Bilateral BBSRC-FAPESP: A "speciomic" toolkit to investigate fatty acid-mediated changes in plasma zinc speciation and their physiological effects

There are 25 chemical elements that are required for mammalian life; 15 of these elements are metals. Zinc, in its ionised form Zn2+, is an essential metal ion in mammals and performs a wide range of important physiological functions by allowing many vital chemical reactions to occur. Zinc plays essential roles in fertility and development, the immune system, ageing, and major diseases such as diabetes, Alzheimer's disease and cancer. The mechanisms that deliver zinc to where it is needed following uptake from the diet and into the bloodstream, are only partially understood. It is known that the protein serum albumin, highly abundant in the blood, plays a vital role in transporting zinc (and other metals including calcium and magnesium) throughout the body. Albumin not only carries zinc through the circulatory system but also mediates its uptake into cells. Factors that control the interaction between zinc and albumin and its uptake into cells are not well studied, but are important to appreciate and influence their impact on health and disease.

Albumin also transports other types of molecules (e.g. fatty acids, hormones), and binding of one molecule can affect binding of another at a separate site. For example, we previously identified that the primary zinc binding site is perturbed by fatty acid-binding elsewhere on the molecule. Physiological events that alter the composition of blood can therefore alter metal transport processes and the ability of albumin to sequester zinc. Such events may be short-term (e.g. fasting, eating, infection, stroke) or long-term (obesity, disease). Long-term alterations in blood chemistry are particularly likely to have serious consequences due to the knock-on effects caused by altered metal binding to various other molecules and delivery to cells. Indeed, we have shown that fatty acid-induced effects on zinc-binding to serum albumin result in other blood proteins, such as those involved in blood clotting, binding more zinc. In addition, we have found that cells take up zinc faster when they are cultured in the presence of fatty acids, which is a consequence of zinc interacting more with the proteins that promote its uptake into cells.

Fatty acid levels in the blood are elevated in disease states, including obesity, type 2 diabetes and fatty liver disease - disorders associated with aberrant insulin signalling (insulin is the hormone responsible for regulating blood sugar levels) and increased tendency to develop blood clots. The fact that both processes are also zinc-dependent has led us to hypothesise that elevated fatty acid levels, particularly in disease states, impair plasma zinc handling and thus negatively affect these processes. As such our specific objectives are to:
(1) Characterise the zinc-binding properties of human serum albumin in the presence of fatty acid mixtures found in normal and disease-related conditions.
(2) Identify and characterise which plasma proteins zinc binds to in the presence of normal and disease-associated concentrations of fatty acids, whilst developing and exploiting new methods to enable such investigations.
(3) Establish whether and how elevated plasma fatty acid levels influence insulin activation.
(4) Examine the effect of fatty acid-albumin interactions on zinc movement and the proteins it binds to in the cells which line the blood vessels.
(5) Determine how fatty acids influence insulin actions in primary endothelial cells via the albumin-zinc link.

To achieve this, we will adopt an integrated approach that includes the use of a variety of methods to probe zinc-protein interactions, state-of-the-art and brand new approaches to investigate which proteins bind zinc, cellular studies to examine zinc movement in primary endothelial cells, in addition to molecular and functional tests to monitor insulin signalling. This work brings together the respective expertise of an interdisciplinary UK-Brazil team best placed to meet these challenges.

Our previous work has shown that an allosteric relationship exists between circulatory NEFA transport and plasma zinc handling through binding to HSA. This has led to our core hypothesis that plasma zinc speciation is influenced by NEFAs in disease states and impacts upon processes including coagulation and insulin signalling. We will examine how binding of complex (patho)physiologically relevant mixtures of NEFAs alter Zn2+ binding to HSA using ITC. This work is based upon our previous measurement of NEFAs in type-II diabetes patients and controls. We will examine how NEFAs influence plasma zinc speciation using an established approach incorporating 2D-PAGE combined with LA-ICP-MS, which will be further developed to include bidimensional separation techniques. We will also develop a bespoke approach incorporating bidimensional separation with quantitative MS and ICP-MS to assess NEFA-induced speciation changes. The zinc-binding properties of interesting proteins identified will be probed using ITC, ESI-MS and IMMS analyses. The effect of NEFA-induced changes in the zinc-binding properties of HSA on insulin hexamer breakdown will be assessed using a FRET-based approach utilising Cy3- and Cy5-labelled insulin. Zinc flux in primary HUVECs will be assessed using our previously established stable Zn isotope-based approach to determine how NEFAs influence cellular zinc homeostasis, with intracellular speciation examined using the 2D-gel electrophoresis/LA-ICP-MS approach detailed above. Combining these two approaches will enable us to identify proteins that load with Zn2+ as influx increases in response to NEFAs. Finally, we will examine how NEFAs influence insulin signalling in HUVECs using molecular and gene expression-based approaches. Activation of PI3K/AKT/eNOS and Ras/Raf/MEK pathways will be assessed using specific antibodies to active forms of these proteins and insulin-dependent gene expression will be examined using a Stat5-dependent luciferase assay.