New insights into the substrate specificities of proton-coupled oligopeptide transporters from E. coli by a pH sensitive assay.

Proton-coupled oligopeptide transporters (POTs) are secondary active transporters that facilitate di- and tripeptide uptake by coupling it to an inward directed proton electrochemical gradient. Here the substrate specificities of Escherichia coli POTs YdgR, YhiP and YjdL were investigated by means of a label free transport assay using the hydrophilic pH sensitive dye pyranine and POT overexpressing E. coli cells. The results confirm and extend the functional knowledge on E. coli POTs. In contrast to previous assumptions, alanine and trialanine appears to be substrates of YjdL, albeit poor compared to dipeptides. Similarly tetraalanine apparently is a substrate of both YdgR and YhiP.

Heterologous expression, characterization and evaluation of the matrix protein from Newcastle disease virus as a target for antiviral therapies.

Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chicken, which poses a real threat to the agriculture industry. Matrix (M) proteins of NDV and many other viruses perform critical functions during viral assembly and budding from the host cell. M-proteins are well conserved and therefore are potential targets for antiviral therapies. To validate this, we expressed the NDV M-protein in its native form in Saccharomyces cerevisiae and in inclusion bodies in Escherichia coli. Proper refolding of the recombinant protein produced in E. coli was verified using circular dichroism and infrared spectroscopies and electron microscopy. Immunization of chickens with the NDV M-protein elicited significant serum antibody titers. However, the antibodies conferred little protection against the ND following lethal viral challenges. We conclude that the M-protein is not exposed on the surface of the host cell or the virus at any stage during its life cycle. We discuss how the conserved M-protein can further be exploited as an antiviral drug target.