CRAC中心酪氨酸突变介导肝细胞胆小管侧膜BSEP膜胆固醇敏感性及转运功能减退的分子机制

Gallstone formation is a complicated process and still remains incompletely elucidated. Substantial evidence indicates that cholesterol oversaturation due to reduced bile acid secretion by the liver is the primary cause of gallstones. The bile salt export pump (BSEP, ABCB11) on the canalicular membrane of hepatocytes is responsible for and a limiting step in bile acid secretion. However, the role of BSEP in cholelithogenesis remains unclear. Membrane cholesterol is efficient regulator of transmembrane protein by cholesterol recognition amino acid consensus (CRAC motif). We have found several CRAC-like motifs in the transmembrane domain of the BSEP from Uniprot database. According to previous studies, we have proposed CRAC-like motifs may be involved in the regulation of BSEP membrane expression and transport activity. Mutation of the central tyrosine to other small amino acids will be shown to alter the cholesterol sensitivity. This project will use recombinant baculovirus transfected Sf9 insect cells, PRL and IRHCs of Bsep-Y+/+ expression transgenosis mice as the research platform, intervention with the mutation of CRACs center tyrosine residue and change of membrane cholesterol content, observation with Bsep membrane expression and bile acid transport activity, to explore the hypothyroidism that CRACs center tyrosine mutation mediated the molecular mechanism of cholesterol sensitivity and the transptor function of BSEP, which provides theoretical and experiment basis for the prevention, treatment and acquired new target for gallstones.

肝细胞胆小管侧膜胆汁酸盐输出泵(BSEP)转运功能下降，胆汁中胆汁酸盐分泌减少是胆固醇结石形成的重要原因。已有研究表明，BSEP在胆小管侧膜的功能表达受细胞膜胆固醇含量正向调节，但具体机制不清；近期研究显示，膜胆固醇可通过与胆固醇识别氨基酸基序（CRACs）结合，调控跨膜蛋白的细胞膜定位及功能表达；研究发现BSEP跨膜区域内存在多个CRACs样氨基酸序列；课题组根据以往研究提出CRACs参与膜胆固醇调控BSEP的细胞膜表达及转运功能，其中心酪氨酸突变影响BSEP对膜胆固醇的敏感性及胆汁酸转运功能，并促进胆固醇结石形成这一论题。课题拟以重组杆状病毒转染Sf9昆虫细胞、Bsep-Y+/+表达小鼠PRL及IRHCs为研究平台，通过诱导Bsep的CRAC中心酪氨酸突变、干预膜胆固醇含量等手段，观察Bsep的膜表达及胆汁酸转运功能等方法进行论证，为获得新的胆结石预防及治疗靶点提供理论和实验依据。

肝细胞胆小管侧膜胆汁酸盐输出泵(BSEP)转运功能下降，胆汁中胆汁酸盐分泌减少是 胆固醇结石形成的重要原因。已有研究表明，BSEP在胆小管侧膜的功能表达受细胞膜胆固醇含量正向调节，但具体机制不清;近期研究显示，膜胆固醇可通过与胆固醇识别氨基酸 基序(CRACs)结合，调控跨膜蛋白的细胞膜定位及功能表达;研究发现BSEP跨膜区域内存在多个CRACs样氨基酸序列;课题组根据以往研究提出CRACs参与膜胆固醇调控BSEP的细 胞膜表达及转运功能，其中心酪氨酸突变影响BSEP对膜胆固醇的敏感性及胆汁酸转运功能，并促进胆固醇结石形成这一论题。研究发现：1、根据反转录法的测序结果提示，AKR小鼠、C57小鼠、C57bl/6小鼠Bsep的氨基酸序列未见明显差异，提示Bsep的表达及转运功能改变与Bsep的一级结构可能关系不大；2、Bsep表达改变受膜胆固醇含量调节，高剂量胆固醇抑制Bsep表达，低剂量胆固醇刺激Bsep表达，降低膜胆固醇含量，Bsep表达下降；3、Cav-1敲减之后，Bsep对胆固醇及MBCD刺激的应答减弱，过表达Cav-1，Bsep对胆固醇及MBCD刺激的应答，提示胆固醇并不是直接调节Bsep表达，而是通过调控Cav-1实现的；4、高胆固醇环境下p-PKC明显增加，且Bsep与Hax-1可共表达，且在低剂量胆固醇刺激下共表达减少，提示Bsep磷酸化及内吞过程参与其膜表达调节。