SNaPe: a versatile method to generate multiplexed protein fusions using synthetic linker peptides for in vitro applications.

Understanding the structure and function of protein complexes and multi-domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon Sortase-mediated and Native chemical ligation using synthetic Peptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps - sortase-mediated and native chemical ligation - together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

More than just recruitment: the X-domain influences catalysis of the first phenolic coupling reaction in A47934 biosynthesis by Cytochrome P450 StaH.

Glycopeptide antibiotic biosynthesis involves a complex cascade of reactions centred on a non-ribosomal peptide synthetase and modifiying proteins acting in trans, such as Cytochrome P450 enzymes. These P450s are responsible for cyclisation of the peptide via cross-linking aromatic amino acid side chains, which are a hallmark of the glycopeptide antibiotics. Here, we analysed the first cyclisation reaction in the biosynthesis of the glycopeptide antibiotic A47934. Our results demonstrate that the P450 StaH is recruited to the NRPS machinery through interaction with the X-domain present in the last A47934 NRPS module. We determined the crystal structure of StaH and showed that it is responsible for the first cyclisation in A47934 biosynthesis and additionally exhibits flexible substrate specificity. Our results further point out that the X-domain has an impact on the efficiency of the in vitro cyclisation reaction: hybrid PCP-X constructs obtained by domain exchange between A47934 and teicoplanin biosynthesis NRPS modules reveal that the X-domain from A47934 leads to decreased P450 activity and alternate stereochemical preference for the substrate peptide. We determined that a tight interaction between StaH and the A47934 X-domain correlates with decreased in vitro P450 activity: this highlights the need for glycopeptide antibiotic cyclisation to be a dynamic system, with an overly tight interaction interfering with substrate turnover in vitro.

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934.

The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

Sox2 is a potent inhibitor of osteogenic and adipogenic differentiation in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are a promising target for cell-based bone regeneration. However, their application for clinical use is limited because hMSCs lose their ability for cell division and differentiation during longer in vitro cultivation. The osteogenic differentiation is regulated through a complex network of molecular signal transduction pathways where the canonical Wnt pathway plays an important role. Sox2, a known key factor for maintenance of cellular pluripotency in stem cells, is supposed to influence the Wnt pathway in osteoblasts. In this study, we overexpressed Sox2 in immortalized hMSCs by lentiviral gene transfer. Sox2 overexpression significantly reduced the osteogenic and adipogenic differentiation potentials. This effect was abolished by knockdown of Sox2 overexpression. In addition, Oct4 and Nanog, other key transcription factors for pluripotency, are strongly upregulated when Sox2 is overexpressed. Furthermore, Dkk1, a target gene of the Sox2-Oct4 heterodimer and a Wnt antagonist, is downregulated. Sox2 overexpression causes higher expression levels of ß-catenin, the central transcription factor of the canonical Wnt pathway. These results suggest that Sox2 keeps hMSCs in an undifferentiated state by influencing the canonical Wnt pathway. Regulated expression of Sox2 may be a promising tool to cultivate hMSCs in sufficient quantities for cell and gene therapy applications.

In vivo mesenchymal stem cell tracking with PET using the dopamine type 2 receptor and 18F-fallypride.

UNLABELLED: Human mesenchymal stem cells (hMSCs) represent a promising treatment approach for tissue repair and regeneration. However, little is known about the underlying mechanisms and the fate of the transplanted cells. The objective of the presented work was to determine the feasibility of PET imaging and in vivo monitoring after transplantation of dopamine type 2 receptor-expressing cells. METHODS: An hMSC line constitutively expressing a mutant of the dopamine type 2 receptor (D2R80A) was generated by lentiviral gene transfer. D2R80A messenger RNA expression was confirmed by reverse transcriptase-polymerase chain reaction. Localization of the transmembrane protein was analyzed by confocal fluorescence microscopy. The stem cell character of transduced hMSCs was investigated by adipogenic and osteogenic differentiation. Migration capacity was assessed by scratch assays in time-lapse imaging. In vitro specific binding of ligands was tested by fluorescence-activated cell sorting analysis and by radioligand assay using (18)F-fallypride. Imaging of D2R80A overexpressing hMSC transplanted into athymic rats was performed by PET using (18)F-fallypride. RESULTS: hMSCs showed long-term overexpression of D2R80A. As expected, the fluorescence signal suggested the primary localization of the protein in the membrane of the transduced cells. hMSC and D2R80A retained their stem cell character demonstrated by their osteogenic and adipogenic differentiation capacity and their proliferation and migration behavior. For in vitro hMSCs, at least 90% expressed the D2R80A transgene and hMSC-D2R80A showed specific binding of (18)F-fallypride. In vivo, a specific signal was detected at the transplantation site up to 7 d by PET. CONCLUSION: The mutant of the dopamine type 2 receptor (D2R80A) is a potent reporter to detect hMSCs by PET in vivo.

Characterization of adipose-derived equine and canine mesenchymal stem cells after incubation in agarose-hydrogel.

Adult stem cells are of particular interest for the therapeutic approach in the field of regenerative medicine. Due to their ease of harvest, adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source that has become increasingly popular. Critical aspects of applied cell therapies are the circumstances of transport from the laboratory towards the site of operation and cell delivery into the desired area. With regard to these issues, agarose-hydrogel was analyzed as a cell carrier matrix of equine and canine ASCs in vitro, which can be used for minimally invasive application. Isolated ASCs were expanded and 2.5 × 10(6) cells were combined with agarose-hydrogel to build a 0.4% hydrogel-cell solution which was stored at two temperatures (room temperature (RT) vs. 37 °C). Cell viability was investigated (live-dead assay) at different time points (0, 1, 6 and 24 h) in order to determine i) the effect of different temperatures on the cell survival as well as ii) the maximum possible time span before implantation. CFU-assay and WST-1 assay were performed after 24 h incubation in agarose-hydrogel and the cells were induced into adipogenic and osteogenic differentiation to analyze the effects of the incubation on the cell behaviour. No negative effect of the agarose-hydrogel incubation was determined on the different species' cell behaviour at either RT or 37 °C with any of the assays used. We can recommend agarose-hydrogel as a cell carrier for cell implantation with a storage period of up to 24 h at room temperature or at 37 °C prior to implantation.