Post-Translational Modifications of FXR; Implications for Cholestasis and Obesity-Related Disorders

The Farnesoid X receptor (FXR) is a nuclear receptor which is activated by bile acids. Bile acids function in solubilization of dietary fats and vitamins in the intestine. In addition, bile acids have been increasingly recognized to act as signaling molecules involved in energy metabolism pathways, amongst others via activating FXR. Upon activation by bile acids, FXR controls the expression of many genes involved in bile acid, lipid, glucose and amino acid metabolism. An inability to properly use and store energy substrates may predispose to metabolic disorders, such as obesity, diabetes, cholestasis and non-alcoholic fatty liver disease. These diseases arise through a complex interplay between genetics, environment and nutrition. Due to its function in metabolism, FXR is an attractive treatment target for these disorders. The regulation of FXR expression and activity occurs both at the transcriptional and at the post-transcriptional level. It has been shown that FXR can be phosphorylated, SUMOylated and acetylated, amongst other modifications, and that these modifications have functional consequences for DNA and ligand binding, heterodimerization and subcellular localization of FXR. In addition, these post-translational modifications may selectively increase or decrease transcription of certain target genes. In this review, we provide an overview of the posttranslational modifications of FXR and discuss their potential involvement in cholestatic and metabolic disorders

N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection

The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety

N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection

The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety

Mechanistic insights into the inhibition of NTCP by myrcludex B

Background & aims: The sodium taurocholate co-transporting polypeptide (NTCP) is the entry receptor for the hepatitis B and delta virus (HBV/HDV) and the main hepatic uptake transporter of conjugated bile acids. Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, blocks HBV/HDV infection and inhibits NTCP-mediated bile acid uptake. In humans this increases systemic bile acid levels, which remain elevated for hours even after Myrcludex B is cleared from the circulation. Here, we investigated the dynamics of Myrcludex B-induced NTCP-mediated bile acid transport inhibition in mice and if/how the duration of this effect relates to NTCP protein turnover. Methods: Plasma bile acids were determined in Myrcludex B-treated OATP1a/1b-deficient mice. In vitro, plasma membrane-resident NTCP was labeled with biotin or fluorescein isothiocyanate (FITC)-labeled Myrcludex B and traced in time using hNTCP-overexpressing U2OS cells. Förster resonance energy transfer by fluorescent lifetime imaging microscopy was used to investigate whether Myrcludex B can transfer to newly synthesized NTCP. Results: Conjugated bile salt levels in plasma peaked 4 h after subcutaneous Myrcludex B administration. After 24 h, plasma bile salt levels were completely normalized, in line with restored NTCP-mediated bile acid transport in vitro. Biotin-labeled NTCP disappeared faster than Myrcludex B-FITC, with almost 40% of FITC signal remaining after 24 h. FITC fluorescence lifetime was strongly decreased upon expression of DY547-labeled acyl carrier protein-tagged NTCP, demonstrating transfer of pre-bound Myrcludex B-FITC to newly formed NTCP. Conclusions: The dynamics of NTCP protein turnover and Myrcludex B-induced plasma bile salt elevations are similar, suggesting that the Myrcludex B:NTCP interaction is very long-lived. Nevertheless, Myrcludex B is not completely degraded together with NTCP and can transfer to newly synthesized NTCP. Lay summary: The experimental drug Myrcludex B binds the sodium taurocholate co-transporting polypeptide (NTCP), the viral entry receptor for the hepatitis B and D virus (HBV/HDV), and thereby prevents infection, but also inhibits hepatic bile salt uptake leading to transiently elevated bile salt levels. This study describes that while the normalization of plasma bile salt levels likely depends on the protein turnover rate of NTCP, Myrcludex B partly escapes co-degradation with NTCP by transferring from one NTCP molecule to another. This is of importance to the HBV/HDV research field as it provides a potential explanation for the distinct kinetics and dose-dependence of Myrcludex B's effects on viral infection versus bile salt transport

Treatment with Bafilomycin but not with MG132 induces expression of NTCP-N5,11Q.

<p>(A,B) Parental HepG2 or cells expressing NTCP-WT or NTCP-N5,11Q were treated with 0.1% DMSO, Bafilomycin (10nM/24h), MG132 (20μM/6h) or a combination of Bafilomycin (10nM/24h) and MG132 (20μM/ for the last 6h). (A) Immunofluorescence microscopy of NTCP-WT and NTCP-N5,11Q. in which NTCP was visualized using anti-HA antibody (green) and Hoechst (blue) was used to visualize the nucleus. (B) Results of TCA uptake in parental HepG2 cells (grey) and HepG2 cells expressing NTCP-N5,11Q (black). (C) Results of TCA uptake in HepG2 cells expressing NTCP-WT (grey) or NTCP-N5,11Q (black). (B,C)The uptake capacity of the NTCP was determined as pmol TCA uptake/well. Bars represent the mean +/- sd of three independent experiments, each performed in quadruplicate. *Indicates significant different from DMSO treated (p <0.05). # indicates significant different between the cell-lines in the same treatment (p<0.05).</p

Effect of N-linked glycosylation on NTCP protein expression.

<p>(A and B) HA-tagged NTCP was immunoprecipitated (IP) from lysates of HepG2 cells stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q. IP samples were subjected to immunoblotting for NTCP (using anti-HA-hrp). (B) IP samples were digested with PNGase F for 1h at 37°C (500 units) prior to immunoblotting for NTCP. (C) NTCP was quantified by image J and expressed relative to NTCP-WT. Molecular mass is given in kDa on the left-hand side. Results are mean +/- sd. *Indicates significant different from NTCP-WT (p <0.05).</p

Effect of NTCP glycosylation on bile acid transport activity.

<p>Taurocholate (TCA) uptake assay in parental HepG2 cells and those stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q. Cells were incubated for 2 minutes with uptake buffer containing 20 mM taurocholate, spiked with [³H]-taurocholate. NTCP specific uptake capacity is defined as pmol TCA uptake/well, where the bars represent the mean +/- sd of three experiments each performed in quadruplicate. *Significantly different from parental cells and # indicates significantly different from NTCP-WT values (p < 0.05).</p

HBV infection of wild type and glycosylation deficient NTCP variants.

<p>HepG2 cells stably expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q or NTCP-N5,11Q were infected with HBV. (A) 3 days post infection, HBeAg production was measured by ELISA. (B) Establishment of HBV infection was determined by quantification of intracellular cccDNA 3 days post-infection. Values are given as mean +/- sd of two independent experiments each performed in triplicate. The dotted line represents the values obtained after infecting parental HepG2 cells that lack NTCP expression. *Significantly different from NTCP-WT values (p < 0.05).</p

Effect of N-linked glycosylation on NTCP plasma membrane expression.

<p>Plasma membrane expression was assessed by surface-biotinylation of parental HepG2 cells or those expressing NTCP-WT, NTCP-N5Q, NTCP-N11Q, NTCP-N5,11Q. (A) Representative immunoblot for NTCP membrane expression and for the Transferrin receptor (TfR), as a loading control. Molecular mass is given in kDa on the left-hand side. (B) NTCP expression at the plasma membrane was semi-quantified by Myrcludex-B-FITC intensity. Fluorescence of the parental cells was subtracted before normalization and the net fluorescence units expressed relative to NTCP-WT. *Indicates significant different from NTCP-WT (p<0,05) (C) Confocal microscopic images of HepG2 cells expressing NTCP-WT or mutant proteins stained with anti-HA (green) and counterstained with Hoechst (blue). Scale bar represents 10μm.</p

Calnexin Depletion by Endoplasmic Reticulum Stress During Cholestasis Inhibits the Na+-Taurocholate Cotransporting Polypeptide

Cholestasis-induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the bile acid importer Na+-taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP-mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis-induced ER stress. Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of calnexin provides an additional mechanism to dampen NTCP-mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis