HEPATOCELLULAR TRANSPORT OF BILIARY PHOSPHOLIPIDS

在人的胆汁形成过程中，平均每小时1克胆汁盐的经肝通量会刺激每天2克胆固醇和11克磷脂的胆汁分泌，这些磷脂富含（大于95%）磷脂酰胆碱。磷脂酰胆碱转移蛋白是一种生理功能未知的胞质蛋白，富集于肝脏，在体外催化磷脂酰胆碱的膜间转移。胶束下胆盐显著提高纯化磷脂酰胆碱转移蛋白的活性，表明该蛋白在肝细胞选择和胆道磷脂转运中起关键作用。最近的cDNA克隆证明了磷脂酰胆碱转移蛋白是一种在脊椎动物中具有保守性的新蛋白。通过检查组织培养中磷脂酰胆碱转移蛋白对细胞分泌和脂质摄取的影响，将收集细胞功能的见解。为了阐明其底物特异性的分子基础，我们将利用x射线晶体学来解析重组表达的磷脂酰胆碱转移蛋白的三维结构。结构数据将被用作通过位点定向诱变进行结构-功能分析的基础。初步研究表明，磷脂酰胆碱转移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。磷脂酰胆碱转移蛋白在肝脏富集的调控元件和转录因子将在组织培养中得到阐明。磷脂酰胆碱转移蛋白基因在人类染色体17q21-22上的遗传定位揭示了与Meckel综合征共定位，Meckel综合征是一种致命的疾病，其特征是多发性先天性异常，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突变，因此将进行系统的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活性和结构的影响。对磷脂酰胆碱转移蛋白的细胞功能、结构生物学、分子调控和遗传学的研究将有助于在分子水平上阐明胆脂分泌，并可能导致胆石症和胆汁淤积的早期干预，以及高胆固醇血症管理的新策略。

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.