Abstract
Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.
Highlights
Multidrug resistance protein 3 (MDR3, ABCB4) deficiency is among those liver disorders associated with impairment of the canalicular transport of bile constituents [1]
We recently reported that the ABCB4 mutations p.G68R, p.G228R, p.D459H, and p.A934T, which we found in children with progressive familial intrahepatic cholestasis type 3 (PFIC3), resulted in ABCB4 protein being trapped in the endoplasmic reticulum (ER) [13,16]
In previous studies we showed that the disease-associated ABCB4 mutants G68R, G228R, D459H, and A934T failed to target the apical membrane and localized in the ER when expressed in polarized MDCK-II cells [13,16]
Summary
Multidrug resistance protein 3 (MDR3, ABCB4) deficiency is among those liver disorders associated with impairment of the canalicular transport of bile constituents [1]. 35 ABCB4 diseasecausing missense variants have been characterized in vitro [7,8,9,10,11,12,13,14,15,16,17] They preclude proper functioning of ABCB4 in various ways, affecting trafficking of the protein to the plasma membrane, protein expression, or PC-translocating activity. Nine out of these 35 variants cause intracellular retention of ABCB4 in the endoplasmic reticulum (ER), leading to mislocalization to the plasma membrane [7,8,11,13,16,17] Such as mutations clearly result in a loss-of-function protein, and biallelic carriers are expected to develop progressive, end-stage liver disease [8,13]
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