FATTY ACID TRANSPORT--STRUCTURAL AND CELL BIOLOGY

缺血时细胞内脂肪酸水平及其直接影响 代谢物酰基肉毒碱和酰基辅酶A升高到异常水平。 正常 运输和酶过程似乎不足以处理 这些高负荷，以及随之而来的增加的分区， 脂质进入膜可以干扰膜相关的细胞功能，如 收缩、兴奋性、节律和细胞活力。 相同的脂质 与脂肪酸遗传性疾病的病理学有关 氧化，这是心肌病和其他疾病的重要原因 使人衰弱的疾病 本项目的总体目标是描述 脂肪酸结合和转运的结构和动力学方面 在细胞膜和细胞质中。 总体战略是 用最先进方法研究天然脂肪酸， 和细胞生物学。 四个具体目标是，首先， 脂肪酸及其直接代谢物（酰基-CoA和 用~（13）C NMR法测定了乙酰肉毒碱（acylcarnitine）与模型膜和生物膜的相互作用 光谱法和滴定量热法。 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射线晶体学和多维NMR研究， 大的白蛋白碎片 第三个具体目标是阐明 细胞内脂肪酸和脂质结合蛋白的分子细节 通过测定它们在有和没有配体的情况下的溶液NMR结构。 的 最后一个具体目标是研究脂肪酸跨膜运动 和通过荧光法监测pH从膜解吸 细胞和囊泡的变化。 我们假设脂肪酸诱导的pH值 通过全细胞荧光和视频检查细胞的变化 在单细胞中成像荧光测量。 这项工作将定义 血浆中脂肪酸的某些重要的分子相互作用 在细胞膜和细胞质中。 它将允许详细的 白蛋白与细胞内脂肪酸结合方式的比较 结合蛋白和更好地了解的功能， 细胞内蛋白质 它将建立脂肪酸的机制， 并提供脂肪酸的真实的时间测定 移动到细胞。 作为重要生理结构的模型 和过程，这些体外系统将有助于形成基础， 阐明了缺血中复杂的病理生理学反应。 这项工作 也将有助于了解脂肪酸紊乱的分子基础， 代谢、糖尿病和肥胖症。

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.