Bilateral BBSRC-FAPESP: A "speciomic" toolkit to investigate fatty acid-mediated changes in plasma zinc speciation and their physiological effects
Lead Research Organisation:
University of St Andrews
Department Name: Sch of Medicine
Abstract
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.
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.
Technical Summary
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.
Organisations
Publications


Arruda MAZ
(2022)
Speciomics as a concept involving chemical speciation and omics.
in Journal of proteomics

Coverdale JPC
(2024)
Recent Advances in Metalloproteomics.
in Biomolecules

Coverdale JPC
(2022)
Albumin-mediated extracellular zinc speciation drives cellular zinc uptake.
in Chemical communications (Cambridge, England)

Dorward AM
(2023)
The role of Zn2+ in shaping intracellular Ca2+ dynamics in the heart.
in The Journal of general physiology

Fritzen R
(2023)
Magnesium Deficiency and Cardiometabolic Disease.
in Nutrients

Leal K
(2024)
Optimizing the performance of single-cell ICP-MS/MS for Fe and Zn determination in human umbilical vascular endothelial cells
in Microchemical Journal

Leal KNDS
(2024)
Metallomic analysis of urine from individuals with and without Covid-19 infection reveals extensive alterations in metal homeostasis.
in Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements (GMS)

Lu J
(2025)
Properties of the major Zn 2+ -binding site of human alpha-fetoprotein, a potential foetal plasma zinc carrier
in Chemical Communications

Polepalli S
(2025)
Protein Based Hybrid Materials of Metal Phosphate Nanoflowers and Gels for Water Remediation: Perspectives and Prospects.
in Chemistry, an Asian journal
Description | One of our key goals was to investigate how zinc binds to albumin, the main protein that transports it in the blood, and how this process changes in the presence of fatty acids. To do this, we created a modified version of albumin that lacks one of its main fatty acid-binding sites. Using a technique called isothermal titration calorimetry, we measured how strongly zinc binds to both normal and modified albumin under different conditions. These experiments provided important new insights and led to a publication in the Journal of Lipid Research. Another major focus has been identifying which other proteins in the blood take up zinc when albumin's binding ability is altered by fatty acids. Through a combination of chromatography and mass spectrometry, we discovered several unexpected proteins that bind zinc in these conditions. Since these proteins are not commercially available, we are now developing methods to produce them in the lab-either by isolating them from blood plasma or by expressing them artificially. We expect to complete this work by April 2025. A key aspect of our research is understanding how fatty acids affect insulin stability. Insulin is stored in the body as clusters of molecules called hexamers held together by zinc, which must break apart into smaller units before insulin can function properly. We developed a novel fluorescence-based method to track how insulin disassembles under different conditions. Our results have been promising, and we are now conducting final experiments using single-molecule techniques to confirm our findings. This work should be completed by early 2025, and we are preparing a manuscript to share these insights with the scientific community. We have also been investigating how fatty acids influence the movement of zinc inside endothelial cells, which line the blood vessels. Using specialised mass spectrometry techniques, we began tracing zinc transport into these cells under different conditions. Early findings highlighted the need to account for other small molecules that interact with zinc, such as histidine, and to carefully measure the fatty acid composition in our experimental system. We are currently addressing these factors and will soon expand our studies to primary human endothelial cells. Meanwhile, our collaborators in Brazil have successfully developed a method to track zinc at the single-cell level, which has already led to a publication in Microchemical Journal. Finally, we have been examining how fatty acids affect insulin signalling within cells. Insulin controls blood sugar by activating specific signalling pathways inside cells, but our initial experiments revealed that one of these pathways was already highly active in our test system. As a result, we are now investigating alternative pathways and working to establish a more physiologically relevant cell model. These experiments will run alongside our zinc transport studies, with results expected in the coming months. |
Exploitation Route | The discoveries made in this study open up several exciting opportunities for further research and potential real-world applications. By shedding light on how fatty acids influence zinc handling and insulin regulation, these findings can be built upon by researchers across various disciplines, from basic biochemistry to clinical medicine. One of the most immediate ways this work can be taken forward is in the study of zinc transport and metabolism. The new insights into how fatty acids disrupt zinc binding to albumin provide a strong foundation for future studies in biochemistry and structural biology. Researchers working in these areas can use this knowledge to refine models of zinc transport in the bloodstream and explore how similar disruptions might occur in other metal-protein interactions. The discovery of alternative zinc-binding proteins in plasma adds another dimension to this field, opening new avenues in metalloproteomics and bioinorganic chemistry, where scientists can further map the role of zinc in circulatory and metabolic health. These findings also have direct implications for diabetes and metabolic disease research. Since zinc plays a key role in insulin function, understanding how fatty acids interfere with zinc availability could help explain why people with conditions like type 2 diabetes often experience disruptions in insulin signalling as well as altered zinc metabolism. This knowledge may contribute to new studies examining whether dietary interventions, lifestyle changes, or pharmacological approaches could help restore normal zinc handling and improve metabolic health. Endocrinologists and diabetes researchers could build on this work by studying the effects of fatty acid levels in patient samples, potentially identifying new biomarkers of disease progression. In addition, this research has potential applications in pharmaceutical development. The discovery that fatty acids influence insulin hexamer breakdown provides valuable insights that could help pharmaceutical scientists improve insulin formulations. By fine-tuning the stability and disassembly of insulin in the bloodstream, drug developers might be able to design new insulin therapies that work more efficiently or have longer-lasting effects. The ability to track zinc movement within cells using mass spectrometry could also prove valuable in drug discovery and development, particularly in designing therapies that target zinc-dependent processes in the body. |
Sectors | Agriculture Food and Drink Healthcare Pharmaceuticals and Medical Biotechnology |
Title | Structural and biochemical characterisation of Co2+-binding sites on serum albumins and their interplay with fatty acids (dataset) |
Description | |
Type Of Material | Database/Collection of data |
Year Produced | 2023 |
Provided To Others? | Yes |
URL | https://risweb.st-andrews.ac.uk/portal/en/datasets/structural-and-biochemical-characterisation-of-co... |
Title | Targeted removal of the FA2 site on human albumin prevents fatty acid-mediated inhibition of Zn2+-binding (dataset) |
Description | Underpinning data for publication. |
Type Of Material | Database/Collection of data |
Year Produced | 2024 |
Provided To Others? | Yes |
URL | https://research-portal.st-andrews.ac.uk/en/datasets/targeted-removal-of-the-fa2-site-on-human-album... |
Description | Talk at the International Conference of Trace Elements and Minerals 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Jordan gave a talk on his work at the conference entitled "Role of plasma fatty acid and zinc dynamics in platelet functioning. Implications for pathological clotting". |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.ukaachen.de/kliniken-institute/ictem/program/ |