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

哺乳动物生命所需的化学元素有25种，其中15种是金属。锌，以其离子化形式Zn 2+，是哺乳动物中的必需金属离子，并通过允许许多重要的化学反应发生来执行广泛的重要生理功能。锌在生育和发育、免疫系统、衰老以及糖尿病、阿尔茨海默病和癌症等重大疾病中起着至关重要的作用。从饮食中摄取锌并进入血液后，将锌输送到需要的地方的机制仅部分了解。众所周知，蛋白质血清白蛋白，在血液中高度丰富，在整个身体中运输锌（和其他金属，包括钙和镁）中起着至关重要的作用。白蛋白不仅携带锌通过循环系统，而且还介导其进入细胞的摄取。控制锌和白蛋白之间的相互作用及其被细胞摄取的因素尚未得到很好的研究，但对于理解和影响它们对健康和疾病的影响很重要。白蛋白还转运其他类型的分子（例如脂肪酸、激素），一种分子的结合可影响另一种分子在不同位点的结合。例如，我们以前确定的主要锌结合位点被干扰的脂肪酸结合在分子上的其他地方。因此，改变血液组成的生理事件可以改变金属转运过程和白蛋白螯合锌的能力。这些事件可能是短期的（如禁食、进食、感染、中风）或长期的（肥胖、疾病）。血液化学的长期改变特别可能具有严重后果，这是由于改变的金属与各种其他分子的结合和向细胞的递送所引起的连锁效应。事实上，我们已经表明，脂肪酸诱导的锌与血清白蛋白结合的影响导致其他血液蛋白，如那些参与血液凝固，结合更多的锌。此外，我们还发现，当细胞在脂肪酸存在下培养时，细胞吸收锌的速度更快，这是锌与促进其吸收进入细胞的蛋白质相互作用更多的结果。血液中的脂肪酸水平在疾病状态下升高，包括肥胖症，2型糖尿病和脂肪肝疾病-与异常胰岛素信号（胰岛素是负责调节血糖水平的激素）和发展血栓的倾向增加有关的疾病。这两个过程也是锌依赖性的事实使我们假设脂肪酸水平升高，特别是在疾病状态下，损害血浆锌处理，从而对这些过程产生负面影响。因此，我们的具体目标是：（1）表征在正常和疾病相关条件下发现的脂肪酸混合物存在下人血清白蛋白的锌结合特性。(2)在正常和疾病相关浓度的脂肪酸存在下，确定和验证锌与哪些血浆蛋白结合，同时开发和利用新的方法来进行此类研究。(3)确定血浆脂肪酸水平升高是否以及如何影响胰岛素活化。(4)检查脂肪酸-白蛋白相互作用对锌运动及其在血管细胞中结合的蛋白质的影响。(5)确定脂肪酸如何通过白蛋白-锌连接影响胰岛素在原代内皮细胞中的作用。为了实现这一目标，我们将采用一种综合方法，包括使用各种方法来探测锌-蛋白质相互作用，最先进的和全新的方法来研究哪些蛋白质结合锌，细胞研究来检查锌在原代内皮细胞中的运动，除了监测胰岛素信号传导的分子和功能测试之外。这项工作汇集了一个跨学科的英国-巴西团队的各自专业知识，最适合应对这些挑战。

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.