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。锌以锌离子的形式存在，是哺乳动物体内的一种必需的金属离子，通过允许发生许多重要的化学反应而发挥着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、阿尔茨海默氏症和癌症等重大疾病中发挥着至关重要的作用。在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只有部分了解。众所周知，血清白蛋白在血液中含量非常丰富，对锌(以及其他金属，包括钙和镁)在体内的运输起着至关重要的作用。白蛋白不仅通过循环系统携带锌，而且还介导锌在细胞中的吸收。控制锌和白蛋白之间的相互作用以及它被细胞摄取的因素还没有得到很好的研究，但对于认识和影响它们对健康和疾病的影响是重要的。白蛋白还运输其他类型的分子(如脂肪酸、激素)，一个分子的结合可以影响另一个分子在不同位置的结合。例如，我们以前发现主要的锌结合位点被分子上其他地方的脂肪酸结合所干扰。因此，改变血液成分的生理事件可以改变金属的运输过程和白蛋白隔离锌的能力。这类事件可能是短期的(如禁食、进食、感染、中风)，也可能是长期的(如肥胖、疾病)。血液化学的长期变化尤其可能产生严重的后果，因为改变的金属与各种其他分子结合并传递到细胞所引起的连锁反应。事实上，我们已经证明，脂肪酸对锌与血清白蛋白结合的影响会导致其他血液蛋白，如那些参与血液凝结的蛋白，结合更多的锌。此外，我们还发现，当细胞在脂肪酸存在的情况下培养时，它们吸收锌的速度更快，这是锌与促进其进入细胞的蛋白质相互作用更多的结果。在疾病状态下，血液中的脂肪酸水平会升高，包括肥胖症、2型糖尿病和脂肪肝--与胰岛素信号异常(胰岛素是负责调节血糖水平的激素)和更容易出现血栓有关的疾病。这两个过程都依赖于锌，这一事实使我们假设，脂肪酸水平的升高，特别是在疾病状态下，会损害血浆锌的处理，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时人血清白蛋白的锌结合特性。(2)识别和表征在存在正常和疾病相关浓度的脂肪酸时锌与哪些血浆蛋白结合。同时开发和开发新的研究方法。(3)确定血浆脂肪酸水平升高是否以及如何影响胰岛素激活。(4)检查脂肪酸-白蛋白相互作用对锌运动的影响以及它在血管细胞中结合的蛋白质。(5)确定脂肪酸如何通过白蛋白-锌连接影响初级内皮细胞中的胰岛素活动。为了实现这一点，我们将采用综合方法，包括使用各种方法来探索锌与蛋白质的相互作用，最新的和全新的方法来研究哪些蛋白质结合锌，除了监测胰岛素信号的分子和功能测试外，还进行细胞研究以检查原代内皮细胞中的锌运动。这项工作汇集了英国-巴西跨学科团队各自的专业知识，最有能力应对这些挑战。

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.