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LIVER STEROL CARRIER PROTEINS--EFFECTS OF ETHANOL

肝脏甾醇载体蛋白——乙醇的影响

基本信息

批准号：
2748451
负责人：
WELLINGTON GIBSON WOOD
金额：
$ 16.54万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-08-01 至 2001-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2748451
关键词：
alcoholic beverage consumption binding proteins chemical binding cholesterol ethanol fatty acid binding protein fluorescent dye /probe intermolecular interaction laboratory mouse lipid transport liver metabolism membrane permeability nuclear magnetic resonance spectroscopy phospholipids protein structure function steroid metabolism transport proteins

项目摘要

DESCRIPTION (from Applicant's Abstract): Both lipids and proteins are sites of ethanol action. There have been an extensive number of studies on effects of ethanol on lipid structure. However, while quite a few studies examined effects of ethanol on protein function, there is little information known on how ethanol actually alters protein activity. It has been previously proposed that ethanol directly binds to proteins, but that conclusion has been largely based on effects of ethanol o protein function. Another issue is if ethanol directly binds to a protein what are the consequences of that binding on protein function. Preliminary data indicate that ethanol indeed directly binds to certain lipid transfer proteins, i.e., sterol carrier binding protein-2 (SCP-2), liver-fatty acid binding protein (L-FABP) and bovine serum albumin (BSA). In addition, we observed that ethanol displaced cis-parinaric acid from some, but not all fatty acid-binding sites on BSA. Thus, not only does ethanol directly bind to some proteins but it selectively binds to certain hydrophobic sites. It is the overall hypothesis of this new application that ethanol directly binds to hydrophobic areas of SCP-2 and L-FABP, two proteins that are affected by chronic ethanol consumption. We further proposed that ethanol inhibits the binding of endogenous ligands to SCP-2 and L-FABP and modifies sterol transport between membranes. These hypotheses will be examined using the powerful combination of fluorescence spectroscopy and 13C NMR relaxation technique. The specific aims of this application are: 1) characteristics of cholesterol phospholipids and fatty acids binding to SCP-2 and L-FABP will be examined in the absence and presence of ethanol in vitro using fluorescence techniques; 2) the binding geometry of ethanol and fatty acids to SCP-2 and L-FABP will be studied using 13C- ethanol NMR relaxation data; and 3) effects of SCP-2 and L-FABP enhanced sterol exchange between liver membranes will be evaluated. Chronic alcohol abuse is the most important cause of morbidity and mortality from liver disease in the United States. Approximately 90 percent of heavy alcohol drinkers develop fatty liver, resulting mainly from marked abnormalities in hepatic lipid metabolism, specifically, from accumulation of triacylglycerols and other lipids. In addition to lipids accumulating in liver with chronic ethanol consumption, proteins accumulate as well. Cytosolic proteins are largely responsible for this increase and L-FABP which binds fatty acids and cholesterol accounted for 22 percent of the total increase in liver cytosolic proteins in chronic ethanol treated rats. Therefore, it is important to understand how ethanol may directly act on L- FABP and SCP-2 with respect to binding, displacement of endogenous ligands, and lipid transport.

描述（来自申请人的摘要）：脂质和蛋白质都是乙醇作用位点。已有大量研究乙醇对脂质结构的影响。然而，虽然相当多研究检查了乙醇对蛋白质功能的影响，但很少有研究关于乙醇实际上如何改变蛋白质活性的已知信息。它之前已经提出乙醇直接与蛋白质结合，但这一结论很大程度上是基于乙醇对蛋白质功能。另一个问题是乙醇是否直接与蛋白质结合对蛋白质功能的影响是什么？初步数据表明，乙醇确实直接与某些物质结合。脂质转移蛋白，即甾醇载体结合蛋白-2 (SCP-2)，肝脂肪酸结合蛋白（L-FABP）和牛血清白蛋白（BSA）。此外，我们观察到乙醇取代了顺式石蜡酸来自 BSA 上某些（但不是所有）脂肪酸结合位点的酸。因此，不乙醇仅直接与某些蛋白质结合，但它选择性地结合与某些疏水位点结合。其总体假设是乙醇直接与疏水区域结合的新应用 SCP-2 和 L-FABP，两种受长期乙醇影响的蛋白质消耗。我们进一步提出乙醇抑制结合 SCP-2 和 L-FABP 的内源性配体并改变甾醇转运膜之间。这些假设将使用强大的荧光光谱与 13C NMR 弛豫相结合技术。该应用程序的具体目标是：1) 胆固醇磷脂和脂肪酸结合的特性 SCP-2 和 L-FABP 将在乙醇不存在和存在的情况下进行检查使用荧光技术进行体外实验； 2) 结合几何形状将使用 13C- 研究乙醇和脂肪酸对 SCP-2 和 L-FABP 的影响乙醇NMR弛豫数据； 3) SCP-2和L-FABP的效果增强将评估肝膜之间的甾醇交换。慢性的酗酒是发病和死亡的最重要原因来自美国的肝病。大约 90% 大量饮酒者会患上脂肪肝，主要是由于明显的肝脏脂质代谢异常，特别是三酰甘油和其他脂质的积累。此外慢性乙醇消耗导致脂质在肝脏中积聚，蛋白质也积累起来。胞浆蛋白对此负有主要责任增加和结合脂肪酸和胆固醇的L-FABP 占肝细胞溶质蛋白总增量的 22% 慢性乙醇治疗的大鼠。因此，了解这一点很重要乙醇如何直接作用于 L-FABP 和 SCP-2 结合、内源配体的置换和脂质转运。