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Published on: September 15, 2018
A structural and mechanistic model for BSEP dysfunction in PFIC2 cholestatic disease
Clémence Gruget1, Bharat G Reddy2, Jonathan M Moore3,4
1Massachusetts Institute of Technology, Cambridge, MA, USA.
Insights
Mutations in Bile Salt Export Pump (BSEP) cause Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC2). Biophysical studies reveal how these mutations destabilize BSEP, impacting bile salt transport and leading to liver disease.
Area of Science:
- Hepatology
- Molecular Biology
- Biophysics
Background:
- Bile Salt Export Pump (BSEP/ABCB11) is crucial for bile salt transport in hepatocytes.
- Biallelic BSEP mutations cause Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC2), a severe pediatric liver disease.
- PFIC2 is often linked to missense mutations affecting BSEP expression, maturation, and trafficking.
Purpose of the Study:
- To investigate the impact of PFIC2-associated mutations on BSEP protein thermodynamic stability.
- To elucidate the molecular mechanisms underlying BSEP dysfunction in cholestatic liver disease.
Main Methods:
- In-cell thermal shift assay (CETSA) was used to measure the stability of 13 BSEP variants.
- High-resolution cryo-electron microscopy (cryo-EM) was employed to determine the structure of BSEP.
Main Results:
- CETSA identified a cluster of residues at the NBD2-ICL2 interface critical for BSEP stability.
- These residues showed significant destabilization in PFIC2 variants compared to wild-type BSEP.
- Cryo-EM structure revealed a novel NBD2-localized mechanism by which severe mutations induce cholestasis.
Conclusions:
- Specific BSEP mutations destabilize the protein, leading to impaired function and cholestatic liver disease.
- Findings provide a structural basis for understanding PFIC2 pathogenesis.
- This research may guide the development of small molecule therapies targeting BSEP trafficking defects.
Abstract:
BSEP (ABCB11) transports bile salts across the canalicular membrane of hepatocytes, where they are incorporated into bile. Biallelic mutations in BSEP can cause Progressive Familial Intrahepatic Cholestasis Type 2 (PFIC2), a rare pediatric disease characterized by hepatic bile acid accumulation leading to hepatotoxicity and, ultimately, liver failure. The most frequently occurring PFIC2 disease-causing mutations are missense mutations, which often display a phenotype with decreased protein expression and impaired maturation and trafficking to the canalicular membrane. To characterize the mutational effects on protein thermodynamic stability, we carried out biophysical characterization of 13 distinct PFIC2-associated variants using in-cell thermal shift (CETSA) measurements. These experiments reveal a cluster of residues localized to the NBD2-ICL2 interface, which exhibit severe destabilization relative to wild-type BSEP. A high-resolution (2.8 Å) cryo-EM structure provides a framework for rationalizing the CETSA results, revealing a novel, NBD2-localized mechanism through which the most severe missense patient mutations drive cholestatic disease. These findings suggest potential strategies for identifying mechanism-based small molecule correctors to address BSEP trafficking defects and advance novel therapies for PFIC2 and other cholestatic diseases.
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