Regulatory roles of the micronutrient zinc in phosphorylations

Lead Research Organisation: King's College London
Department Name: Diabetes & Nutritional Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Bellomo E (2016) The metal face of protein tyrosine phosphatase 1B. in Coordination chemistry reviews

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Bellomo E (2014) Zinc ions modulate protein tyrosine phosphatase 1B activity. in Metallomics : integrated biometal science

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Kasana S (2015) Genetic causes and gene-nutrient interactions in mammalian zinc deficiencies: acrodermatitis enteropathica and transient neonatal zinc deficiency as examples. in Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS)

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Krezel A (2016) The biological inorganic chemistry of zinc ions. in Archives of biochemistry and biophysics

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Maret W (2013) Zinc and the zinc proteome. in Metal ions in life sciences

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Maret W (2016) The Metals in the Biological Periodic System of the Elements: Concepts and Conjectures. in International journal of molecular sciences

 
Title Cover Figure for Journal 
Description One of my students is an accomplished artist but she had not training in scientific presentations. Instead of computer drawn graphics, she prepared artistic presentation for the role of zinc transporters involved in genetic diseases. I am mentioning this activity because one figure from our 2015 publication in the Journal of Trace Elements in Medicine and Biology was chosen for the cover of the journal in 2016. 
Type Of Art Artwork 
Year Produced 2015 
Impact Wide distribution of our work through artistic presentation of scientific illustrations. 
 
Description We have determined a new mechanism of inhibition of enzymes. Protein tyrosine phosphatases were not known to be metalloenzymes. We demonstrated that they bind both metal cations and metal anions, resulting in the modulation of their activity. Our findings have wide applications for cell biology (regulation and signal transduction), nutrition, toxicology, and pharmacology.
Exploitation Route Our data will influence a great number of researchers that study phosphorylation signalling. Inhibition of protein tyrosine phosphatases by metal complexes has been investigated extensively. Our research provides a new basis for the work and has the potential for new drug development for these enzymes involved in cancer, diabetes, neurodegeneration and a lot of other most intractable diseases.
Sectors Agriculture, Food and Drink,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Workshop
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact This workshop was organized by the Ernst Struengmann Foundation. The topic was "Trace Metals in Infectious Diseases". I was a rapporteur of one section on advances in analytical instrument for studying metals in biology. The results of this workshop are now published by MIT Press, Cambridge (Struengmann Forum Reports 16) under the Title Trace Metals in Infectious Disease, J.O. Nriagu and E.P. Skaar, eds. This text is used for purposes of grant writing in many parts of the world and therefore is rather influential in setting policies and practice in this area.
 
Description Newton Bhaphta Fund
Amount £4,500 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 11/2016
 
Description Computational studies of metalloproteins 
Organisation National University of Singapore
Department Department of Chemistry
Country Singapore 
Sector Academic/University 
PI Contribution We have provided new questions regarding metal-protein interactions to be addressed by computational studies.
Collaborator Contribution Our partner has the skills in molecular dynamics calculations and access to the computational facilities to study the binding of metal ions to proteins and to investigate reaction pathways of enzymes.
Impact We have generated new models of how metal ions bind to reaction intermediates of protein tyrosine phosphatases and determined energetic profiles for the reaction in the presence and absence of activating and inhibiting metal ions.
Start Year 2015
 
Description Speciation of Metal Ions in the Biological Environment 
Organisation Polish Academy of Sciences
Country Poland 
Sector Public 
PI Contribution We are investigating the participation of low molecular weight biological molecules and buffer components in the functioning of proteins.
Collaborator Contribution Our collaborators have unique experience in inorganic chemistry of solutions regarding speciation and chemcial structure of metal ion complexes.
Impact The partners have been instrumental in supporting our work in terms of some very specific aspects of biological inorganic biochemistry. It has included the hosting of a student under the Erasmus exchange program.
 
Description X-ray crystallography of proteins 
Organisation University of Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution We started to work on the crystallization of protein and the determination of 3D structures. We are obtaining expressed proteins and characterize them in terms of purity and functional properties.
Collaborator Contribution The collaboration is extremely helpful as our partners have robotic facilities for fast screening of conditions for crystallization and access to synchrotron facilities for collecting difraction data.
Impact We have obtained a crystal structure of expressed human protein phosphatase 1B which essentially confirms the published structure. We employ this structure as a platform for preparation of various complexes to investigated the binding of metal ions.
Start Year 2015
 
Description A focussed discussion group of invited international experts on "zinc and diabetes" sponsored by the Friends of Israel Educational Foundation In Israel 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The organizers brought together several experts from Israel and the UK to discuss the role of zinc in diabetes and obesity. There were additional presentation on how type 1 diabetes is (or is not) addressed in the developing world, and, e.g. on the role of alpha1 antitrypsin deficiency.
Year(s) Of Engagement Activity 2016
 
Description Analytical Proteomics Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary speaker (Out of 4) to present the importance of zinc for the human proteome.
Year(s) Of Engagement Activity 2015
 
Description COST-Zinc-NET 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The workshop in Granada Spain focused on biomarker discoveries. It is hosted by a European COST action that focuses on networking/dissemination/education/raising awareness in different communities about the role of zinc in biology.
Year(s) Of Engagement Activity 2015
 
Description Chair of the Editorial Board 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact I am serving as the chair of the Editorial Board of the journal Metallomics of the Royal Society of Chemistry. There are usually two board meetings per year where the strategies of the journal are discussed and developed.
Year(s) Of Engagement Activity 2015
 
Description Editor's Symposium 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Every 2-3 years the Publishing section of the Royal Society of Chemistry convenes to discuss the strategies of their journal. Since the Royal Society of Chemistry is now the largest scientific publisher in chemistry in the world, it is a very important event. There are a lot of presentations on recent developments in scientific publishing, all of which are extremely informative. Board meeting are held and there are intense 2-3 days discussions about the future of scientific publishing.
Year(s) Of Engagement Activity 2016
 
Description Honorary symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invitation to present our work on zinc in biology at a workshop on bioinorganic chemistry to celebrate the life-time achievments of an esteemed colleague. The proceedings are being collected in a high impact journal (Coordination Chemistry Reviews).
Year(s) Of Engagement Activity 2015
 
Description Masterclass on Diet & Health, teaching two days at the R&D Unilever Campus in Bangalore, India in a partnership between King's College London, King's International 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact King's College London has agreed to a partnership with Unilever. As part of King's International there is a programme to teach aspects of Health, Diet, Nutrition, Healthspan, and Wellness to a participants from India with various academic backgrounds and practioners. Teaching is in the form of lectures, structured discussion and interactions with various stakeholders and involvement of the Royal Society of Chemistry.
Year(s) Of Engagement Activity 2016
 
Description Metallomics Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited keynote lecture in Beijing, China. This conference is instrumental in developing a new field, integrated biometal sciences, namely studying the role of metal ions biological systems. There is significant interest from the business sector, i.e. analytical instrumentation.
Year(s) Of Engagement Activity 2015
 
Description Visit to Trade Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact This was a trade fair in Hyderabad India, where a international speakers were selected to talk about advances in instrument development. My presentation was unique as it introduced the audience to the role of zinc in biology, including any challenges in analytical sciences.
Year(s) Of Engagement Activity 2015
 
Description a focused workshop on Wellness "The 6th industrialization of Health and Beauty" at the Pyeongchang Campus, Seoul National University, South Korea 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Novel approaches and technologies used in laboratories as well as innovative preclinical science and clinical trial result for wellness life.
Year(s) Of Engagement Activity 2016