FATTY ACID TRANSPORT--STRUCTURAL AND CELL BIOLOGY

脂肪酸运输——结构和细胞生物学

基本信息

批准号：
6109586
负责人：
JAMES Athur HAMILTON
金额：
$ 34.7万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 1999-12-31
项目状态：
已结题

项目摘要

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.

缺血时细胞内脂肪酸水平及其直接影响代谢物酰基肉碱和酰基辅酶A上升至异常水平。普通的运输和酶促过程似乎不足以处理这些高负载，以及随之而来的这些分区的增加脂质进入膜会扰乱膜相关的细胞功能，例如收缩、兴奋性、节律和细胞活力。相同的脂质与遗传相关的脂肪酸疾病的病理学有关氧化是导致心肌病和其他疾病的重要原因使人衰弱的疾病。该项目的总体目标是描述脂肪酸结合和运输的结构和动态方面血浆、细胞膜和细胞质中。总体策略是使用最先进的方法研究天然脂肪酸的结构和细胞生物学。这四个具体目标是：首先，脂肪酸及其直接代谢物（酰基辅酶A和酰基辅酶A）的相互作用酰基肉碱）与模型膜和生物膜（13/C NMR）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 射线晶体学和多维核磁共振研究白蛋白的大片段。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确定它们在有或没有配体的情况下的溶液核磁共振结构。这最终的具体目标是研究脂肪酸跨膜的运动通过荧光方法监测 pH 值并从膜上解吸细胞和囊泡的变化。我们对脂肪酸诱导 pH 值的假设通过全细胞荧光和视频检查细胞的变化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的分子相互作用室、膜和细胞质中。它将允许详细白蛋白与细胞内脂肪酸结合方式的比较结合蛋白并更好地了解其功能细胞内蛋白质。它将建立脂肪酸的机制渗透膜并提供脂肪酸的实时测定运动进入细胞。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缺血中复杂的病理生理反应。这部作品还将有助于了解脂肪酸紊乱的分子基础新陈代谢、糖尿病和肥胖。