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Title: | 膽汁傳輸蛋白之肝細胞分布與膽汁滯留疾病之相關機轉 The subcellular trafficking and targeting of bile salt export pump in hepatocytes and related mechanisms in cholestatic liver diseases |
Authors: | Shang-Hsin Wu 吳上欣 |
Advisor: | 張美惠(Mei Hwei Chang) |
Keyword: | 膽汁傳輸蛋白,ESCRT,胞內體,後高基氏體傳送,PLEC, BSEP,ESCRT,early/sorting endosome,post-Golgi trafficking,plectin, |
Publication Year : | 2019 |
Degree: | 博士 |
Abstract: | 人類的膽汁傳輸蛋白(Bile salt export pump,BSEP;基因為ABCB11)是一個具有十二個穿膜區塊(transmembrane domain)的膜蛋白,主要分布在肝細胞的膽小管膜(canalicular/apical membrane)上將膽鹽自肝細胞輸送進入膽道系統(biliary system)。相較於大、小鼠有其他的分子能代償其膽汁傳輸蛋白的功能缺損,人類的膽汁傳輸蛋白是膽小管膜上唯一能傳輸膽鹽的蛋白質。當人類的膽汁傳輸蛋白異常或功能缺損時,會造成一系列不同嚴重程度的膽汁滯留症(cholestasis),包括:第二型進行性家族性肝內膽汁滯留(progressive familial intrahepatic cholestasis type 2,PFIC2)、第二型良性反覆性肝內膽汁滯留(benign recurrent intrahepatic cholestasis type 2,BRIC2)、妊娠期肝內膽汁滯留症(intrahepatic cholestasis in pregnancy,ICP),以及藥物引起的肝臟受損(drug-induced liver injuries)。在許多藥物開發過程中,若會造成膽汁傳輸蛋白功能受到抑制或是影響膽汁傳輸蛋白在膽小管膜上表現時,也是造成該藥物開發失敗的主因之一。然而目前關於膽汁傳輸蛋白造成膽汁滯留的機制所知不多,特別是在臨上可以發現許多病人的膽汁傳輸蛋白無法在膽小管膜上表現,但是卻沒有膽汁傳輸蛋白基因上的突變。此外,在人類胎兒肝臟裡的膽汁傳輸蛋白分布與成人有顯著地不同。相較於成人肝細胞內的膽汁傳輸蛋白主要分布在膽小管膜上,胎兒肝細胞裡的膽汁傳輸蛋白卻只有部份在膽小管膜上,其他則散布在細胞質中。由這些觀察可以推測有些潛在的因子能直接或間接地調控膽汁傳輸蛋白在細胞內的分布與膽小管膜的定位(canalicular/apical targeting)。但是在這部分的相關研究相當有限。
本論文試圖探索能調控膽汁傳輸蛋白於細胞內的移動(subcellular trafficking)可能的膽汁傳輸蛋白交互作作用蛋白分子(interacting proteins),同時也企圖從疑似患有進行性家族性肝內膽汁滯留的病人檢體來找尋潛在的致病基因。在找尋可能的膽汁傳輸蛋白交互作用蛋白分子部份,利用人類膽汁傳輸蛋白的片段分子於人類胎兒肝臟cDNA基因庫(human fetal liver cDNA library)中進行酵母菌雙雜交系統分析,進而找到CHMP5為可能的膽汁傳輸蛋白交互作用分子。CHMP5已知參與細胞內endosomal sorting complex required for transport(ESCRT)subcomplex-III的組成。在體外培養細胞株以及小鼠中進行研究,同時以人類的胎兒肝臟與患有膽汁滯留的肝臟染色發現:膽汁傳輸蛋白若是與ESCRT的交互作用異常,很可能是導致膽汁傳輸蛋白於細胞質內異常停滯進而造成膽汁滯留疾病。同時這也是首次發現ESCRT參與膽汁傳輸蛋白於後高基氏體的傳送(post-Golgi trafficking),而且是在已知的Rab11上游。更進一步地發現肝細胞內已知膽汁傳輸蛋白所停留的胞器(subapical compartments,SACs),除了會有CHMP5以及另一個ESCRT-III分子LIP5外,這個仍諸多未知的胞器同時也有Rab5及Rab11的表現,所以這個胞器極有可能是所謂的早期/轉運胞內體(early/sorting endosome)。因此可以推測ESCRT所參與的膽汁傳輸蛋白於細胞內的移動是在膽汁傳輸蛋白離開高基氏體至轉運胞內體之間。 另一方面在找尋可能造成進行性家族性肝內膽汁滯留的基因部分,自一對膽汁滯留手足中發現一個全新的膽汁滯留基因PLEC。Plectin(PLEC,基因為PLEC)是一個細胞骨架連接蛋白(cytoskeleton linker protein)。膽汁傳輸蛋白在細胞內的運輸已知會受到細胞骨架以及細胞骨架上相關蛋白分子(cytoskeleton-associated proteins)影響。Plectin能聯結不同的細胞骨架(例如:角質蛋白)以維持肝細胞的結構。在帶有PLEC突變的膽汁滯留病人肝臟中發現其膽汁傳輸蛋白在膽小管膜上的表現嚴重受到影響,同時PLEC與角質蛋白第八型(Keratin 8,K8)的共同分布(co-localization)也降低。由膽汁傳輸蛋白於膽小管膜上的分布異常,同時血清中的膽鹽濃度異常增加,意味著PLEC突變可能藉由影響膽汁傳輸蛋白的膽小管膜定位或是可能扮演疾病修飾角色,進而造成膽汁滯留。 膽汁傳輸蛋白於膽小管膜上表現對其功能極為重要。本論文所運用的一些研究方式亦能應用於其他的膜蛋白的細胞內運輸與相關疾病的機制探索。同時,本論文中所發現的一些其他膽汁傳輸蛋白交作用分子及PLEC對膽汁滯留的機制值得更深入的探討與研究。 The bile salt export pump (BSEP), encoded by the ABCB11 gene, is an apical/canalicular protein with 12 transmembrane domains and mediates bile salts from hepatocytes into the biliary system. BSEP is the only apical transporter for the bile salts in humans and, unlike rodents, no other genes could compensate for the loss of BSEP functions. Abnormalities of BSEP could lead to a spectrum of cholestatic diseases including the progressive familial intrahepatic cholestasis type 2, benign recurrent intrahepatic cholestasis, intrahepatic cholestasis in pregnancy, and drug-induced liver injuries. Defects in BSEP function and canalicular expression, such as cytoplasmic accumulation, are also pivotal events that have caused the failure of many newly developed drugs. However, the mechanism of BSEP-associated cholestasis is poorly understood, especially in patients with no detected BSEP mutations in the coding regions but having impaired apical targeting of BSEP. Moreover, BSEP in human fetal livers partially expressed at the canalicular membrane and in the cytoplasm, rather than majorly at the canalicular membrane in adult. These data suggested some underlying factors regulate the distribution and apical targeting of BSEP directly or in an indirect manner. In this dissertation, I aimed to discover BSEP-interacting proteins that modulates the subcellular trafficking of BSEP and to search novel PFIC-associated genes. I used a human BSEP polypeptide as a bait to screen the human fetal liver cDNA library through yeast two-hybrid systems. Several BSEP-interacting candidates were uncovered. One of the BSEP-interacting candidates is charged multivesicular body protein 5 (CHMP5), a key endosomal protein complex required for transport subcomplex-III (ESCRT-III). Using in vitro and in vivo modals, cholestatic, and developmental human liver samples, BSEP aberrantly associating with ESCRTs may cause cytoplasmic retention of BSEP at the subapical compartments (SACs) in cholestatic diseases. These BSEP locating SACs had the expression of not only ESCRT molecules but also the two endosomal proteins Rab5 and Rab11. These findings suggested that these SACs are the early/sorting endosomes. Moreover, I provided the first example and new function of ESCRTs, in which ESCRTs involved in the post-Golgi trafficking of BSEP, which was upstream of Rab11 regulated apical cycling of BSEP. Therefore, ESCRTs mediated BSEP sorting from the trans-Golgi to the sorting endosomes. The last part of the dissertation described the discovery of a novel cholestatic gene, PLEC, associated by studying the samples from a pair of cholestasis siblings. Plectin (PLEC), encoded by PLEC, is a cytoskeleton linker protein, which linked different cytoskeletons to sustain the architecture of hepatocytes. PLEC mutated patients’ liver samples revealed impaired canalicular expression of BSEP and reduced co-localization of PLEC and the keratin 8 (K8). The impaired BSEP canalicular expression and elevated bile acid levels suggested that the PLEC mutations may either affect BSEP targeting and cause cholestasis, or be a disease modifier gene. Canalicular expression of BSEP is critical to BSEP function. The modus operandi of my work on the trafficking mechanism of BSEP could be easily applied to study all other transmembrane proteins for uncovering the etiologies of other important diseases. Besides, several BSEP-interacting candidates and the diseased mechanism of impaired BSEP trafficking in PLEC mutations are worthy to further study. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76492 |
DOI: | 10.6342/NTU201904328 |
Fulltext Rights: | 同意授權(全球公開) |
metadata.dc.date.embargo-lift: | 2022-03-13 |
Appears in Collections: | 臨床醫學研究所 |
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