The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins. A n intein is an intervening protein domain that undergoes a unique posttranslational autoprocessing event, termed protein splicing. In this spontaneous process, the intein excises itself from the host protein and, in the process, ligates together the flanking N-and C-terminal residues (exteins) to form a native peptide bond (SI Appendix, Fig. S1A) (1, 2). Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing (SI Appendix, Fig. S1B). Unlike many well-characterized contiguous inteins that splice slowly, several naturally split inteins demonstrate rapid splicing kinetics (3-6). Indeed, the discovery of these ultrafast split inteins has enabled the development of numerous tools for both synthetic and biological applications (1).A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. In addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N-and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods (3, 7-11). Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. Although efforts have previously been made to engineer promiscuous inteins (12, 13), these have not focused on naturally split inteins, which have superior fragment association and splicing kinetics (4-6).Here, we report engineered versions of naturally split inteins that possess greatly improved extein tolerance. Guided by our understanding of active site interactions critical for efficient protein splicing, we carried out targeted saturation mutagenesis of an ultrafast split intein and then used a cell-based selection system to...
Studies of the Hepatitis C virus (HCV) life-cycle rely heavily upon Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here, we demonstrate that Huh7.5 cells, when compared to Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers and markedly upregulated Hh pathway activity: Shh, >100 fold, Gli1, >30 fold, Ptc, 2 fold. In Huh7.5 cells, we found that cyclopamine, a Hh pathway antagonist, reduced HCV RNA levels by 50% compared to vehicle and inactive isomer controls. Moreover, in Huh7 cells, treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2 fold and 4 fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose response manner. Conclusions We have identified a relationship between HCV and Hh signaling where upregulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.
The sugar capsule Capsular Polysaccharide A (CPSA), which coats the surface of the mammalian symbiont Bacteroides fragilis, is a key mediator of mammalian immune system development. In addition, this sugar polymer has shown therapeutic potential in animal models of Multiple Sclerosis and other autoimmune disorders. The structure of the CPSA polymer includes a rare stereoconfiguration sugar acetamido-4-amino-6-deoxy-galactopyranose (AADGal) that we propose is the first sugar linked to a bactoprenyl diphosphate scaffold in the production of CPSA. In this report we have utilized a heterologous system to reconstitute bactoprenyl-diphosphate-linked AADGal production. Construction of this system included a previously reported Campylobacter jejuni dehydratase, PglF, coupled to a B. fragilis encoded aminotransferase (WcfR) and initiating hexose-1-phosphate transferase (WcfS). Function of the aminotransferase was confirmed by capillary electrophoresis and a novel high performance liquid chromatography (HPLC) method. Production of the rare uridine diphosphate (UDP)-AADGal was confirmed through a series of 1D and 2D nuclear magnetic resonance experiments and high resolution mass spectrometry (HR-MS). A spectroscopically unique analogue of bactoprenyl phosphate was utilized to characterize the transfer reaction catalyzed by WcfS, and allowed HPLC based isolation of the isoprenoid-linked sugar product. Importantly, the entire heterologous system was utilized in a single pot reaction to biosynthesize the bactoprenyl-linked sugar. This work provides the first critical step in the in vitro reconstitution of CPSA biosynthesis.
Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.
Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme critical to the production of complex polysaccharides in bacteria, as it produces the crucial bactoprenol scaffold on which these materials are assembled. Methods to characterize the systems associated with polysaccharide production are non-trivial, in part due to the lack of chemical tools to investigate their assembly. In this report, we develop a new fluorescent tool using UPPS to incorporate a powerful fluorescent anthranilamide moiety into bactoprenol. The activity of this analogue in polysaccharide biosynthesis is then tested with the initiating hexose-1-phosphate transferases involved in Capsular Polysaccharide A biosynthesis in the symbiont Bacteroides fragilis and the asparagine-linked glycosylation system of the pathogenic Campylobacter jejuni. In addition, it is shown that the UPPS used to make this probe is not specific for E-configured isoprenoid substrates and that elongation by UPPS is required for activity with the downstream enzymes.
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