Shang-Hsin Wu1, Mei-Hwei Chang1,2,3, Ya-Hui Chen2, Hui-Lin Wu1,3, Huey-Huey Chua2, Chin-Sung Chien1, Yen-Hsuan Ni2,3,4, Hui-Ling Chen5, Huey-Ling Chen6,7,8. 1. Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, 100, Taiwan. 2. Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan. 3. Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan. 4. Medical Microbiota Center of the First Core Laboratory, National Taiwan University College of Medicine, Taipei, 100, Taiwan. 5. Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan. hlchen9@ntu.edu.tw. 6. Department of Pediatrics, National Taiwan University College of Medicine and National Taiwan University Children's Hospital, Taipei, 100, Taiwan. hueyling@ntu.edu.tw. 7. Hepatitis Research Center, National Taiwan University Hospital, Taipei, 100, Taiwan. hueyling@ntu.edu.tw. 8. Department and Graduate Institute of Medical Education and Bioethics, National Taiwan University College of Medicine, Taipei, 100, Taiwan. hueyling@ntu.edu.tw.
Abstract
BACKGROUND: The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. METHODS: The human BSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. RESULTS: We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. Cholestatic BSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. CONCLUSIONS: Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.
BACKGROUND: The bile salt export pump (BSEP) is a pivotal apical/canalicular bile salt transporter in hepatocytes that drives the bile flow. Defects in BSEP function and canalicular expression could lead to a spectrum of cholestatic liver diseases. One prominent manifestation of BSEP-associated cholestasis is the defective canalicular localization and cytoplasmic retention of BSEP. However, the etiology of impaired BSEP targeting to the canalicular membrane is not fully understood. Our goal was to discover what molecule could interact with BSEP and affect its post-Golgi sorting. METHODS: The humanBSEP amino acids (a.a.) 491-630 was used as bait to screen a human fetal liver cDNA library through yeast two-hybrid system. We identified a BSEP-interacting candidate and showed the interaction and colocalization in the co-immunoprecipitation in hepatoma cell lines and histological staining in human liver samples. Temperature shift assays were used to study the post-Golgi trafficking of BSEP. We further determine the functional impacts of the BSEP-interacting candidate on BSEP in vitro. A hydrodynamically injected mouse model was established for in vivo characterizing the long-term impacts on BSEP. RESULTS: We identified that charged multivesicular body protein 5 (CHMP5), a molecule of the endosomal protein complex required for transport subcomplex-III (ESCRT-III), interacted and co-localized with BSEP in the subapical compartments (SACs) in developing human livers. CholestaticBSEP mutations in the CHMP5-interaction region have defects in canalicular targeting and aberrant retention at the SACs. Post-Golgi delivery of BSEP and bile acid secretion were impaired in ESCRT-III perturbation or CHMP5-knockdown hepatic cellular and mouse models. This ESCRT-III-mediated BSEP sorting preceded Rab11A-regulated apical cycling of BSEP. CONCLUSIONS: Our results showed the first example that ESCRT-III is essential for canalicular trafficking of apical membrane proteins, and provide new targets for therapeutic approaches in BSEP associated cholestasis.
Entities:
Keywords:
Apical trafficking; Bile salt export pump; CHMP5; Cholestasis; ESCRT; Liver development; Post-Golgi trafficking; Rab11; Subapical compartment
Authors: T Gerloff; B Stieger; B Hagenbuch; J Madon; L Landmann; J Roth; A F Hofmann; P J Meier Journal: J Biol Chem Date: 1998-04-17 Impact factor: 5.157
Authors: S S Strautnieks; L N Bull; A S Knisely; S A Kocoshis; N Dahl; H Arnell; E Sokal; K Dahan; S Childs; V Ling; M S Tanner; A F Kagalwalla; A Németh; J Pawlowska; A Baker; G Mieli-Vergani; N B Freimer; R M Gardiner; R J Thompson Journal: Nat Genet Date: 1998-11 Impact factor: 38.330