the zinc-binding site on PTP, bioinformatics approaches to search for analogous binding sites, kinetic analyses of isolated PTPs to determine the strength and type of inhibition, and last but not least investigations in cultured human cells to examine physiological significance.
Protein phosphorylations and zinc concentrations are both exquisitely controlled. Perturbation of this control is a major cause for diseases, such as cancer, diabetes and impaired wound healing. Accordingly, PTPs are coveted targets for drug design. Our investigations demonstrate that zinc(II) ions bind more tightly to some PTPs than any experimental drug and hence suggest new avenues for therapeutic interventions. One approach is to control the cellular zinc status as it turns out to be important for proper functioning of hormones, such as insulin. It is well known that zinc deficiency or perturbations of zinc metabolism caused by genetic or environmental factors impair growth and development and cause disease, but the molecular mechanisms are virtually unknown. Our observations provide a testable hypothesis for this long-standing issue with the potential of defining a major pathway that determines healthy ageing and the balance between health and disease. The results forthcoming from our work will demonstrate that zinc is not just a permanent cofactor of proteins but participates dynamically in cellular metabolism. They are expected to bring about an integration of the two research fields of zinc biology and biological phosphorylations.

Nutritionally essential transition metal ions are structural and catalytic cofactors of proteins. A new paradigm positions that zinc(II) ions have functions as intracellular messengers. Stimulation of some cell surface receptors causes release of zinc(II) ions from an endoplasmic reticulum store. The molecular targets of the resulting cytosolic zinc(II) ion transients have not been identified, though an effect on phosphorylation has been documented widely. We have now shown that zinc(II) ions inhibit some protein tyrosine phosphatases (PTPs) at remarkably low picomolar concentrations. This finding is an exciting development as it suggests a new mode of regulation of these critically important enzymes.
We have developed enabling methodologies to address the role of cellular zinc(II) ions quantitatively at their extremely low concentrations. They include measurement of zinc(II) ions with fluorescent chelating agents, control of zinc(II) ion concentrations with metal-buffering techniques, and kinetic inhibition analyses of PTPs. Employing isolated PTPs, we plan to investigate which PTPs zinc inhibits and to characterize the structure of the inhibitory zinc site with NMR spectroscopy. We will then address the physiological significance in cultured cells. Our results are expected to demonstrate a novel control of biological processes involving PTPs and zinc. We will focus on two enzymes in particular, PTP-1B, which controls insulin and leptin signalling, and RPTP-beta, which is involved in angiogenesis. The results will have the widest possible impact as PTPs and zinc are involved in yet many other fundamental cellular processes and chronic diseases.
The zinc inhibition we have discovered is stronger than that of any synthetic inhibitors developed for these enzymes. We believe our results will convince the pharmaceutical industry, which has a keen interest in PTPs, that a properly functioning cellular zinc metabolism is a key to health and disease.

We expect to generate major new insights into the regulation of protein tyrosine phosphatases and the functions of zinc(II) ions in cellular information transfer. Demonstrating a relationship between phosphorylations and zinc metabolism will have significant societal and economic impact for human and animal health and healthy ageing. About 30% of the world's population is believed to be zinc-deficient, a problem with at least the scope of iron deficiency. Among the groups at risk, the elderly are prone to become zinc deficient. The World Health Organization (WHO) has identified zinc deficiency as the fifth most important risk factor for morbidity and mortality in developing countries (11th worldwide) (World Health Report 2002 - reducing risks, promoting healthy life. Geneva: WHO 2002). The figure translates into 3.2% of all lost disability-adjusted life years (DALYs). These estimates for the human health implication of zinc are derived primarily from the incidence of infectious and parasitic diseases due to compromised immune functions in zinc deficiency. Clearly, this measured outcome is far from inclusive. The estimates do not take into account the functions of zinc in human memory acquisition and storage and behavior, growth retardation, delayed wound healing, the effects of environmental exposures ("zinc overload"), or the role of zinc in ageing and in chronic diseases, such as cancer, neurodegeneration, and diabetes. Thus, a balanced zinc status is critical. Understanding the functions of zinc(II) ions in cellular control will lead to a new level of appreciation of the importance of this micronutrient, which in turn could lead to new preventative interventions and treatments. We strongly believe that the role of zinc in biology is being underestimated. A new multi-authored text (L. Rink, ed. Zinc in Human Health and Disease, IOS Press, Amsterdam, 2011) summarizes the vast knowledge that accumulated since the field was last reviewed decades ago. We also believe that monitoring and restoring zinc status, which perhaps could be achieved by assaying regulatory functions of zinc(II) ions, has a major societal and economic impact. Past training of the PI at the University of Texas Medical Branch has taught him the economic benefits of a preventive approach when compared to the costs of therapy in an economically sustainable world. He is already engaged in reaching the non-scientific community as further explained in the pathways to impact section. With regard to economic impact, we are already collaborating with a company specializing in the role of zinc in improving pancreatic beta-cell function (Mellitech, Grenoble). We will explore any commercial aspect of our research with King's Business and other stakeholders. The PI had been invited to discuss the scientific basis for zinc supplementation (Maret, W. and Sandstead, H.H. Zinc requirements and the risks and benefits of zinc supplementation. J. Trace Elem. Med. Biol. 20, l3, 2006), while Prof. Christer Hogstrand is a member of an expert panel of the European Food Safety Authority (EFSA), providing advice on trace elements since 2006. In summary, the fundamental biochemical issues that we are addressing in this application have significant economic, social, and political impact.