Molecular targets in cholestasis caused by bile salt export pump deficiency

项目总结/摘要 在人类中，ABCB 11基因突变损害胆盐输出泵（BSEP）功能，并导致 进行性家族性肝内胆汁淤积II型（PFIC 2），这是一种快速进展且经常致命的肝脏疾病， 婴儿期的疾病肝移植仍然是许多PFIC 2患者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 在不存在BSEP的情况下恢复胆汁排泄。我们的长期目标是发现分子靶点， 治疗胆汁淤积性肝病。本申请的总体目标是确定能够 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abcb 11b突变体斑马鱼产生的，中心假设是替代转运蛋白可以是 促使BSEP缺陷肝细胞分泌胆汁酸。拟议研究的基本原理是 找到替代的胆盐转运体并描绘触发其胆汁的分子机制， 排泄功能将有助于设计治疗BSEP引起的胆汁淤积的靶向治疗方法 缺陷确定调节胆汁分泌的新策略也将使患有其他疾病的患者受益。 胆汁淤积性肝病中心假设将通过三个实验中提出的实验进行检验。 补充的具体目标：1）确定可促使胆汁排泄的替代转运蛋白 BSEP缺陷肝细胞中的酸; 2）定义可以被激活的细胞和分子机制 恢复BSEP缺陷肝细胞中的胆汁排泄;和3）鉴定恢复胆汁分泌的遗传修饰剂 BSEP缺陷肝细胞中的排泄。第一个目标将测试恢复 另一种ABC转运蛋白MDR 1定位于胆小管，使其承担胆汁排泄 在BSEP缺陷肝细胞中的功能。第二个目标将调查是否增加 自噬是BSEP缺乏时肝细胞胆汁分泌恢复的一种新机制。 第三个目标将利用无偏倚的全基因组测序方法来鉴定遗传修饰剂， BSEP缺陷表型严重程度。本申请中提出的研究具有高度创新性， 因为它使用了斑马鱼模型，为研究胆汁排泄提供了独特的优势， 突变斑马鱼是第一个BSEP缺失导致胆汁严重扰动的动物模型 在PFIC患者中观察到的排泄2。这项研究意义重大，因为它有望 揭示恢复BSEP缺陷肝细胞中胆汁排泄的策略，并发现新的治疗方法 用于治疗胆汁淤积性肝病的靶点。

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.