NisC Binds the FxLx Motif of the Nisin Leader Peptide

Nisin is a model system for lantibiotics, a class of peptides displaying antimicrobial activity against various Gram-positive bacteria. After ribosomal synthesis, the precursor peptide is modified in two steps, of which the last one involves consecutive cyclization reactions mediated by the cyclase NisC. Here, we present a detailed <i>in vitro</i> study of the interaction between NisC and the nisin precursor peptide. Our results unravel a specific interaction of NisC with the leader peptide independent of the maturation state. Furthermore, mutagenesis studies identified a specific binding sequence within the leader. Two amino acids (F_–18 and L_–16) within the highly conserved -FNLD- box of class I lantibiotics are essential for binding. They represent a potential general binding motif between leader peptides of a group of lantibiotics with their cyclase family. In summary, these <i>in vitro</i> data provide a new perception on the complexity of the lantibiotic modification machineries

Analysis of the Bile Salt Export Pump (<i>ABCB11</i>) Interactome Employing Complementary Approaches

<div><p>The bile salt export pump (BSEP, <i>ABCB11</i>) plays an essential role in the formation of bile. In hepatocytes, BSEP is localized within the apical (canalicular) membrane and a deficiency of canalicular BSEP function is associated with severe forms of cholestasis. Regulation of correct trafficking to the canalicular membrane and of activity is essential to ensure BSEP functionality and thus normal bile flow. However, little is known about the identity of interaction partners regulating function and localization of BSEP. In our study, interaction partners of BSEP were identified in a complementary approach: Firstly, BSEP interaction partners were co-immunoprecipitated from human liver samples and identified by mass spectrometry (MS). Secondly, a membrane yeast two-hybrid (MYTH) assay was used to determine protein interaction partners using a human liver cDNA library. A selection of interaction partners identified both by MYTH and MS were verified by <i>in vitro</i> interaction studies using purified proteins. By these complementary approaches, a set of ten novel BSEP interaction partners was identified. With the exception of radixin, all other interaction partners were integral or membrane-associated proteins including proteins of the early secretory pathway and the bile acyl-CoA synthetase, the second to last, ER-associated enzyme of bile salt synthesis.</p></div

Venn diagram of potential BSEP interaction partners obtained with MYTH and co-IP / MS/MS.

<p>The numbers represent the potential BSEP interaction partners identified by MYTH and co-IP / MS/MS and the overlapping set of proteins found in both screens.</p

Functional control assay for the membrane yeast two-hybrid with BSEP and self-activation control.

<p>The yeast strain NMY51 was transformed with the BSEP bait construct and control plasmids coding either for a nonsense peptide with the NubI (wild-type ubiquitin) or the NubG (mutated ubiquitin) -tag. Due to its affinity to its C-terminal half the NubI moiety activates the system regardless of bait interaction. Both controls show equal transformation efficiency on selective media (SD-LW) for the plasmids (upper panels). The positive control shows yeast growth on selective medium (SD-LWH) due to the affinity of the wild-type ubiquitin moieties. This confirms expression of the BSEP fusion protein. In contrast, the negative control shows no reporter gene activation based on unspecific interaction of BSEP with the NubG-nonsense peptide. Additionally, no self-activation of the MYTH system with BSEP was detected in a library-scale transformation of the empty pPR3-N library vector.</p

Proteins identified as interaction partners of BSEP in MYTH and co-IP / MS/MS screens.

<p>Proteins identified as interaction partners of BSEP in MYTH and co-IP / MS/MS screens.</p

Bait dependency test with BSEP interaction partners in the early secretory pathway.

<p>To confirm the interaction with prey proteins from the initial MYTH screen, reporter gene activation was tested individually for each prey against the bait, BSEP, or a non-interacting control bait, large T antigen. The figure shows one representative result for the positively tested preys, which are denoted by their respective gene name.</p

co-IP / MS/MS identifies BSEP interaction partners.

<p><b>(A)</b> Immunoprecipitation of BSEP coupled to complex mass spectrometry reveals new interaction partners of BSEP in human liver. Crude canalicular membrane preparations were solubilized in either digitonin or Triton X-100, and immunoprecipitated samples were subjected to MS/MS. For each detergent, protein frequencies from two co-IPs with BSEP antibody are plotted against the respective negative control with naïve mouse IgG. Interaction partners of interest are labeled. For the sake of clarity, proteins found exclusively in either the BSEP or control co-IP are depicted with an MS-score of one instead of zero on the other axis. (<b>B)</b> Immunoblot analysis of co-IPs shown inA.</p

BSEP interacts with radixin and the bile acyl-CoA synthetase.

<p><b>(A)</b> MYTH bait dependency test of radixin_1-318 and BACS_640-690 against BSEP or a non-interacting control bait. (<b>B)</b> Pull-down of BSEP with radixin_1-318, BACS_77-690 and AP-2 μ1. Protein interaction partners were immobilized as bait on Strep-Tactin Sepharose and purified BSEP was added. BSEP and the interaction partners were detected by immunoblot analysis with monoclonal antibodies against BSEP or the Strep-tag, respectively. Strep-Tactin Sepharose without bait protein served as negative control. (<b>C)</b> Tag-less BSEP can be pulled down with radixin_1-318, but not with full-length, non-activated radixin. Protein interaction partners were immobilized on Strep-Tactin Sepharose. Purified, tag-less BSEP was added to the beads and the complexes were eluted after washing. BSEP and the interacting proteins were detected by immunoblot analysis with monoclonal antibodies against BSEP or the Strep-tag, respectively.</p

Schematic diagram of the membrane yeast two-hybrid system (MYTH) to screen for interaction partners of the ABC-transporter BSEP.

<p>The MYTH system is based on a split-ubiquitin approach. The bait, BSEP, is fused to the C-terminus of ubiquitin (Cub) and a transcription factor (LexA-VP16). The preys are soluble or membrane-associated liver proteins, introduced via a cDNA library. They are fused to the N-terminus of ubiquitin (NubG). Upon interaction of BSEP and the liver protein the ubiquitin moieties, which have a low affinity for each other due to a mutation in the N-terminus, come into close proximity. The reassembled ubiquitin is recognized by endogenous ubiquitin specific proteases (UBP). The transcription factor is cleaved off and activates the reporter genes (<i>HIS3</i>, <i>ADE2</i>, <i>lacZ</i>).</p