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Identification of mutation-prone points in bile salt export pump.

ABSTRACT: BACKGROUND: The bile salt export pump mediates uphill canalicular bile acid secretion. Inherited dysfunction of the bile salt export pump causes a progressive and a benign form of familial intrahepatic cholestasis. Mutations within the bile salt export pump gene are reported. Presently, prediction of protein nanostructure and function is a great challenge in the proteomics and structural genomics era. Identification of the point vulnerable to mutation is a new trend to expand knowledge on disorders at the genomic and proteomic level of diseases. MATERIALS AND METHODS: A bioinformatic analysis was performed to study the positions that determine peptide motifs in the amino acid sequence of the bile salt export pump. To identify the weak linkage in bile salt export pump, a new bioinformatic tool named GlobPlot was used. RESULTS: The positions 16-34, 119-127, 451-459, 550-561, 1084-1099, 1108-1118, 1135-1140, 1211-1217, and 1314-1321 were identified as the positions prone to mutation. CONCLUSION: Based on this study, the weak linkages in the bile salt export pump can be identified and can provide good information for expectation of possible new mutations that can lead to cross species jumping. In addition, the results from this study provide useful information for further research on the bile salt export pump.

SUBMITTER: Wiwanitkit V

PROVIDER: S-EPMC2215358 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Identification of mutation-prone points in bile salt export pump.

Wiwanitkit Viroj V

HPB : the official journal of the International Hepato Pancreato Biliary Association 20070101 6

<h4>Background</h4>The bile salt export pump mediates uphill canalicular bile acid secretion. Inherited dysfunction of the bile salt export pump causes a progressive and a benign form of familial intrahepatic cholestasis. Mutations within the bile salt export pump gene are reported. Presently, prediction of protein nanostructure and function is a great challenge in the proteomics and structural genomics era. Identification of the point vulnerable to mutation is a new trend to expand knowledge on ...[more]

PMID: 18345292

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Project description:To investigate the relation of two different mutations to the outcome of partial external biliary diversion (PEBD) in severe bile salt export pump (BSEP) deficiency.Mutations in the gene encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney (HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatography-tandem mass spectrometry. Wild-type and mutant BSEP transport of [3H]-labeled taurocholate (TC) and taurochenodeoxycholate (TCDC) was assessed by vesicular transport assays.A girl (at 2 mo) presented with pruritus, jaundice and elevated serum bile salts (BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919del and the nonsense mutation p.R1235X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy (age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives (< 5%). The patients' native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919del and p.G1032R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively.In summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2 (PFIC-2).

Project description:BackgroundThe 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.MethodsThe 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.ResultsWe 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.ConclusionsOur 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.

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Identification of mutation-prone points in bile salt export pump.

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Identification of mutation-prone points in bile salt export pump.

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