FATTY ACID TRANSPORT--STRUCTURAL AND CELL BIOLOGY

缺血时细胞内脂肪酸水平及其即刻变化 代谢物，酰卡尼汀和酰辅酶A，上升到异常水平。正常 运输和酶过程似乎不足以处理 这些高负载以及随之而来的增加的分区 脂类进入细胞膜会扰乱与膜相关的细胞功能，例如 收缩、兴奋性、节律和细胞活力。同样的脂类 与遗传性脂肪酸病的病理有关 氧化，这是心肌病和其他疾病的重要原因 使人衰弱的疾病。这个项目的总体目标是描述 细胞内脂肪酸结合和运输的结构和动力学方面 胞浆、细胞膜和胞浆中。总体战略是 用最先进的方法研究天然脂肪酸在结构上 和细胞生物学。四个具体目标是，首先，描述 脂肪酸及其直接代谢物(酰辅酶A和辅酶A)的相互作用 模型膜和生物膜的~(13)C核磁共振研究 光谱分析和滴定量热法。第二个具体目标是 阐明脂肪酸与白蛋白相互作用的分子细节， 主要通过X射线结晶学和多维核磁共振研究 大块白蛋白碎片。第三个具体目标是澄清 细胞内脂肪酸和脂结合蛋白的分子细节 通过测定含和不含配体的溶液的核磁共振结构。这个 最终的具体目标是研究脂肪酸在膜上的移动。 用荧光法监测pH值并从膜上解吸 细胞和小泡的变化。我们的脂肪酸诱导pH假说 细胞的变化将通过全细胞荧光和视频进行检查 单细胞成像荧光测量。这项工作将定义 血浆中脂肪酸的某些重要分子相互作用 在隔室、膜和细胞质中。它将允许详细的 白蛋白与胞内脂肪酸结合方式的比较 结合蛋白和更好地理解蛋白的功能 胞内蛋白质。它将建立脂肪酸的作用机制 并提供脂肪酸的实时检测 进入细胞的运动。作为生理上重要结构的模型 和过程，这些体外系统将有助于形成一个基础 阐明缺血时复杂的病理生理反应。这部作品 也将有助于理解脂肪酸紊乱的分子基础 新陈代谢、糖尿病和肥胖。

In ischemia intracellular levels of fatty acids and their immediate metabolites, acylcarnitine and acyl-CoA, rise to abnormal levels. Normal transport and enzymatic processes appear to be insufficient to handle these high loads, and the consequent increased partitioning of these lipids into membranes can disturb membrane-related cell functions, such as contraction, excitability, rhythm, and cell viability. The same lipids are implicated in the pathology of genetic-linked disorders of fatty acid oxidation, which are important causes of cardiomyopathy and other debilitating diseases. The overall aim of this project is to describe structural and dynamic aspects of fatty acid binding and transport in plasma, in cell membranes, and in the cytosol. The overall strategy is to study native fatty acids with state-of-the-art methods in both structural and cell biology. The four specific aims are, first, to characterize interactions of fatty acids and their immediate metabolites (acyl-CoA and acylcarnitine) with model membranes and biological membranes by 13/C NMR spectroscopy and by titration calorimetry. The second specific aim is to elucidate molecular details of fatty acid interactions with albumin, primarily by X-ray crystallographic and multidimensional NMR studies of large fragments of albumin. The third specific aim is to elucidate the molecular details of intracellular fatty acid and lipid binding proteins by determining their solution NMR structure with and without ligands. The final specific aim is to study movement of fatty acids across membranes and desorption from membranes by fluorescence methods monitoring pH changes in cells and in vesicles. Our hypothesis of fatty acid-induced pH changes in cells will be examined by whole cell fluorescence and video imaging fluorescence measurements in single cells. This work will define certain important molecular interactions of fatty acids in the plasma compartment, in membranes and in the cytosol. It will permit a detailed comparison of the binding modes of albumin and intracellular fatty acid binding proteins and a better understanding of the functions of intracellular proteins. It will establish mechanisms of fatty acid permeation through membranes and provide a real time assay of fatty acid movement into cells. As models of physiologically important structures and processes, these in vitro systems will help form a basis for clarifying complex pathophysiological responses in ischemia. This work will also help understand the molecular basis of disorders of fatty acid metabolism, diabetes, and obesity.