HEPATOCELLULAR TRANSPORT OF BILIARY PHOSPHOLIPIDS

在人类胆汁形成过程中，平均每小时1克胆盐的跨鞘流量刺激每日胆汁分泌2克胆固醇和11克磷脂，磷脂富含（大于95%）磷脂酰胆碱。 磷脂酰胆碱转运蛋白是一种在肝脏中富集的、在体外催化磷脂酰胆碱跨膜转运的胞浆蛋白，其生理功能尚不清楚。 亚胶束胆汁盐显着增强纯化的磷脂酰胆碱转移蛋白的活性，并建议在肝细胞的选择和胆汁磷脂的运输这种蛋白质的关键作用。最近的cDNA克隆表明，磷脂酰胆碱转运蛋白是新的和保守的脊椎动物。 通过在组织培养中研究磷脂酰胆碱转运蛋白对细胞分泌和摄取脂质的影响，将深入了解细胞功能。 为了阐明其底物特异性的分子基础，X射线晶体学将被用来解决重组表达的磷脂酰胆碱转移蛋白的三维结构。 结构数据将用作通过定点诱变进行结构-功能分析的基础。初步研究表明，磷脂酰胆碱转运蛋白基因启动子中的顺式作用元件负责肝脏特异性转录调控。负责肝脏富集磷脂酰胆碱转移蛋白的调控元件和转录因子将在组织培养中阐明。 磷脂酰胆碱转移蛋白基因在人类染色体17 q21 -22的遗传定位揭示了与Meckel综合征的共定位，Meckel综合征是一种致命的疾病，其特征在于多种先天性异常，包括门静脉周围肝纤维化和子宫内胆管增生。 由于初步实验已经揭示了Meckel综合征患者编码磷脂酰胆碱转移蛋白的cDNA中的突变，因此将进行系统研究以检测一系列患者的基因突变。将评估突变对重组蛋白的活性和结构的影响。 对磷脂酰胆碱转运蛋白的细胞功能、结构生物学、分子调控和遗传学的研究有助于在分子水平上阐明胆汁脂质分泌，并可能导致胆石症和胆汁淤积的早期干预，以及管理高胆固醇血症的新策略。

During bile formation in humans, a transhepatic flux of an average of one gram of bile salt per hour stimulates the daily biliary secretion of two grams of cholesterol together with eleven grams of phospholipids, which are enriched (greater than 95 percent) in phosphatidylcholines. Phosphatidylcholine transfer protein is a cytosolic protein of unknown physiological function that is enriched in liver and in vitro catalyzes intermembrane transfer of phosphatidylcholines. Submicellar bile salts markedly enhance activity of purified phosphatidylcholine transfer protein and suggest a critical role for this protein in hepatocellular selection and transport of biliary phospholipids. Recent cDNA cloning has demonstrated phosphatidylcholine transfer protein to be novel and conserved among vertebrates. Insights into cellular function will be gleaned by examining in tissue culture the influence of phosphatidylcholine transfer protein on cellular secretion and uptake of lipid. To elucidate the molecular basis for its substrate specificity, x-ray crystallography will be utilized to solve the three dimensional structure of recombinant expressed phosphatidylcholine transfer protein. Structural data will be employed as a basis for structure-function analysis by site-directed mutagenesis. Preliminary studies suggest that cis acting elements in the phosphatidylcholine transfer protein gene promoter are responsible for liver-specific transcriptional regulation. Regulatory elements and transcription factors responsible for hepatic enrichment with phosphatidylcholine transfer protein will be elucidated in tissue culture. Genetic mapping of the phosphatidylcholine transfer protein gene to human chromosome 17q21-22 has revealed co-localization with Meckel syndrome, a lethal disorder characterized by multiple congenital anomalies including periportal hepatic fibrosis and bile duct proliferation in utero. Because preliminary experiments have revealed mutations in cDNAs encoding phosphatidylcholine transfer protein from patients with Meckel syndrome, a systematic study will be undertaken to test for genetic mutations in a series of patients. Influence of mutations on activity and structure of recombinant protein will be assessed. Studies of the cellular function, structural biology, molecular regulation and genetics of phosphatidylcholine transfer protein should help to elucidate biliary lipid secretion at a molecular level and potentially lead to early interventions in cholelithiasis and cholestasis, as well as new strategies for management of hypercholesterolemia.