Jan Stindt

Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.

BACKGROUND & AIMS Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. METHODS To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. RESULTS Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type-related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. CONCLUSIONS Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.

The bile salt export pump (BSEP) in health and disease

The bile salt export pump (BSEP) is the major transporter for the secretion of bile acids from hepatocytes into bile in humans. Mutations of BSEP are associated with cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 2 (PFIC-2), benign recurrent intrahepatic cholestasis type 2 (BRIC-2) and genetic polymorphisms are linked to intrahepatic cholestasis of pregnancy (ICP) and drug-induced liver injury (DILI). Detailed analysis of these diseases has considerably increased our knowledge about physiology and pathophysiology of bile secretion in humans. This review focuses on expression, localization, and function, short- and long-term regulation of BSEP as well as diseases association and treatment options for BSEP-associated diseases.

Multidrug efflux pumps: substrate selection in ATP-binding cassette multidrug efflux pumps--first come, first served?

Multidrug resistance is a major challenge in the therapy of cancer and pathogenic fungal infections. More than three decades ago, P‐glycoprotein was the first identified multidrug transporter. It has been studied extensively at the genetic and biochemical levels ever since. Pdr5, the most abundant ATP‐binding cassette transporter in Saccharomyces cerevisiae, is highly homologous to azole‐resistance‐mediating multidrug transporters in fungal pathogens, and a focus of clinical drug resistance research. Despite functional equivalences, P‐glycoprotein and Pdr5 exhibit striking differences in their architecture and mechanisms. In this minireview, we discuss the mechanisms of substrate selection and multidrug transport by comparing the fraternal twins P‐glycoprotein and Pdr5. We propose that substrate selection in eukaryotic multidrug ATP‐binding cassette transporters is not solely determined by structural features of the transmembrane domains but also by their dynamic behavior.

Easy and Rapid Purification of Highly Active Nisin

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1 M NaCl. Here, we describe an optimized purification protocol using a five-step NaCl elution to remove contaminants. The obtained nisin is devoid of impurities and shows high bactericidal activity against the nisin-sensitive L. lactis strain NZ9000. Purified nisin exhibits an IC50 of ~3 nM, which is a tenfold improvement as compared to nisin obtained via the one-step elution procedure.

Human Cytomegalovirus Disrupts the Major Histocompatibility Complex Class I Peptide-Loading Complex and Inhibits Tapasin Gene Transcription

ABSTRACT Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8+ T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and β2-microglobulin (β2m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8+ T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMV-infected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.

Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

Bile salt export pump‐reactive antibodies form a polyclonal, multi‐inhibitory response in antibody‐induced bile salt export pump deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

A novel mutation within a transmembrane helix of the bile salt export pump (BSEP, ABCB11) with delayed development of cirrhosis.

The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC‐2) or progressive familial intrahepatic cholestasis (PFIC‐2).

Bile Salt Export Pump‐reactive Antibodies Form a Polyclonal, Multi‐inhibitory Response in Antibody‐induced BSEP Deficiency

Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

Background: A mutation of the extended X loop of MDR3 caused hereditary liver cholestasis. Results: Wild type MDR3 exhibited PC-induced ATPase activity, but the Q1174E mutant displayed no stimulation. Conclusion: The glutamine preceding the ABC signature motif communicates substrate binding within the TMD to the extended X loop of the NBD. Significance: This study provides evidence for a transmission interface coupling ATP hydrolysis to substrate transport. The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.