Endothelial lipase: a modulator of HDL metabolism and atherosclerosis in humans

描述(申请人提供)：内皮脂肪酶(EL)是一种细胞外蛋白质，对高密度脂蛋白具有显著的水解活性。在模式生物中的研究表明，EL在高密度脂蛋白代谢中很重要；然而，在人类高密度脂蛋白代谢中，EL的意义尚不清楚。这项建议的目的是调查人类EL的变异以及这种变异对高密度脂蛋白水平的影响。对高密度脂蛋白受试者的EL进行深度重测序，已经发现了潜在的功能非同义和启动子变体。我们假设罕见的EL编码变体通过降低EL活性来增加高密度脂蛋白。我们还假设，EL启动子变体通过改变转录效率来影响EL蛋白的水平，从而调节高密度脂蛋白的水平。具体目标1：确定EL的编码变体是否通过降低EL的活性来增加高密度脂蛋白。为了测试活性的降低，将通过定点突变在EL表达载体中重新创建编码变体，在293细胞中表达，并在体外检测它们对合成磷脂底物和天然高密度脂蛋白的特定活性。为了测试对体内高密度脂蛋白水平的影响，编码变体将被克隆到基于AAV的载体中，注射到EL-KO小鼠中，并在6周内评估高密度脂蛋白水平。变异与人类高密度脂蛋白水平的关联性将通过比较高密度脂蛋白极端受试者之间的频率，以及通过分析家庭内高密度脂蛋白水平的协同隔离来进行统计检验。特定目的2：确定EL的启动子变体是否通过影响EL基因的转录速率来调节高密度脂蛋白水平。为了测试转录速度的变化，将通过定点突变在由EL启动子驱动的萤火虫荧光素酶表达载体中重新产生启动子变体，并将其导入HUVEC细胞。表达结果将通过对杂合子受试者分离的内皮细胞进行等位基因特异性单链构象分析来验证。变异与人类高密度脂蛋白水平的关联性将通过比较高密度脂蛋白极端受试者之间的频率，以及通过分析家庭内高密度脂蛋白水平的协同隔离来进行统计检验。高密度脂蛋白水平与预防冠心病相关，冠心病是导致男性和女性死亡的主要原因。这项建议将试图确定内皮脂肪酶在人类高密度脂蛋白代谢中的意义，并验证其作为药物抑制提高高密度脂蛋白水平的靶点。

DESCRIPTION (provided by applicant): Endothelial lipase (EL) is an extracellular protein with significant hydrolysis activity against HDL. Studies in model organisms suggest that EL is important in HDL metabolism; however, the significance of EL in human HDL metabolism is unknown. The objective of this proposal is to investigate variation in human EL and the effects of this variation on HDL levels. Deep resequencing of EL in subjects with high HDL has identified potentially functional nonsynonymous and promoter variants. We hypothesize that rare EL coding variants increase HDL by decreasing EL activity. We also hypothesize that EL promoter variants modulate HDL levels by affecting levels of EL protein via alterations in transcriptional efficiency. Specific Aim 1: To determine if coding variants of EL increase HDL by decreasing EL activity. To test for decreased activity, coding variants will be recreated in an EL expression plasmid via site-directed mutagenesis, expressed in 293 cells, and their specific activities assayed in vitro against synthetic phospholipids substrates and native HDL. To test for an influence on HDL levels in vivo, the coding variants will be cloned into AAV-based vectors, injected into EL-KO mice, and HDL levels assessed over 6 weeks. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. Specific Aim 2: To determine if promoter variants of EL modulate HDL levels through their influence on the rate of transcription of the EL gene. To test for altered rates of transcription, promoter variants will be recreated via site-directed mutagenesis in a firefly luciferase expression plasmid driven by the EL promoter, and transfected into HUVEC cells. Expression results will be validated by allele-specific HaploChIP analysis on endothelial cells isolated from heterozygous subjects. Association of the variants with human HDL levels will be tested statistically by comparing frequencies among subjects from HDL extremes and by analyzing cosegregation with HDL levels within families. HDL levels correlate with protection from coronary heart disease, the leading cause of mortality among men and women. This proposal will attempt to determine the significance of endothelial lipase in human HDL metabolism, and validate it as a target for pharmacologic inhibition to raise HDL levels.