Molecular targets in cholestasis caused by bile salt export pump deficiency

项目摘要/摘要 在人类中，ABCB11基因的突变损害了胆盐输出泵(BSEP)的功能和原因 进展性家族性肝内胆汁淤积症II型(PFIC2)，进展迅速且往往致命 婴儿期的疾病。肝移植仍然是许多PFIC2患者的唯一可治愈的治疗方法。 开发替代疗法的主要挑战是没有已知的机制允许 无BSEP时恢复胆汁排泄。我们的长期目标是发现分子靶标 治疗胆汁淤积性肝病。此应用程序的总体目标是确定可以 在BSEP缺乏的情况下被激活以恢复胆汁排泄。根据初步数据 使用abcb11b突变体斑马鱼产生的，中心假设是替代转运蛋白可以是 提示BSEP缺陷肝细胞分泌胆汁酸。建议进行这项研究的理由是 找到了替代的胆盐转运体，并描绘了触发胆汁的分子机制 排泄功能将有助于设计治疗BSEP所致胆汁淤积的靶向治疗方法 缺乏症。发现调节胆汁分泌的新策略也将使患有其他疾病的患者受益 胆汁淤积性肝病。核心假设将通过三个实验中提出的实验来检验 互补的具体目标：1)确定可促使胆汁排出的替代转运体 BSEP缺陷肝细胞中的酸；2)确定可被激活的细胞和分子机制 恢复BSEP缺陷肝细胞的胆汁排泄；以及3)确定恢复胆汁的遗传修饰物 BSEP缺陷肝细胞的排泄。第一个目标将检验这样的假设，即恢复 另一种ABC转运蛋白MDR1定位于胆小管，使其能够承担胆汁排泄 在BSEP缺陷的肝细胞中的功能。第二个目标将调查是否扩大了 自噬是一种恢复BSEP缺陷肝细胞胆汁排泄的新机制。 第三个目标将利用一种无偏见的全基因组测序方法来识别 BSEP缺乏表型严重程度。本申请中提出的研究具有很高的创新性， 因为它使用了斑马鱼模型，这为研究胆汁排泄提供了独特的优势，并且abcb11b 突变斑马鱼是第一个BSEP丢失导致胆汁严重紊乱的动物模型 PFIC2患者的排泄物。这项拟议的研究意义重大，因为预计它将 揭示恢复BSEP缺陷肝细胞胆汁排泄的策略和发现新的治疗方法 胆汁淤积性肝病的治疗靶点。

PROJECT SUMMARY/ABSTRACT In humans, mutations in the ABCB11 gene impair bile salt export pump (BSEP) function and cause progressive familial intrahepatic cholestasis type II (PFIC2), which is a fast-progressing and often-fatal liver disease in infancy. Liver transplantation remains as the only curable treatment for many PFIC2 patients. The main challenge for developing alternative therapy is that there is no known mechanism allowing restoration of bile excretion in the absence of BSEP. Our long-term goal is to discover molecular targets to treat cholestatic liver diseases. The overall objective for this application is to identify mechanisms that can be activated to restore bile excretion in the context of BSEP deficiency. Based on the preliminary data generated using the abcb11b mutant zebrafish, the central hypothesis is that alternative transporters can be prompted to excrete bile acids in BSEP-deficient hepatocytes. The rationale for the proposed research is that, finding the alternative bile salt transporter and delineating the molecular mechanisms triggering its bile excretion function will facilitate the designing of targeted therapies to treat cholestasis caused by BSEP deficiency. Identifying novel strategies to modulate bile secretion will also benefit the patients with other cholestatic liver diseases. The central hypothesis will be tested by the experiments proposed in three complementary specific aims: 1) Determine the alternative transporter that can be prompted to excrete bile acids in BSEP-deficient hepatocytes; 2) Define the cellular and molecular mechanisms that can be activated to restore bile excretion in BSEP-deficient hepatocytes; and 3) Identify genetic modifiers that restore bile excretion in BSEP-deficient hepatocytes. The first aim will test the hypothesis that recovering the localization of another ABC transporter MDR1 to the bile canaliculus allows it to assume bile excretion function in BSEP-deficient hepatocytes. The second aim will investigate whether augmentation of autophagy represents a novel mechanism to restore bile excretion in hepatocytes with BSEP deficiency. The third aim will utilize an unbiased whole-genome sequencing approach to identify genetic modifiers of BSEP-deficiency phenotype severity. The research proposed in this application is highly innovative, because it uses the zebrafish model that offers unique advantages for studying bile excretion, and abcb11b mutant zebrafish is the first animal model in which loss of BSEP results in severe perturbation of bile excretion as seen in patients with PFIC2. The proposed research is significant, because it is expected to reveal strategies to restore bile excretion in BSEP-deficient hepatocytes and uncover new therapeutic targets for treating cholestatic liver diseases.