ALCOHOL, APOLIPOPROTEIN-J BIOCHEMISTRY, AND HDL FUNCTION

酒精、载脂蛋白-J 生物化学和 HDL 功能

• 批准号: 2748435
• 负责人: PRADEEP GHOSH
• 金额: $ 11.62万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2748435
• 关键词: SDS polyacrylamide gel electrophoresis; aminoacid; apolipoproteins; biomarker; blood lipoprotein metabolism; ethanol; gas chromatography; glycosylation; high density lipoproteins; high performance liquid chromatography; laboratory rat; posttranslational modifications; protein purification; protein structure function; protein transport; secretion; sulfated glycoprotein 2

The pathogenesis of alcohol-induced liver injury is normally accompanied by profound changes in the composition and concentration of lipids and lipoproteins at each stage. Plasma high density lipoproteins (HDL) is a normal constituent in both rat and human, and its role in the regulation of body cholesterol via reverse cholesterol transport is well documented. Apoprotein (apo)A and E, major components of HDL play important regulatory roles in cholesterol homeostasis. Recently, Apo J, a 70-kDa protein has been identified as an integral component of human plasma HDL and its role in the reverse cholesterol transport has been suggested. Sulfated glycoprotein-2 (SGP-2), is the major secretory protein product of sertoli cells in rat and has ben suggested as a homolog of human plasma apo J. [We have previously shown that chronic ethanol affects the synthesis, glycosylation and secretion of many N- and O-glycosylated proteins including HDL-apo E and transferrin. The physiological role of Apo J remains unclear]. No studies have been done on Apo J as an integral component of HDL in rats. Our preliminary results clearly established the presence of Apo J in rat liver, plasma and plasma HDL fractions and its involvement in the [reverse cholesterol transport] process. Further, our results suggest that Apo J is strongly susceptible to deleterious actions of chronic ethanol [as evidenced by the loss of sialic acid residues from Apo J]. These facts provide a compelling rationale for studying the effects of chronic ethanol on the structure and function of Apo J in rats. The specific aims of this proposal, using rat as a model are; 1. to determine plasma Apo J levels and its distribution in HDL subspecies and effects of chronic ethanol treatment on the normal distribution profile. 2. to study how chronic ethanol affects the synthesis, post-translational modifications and secretion of Apo J, 3. to investigate the microheterogeneity pattern of Apo J [and effects of ethanol on sialic acid composition of apo J, thus, exploring its potential as a viable marker for alcohol consumption]. 4. to evaluate the participation of Apo J in HDL assembly and its role in the 'Reverse cholesterol transport' and alterations, if any, induced by chronic ethanol, and 5. to explore the structural designing of rat HDL=Apo J, its similarities and differences with rat SGP-2 and human apo J and effects of chronic ethanol on the structural framework of rat HDL-Apo J. The systems to be used to accomplish the above specific aims: (i) the macrophage and hepatocyte system to study reverse cholesterol transport, (ii) Apo J purification by SDS-PAGE and immunoaffinity columns, (iii) hepatocyte study reverse cholesterol transport, (ii) Apo J purification by SDS-PAGE and immunoaffinity columns, (iii) hepatocyte system for the synthesis, glycosylation and subcellular transit studies on Apo J,(v) iso- electric focussing for microheterogeneity studies, and (vi) HPLC and gas chromatography for studies on structural aspects of rat Apo J. An animal model provides a good tool to explore the metabolic and physiological activities of biological systems. Studies on alcohol and HDL remain unclear. The results of this study may provide a new insight into HDL metabolism and role of Apo J under alcoholic conditions. [More significantly, our recent findings suggest the viability of the index of the loss of sialic acid residues from Apo J as a clinical marker for chronic alcohol consumption. This method may prove to be simple, improved and cost-effective compared to those currently used in clinics.

酒精引起的肝损伤的发病机理通常伴随着 脂质的组成和浓度的深刻变化 脂蛋白在每个阶段。 血浆高密度脂蛋白（HDL）是一个 大鼠和人类的正常成分及其在调节中的作用 通过反向胆固醇运输的身体胆固醇有充分的文献记载。 载脂蛋白（APO）A和E，HDL的主要组成部分发挥重要调节性 在胆固醇稳态中的角色。 最近，Apo J，一种70 kDa蛋白 被确定为人血浆HDL的组成部分及其作用 在反向胆固醇中，已经提出了。 硫化 糖蛋白-2（SGP-2）是Sertoli的主要分泌蛋白产物 大鼠中的细胞，并提出是人血浆Apo J.的同源物。[我们 以前表明慢性乙醇会影响合成， 许多N和O-糖基化蛋白的糖基化和分泌 包括HDL-APO E和转铁蛋白。 Apo J的生理作用 仍然不清楚]。 尚未对APO J进行研究 大鼠HDL的成分。 我们的初步结果清楚地确定了 大鼠肝脏，等离子体和血浆HDL级分中的APO J及其存在 参与[反向胆固醇运输]过程。 此外，我们的 结果表明，Apo J非常容易受到有害行动的影响 慢性乙醇的[如从 apo j]。 这些事实为研究 慢性乙醇对大鼠Apo J的结构和功能的影响。 该提案的具体目的，使用大鼠作为模型； 1 确定血浆apo j级别及其在HDL亚种中的分布和分布 慢性乙醇处理对正态分布的影响。 2。 研究慢性乙醇如何影响综合，翻译后 Apo J的修改和分泌，3。 Apo J的微观分子模式[乙醇对唾液酸的影响 因此，Apo J的组成，探索其作为可行标记的潜力 饮酒]。 4。评估Apo J在HDL中的参与 组装及其在“反向胆固醇运输”中的作用 慢性乙醇引起的改变（如果有的话），5。 大鼠hdl = apo j的结构设计，其相似性和差异 用大鼠SGP-2和人类APO J以及慢性乙醇对 大鼠HDL-APO J.的结构框架。 用于完成上述特定目的的系统：（i） 巨噬细胞和肝细胞系统研究反向胆固醇的运输， （ii）SDS-PAGE和免疫亲和力柱的Apo J纯化，（III） 肝细胞研究反向胆固醇转运，（ii）Apo J纯化通过 SDS-PAGE和免疫亲和力柱，（III）肝细胞系统 对APO J，（V）等二的合成，糖基化和亚细胞运输研究 电力集中于微观分别研究，以及（vi）HPLC和气体 用于研究大鼠Apo的结构方面的色谱。 动物模型为探索新陈代谢和 生物系统的生理活动。 关于酒精和HDL的研究 保持不清楚。 这项研究的结果可能会提供新的见解 HDL代谢和Apo J在酒精条件下的作用。 [更多的 重要的是，我们最近的发现表明该指数的生存能力 apo j的唾液酸残基的损失是作为临床标记的 长期饮酒。 此方法可能被证明很简单，改进了 与当前在诊所中使用的人相比，具有成本效益。