SummaryMammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

The insect GABA receptor, RDL, is the target of several classes of pesticides. The peptide sequences of RDL are generally highly conserved between diverse insects. However, RNA A-to-I editing can effectively alter amino acid residues of RDL in a species specific manner, which can affect the potency of GABA and possibly insecticides. We report here that RNA A-to-I editing alters the gene products of Rdl in three mosquito disease vectors, recoding five amino acid residues in RDL of Aedes aegypti and six residues in RDLs of Anopheles gambiae and Culex pipiens, which is the highest extent of editing in RDL observed to date. Analysis of An. gambiae Rdl cDNA sequences identified 24 editing isoforms demonstrating a considerable increase in gene product diversity. RNA editing influenced the potency of the neurotransmitter, GABA, on An. gambiae RDL editing isoforms expressed in Xenopus laevis oocytes, as demonstrated by ECs ranging from 5 ± 1 to 246 ± 41 μM. Fipronil showed similar potency on different editing isoforms, with ICs ranging from 0.18 ± 0.08 to 0.43 ± 0.09 μM. In contrast, editing of An. gambiae RDL affected the activating, potentiating and inhibiting actions of ivermectin. For example, ivermectin potentiated currents induced by GABA at the EC concentration in the unedited isoform but not in the fully edited variant. Editing of a residue in the first transmembrane domain or the cys-loop influenced this potentiation, highlighting residues involved in the allosteric mechanisms of cys-loop ligand-gated ion channels. Understanding the interactions of ivermectin with molecular targets may have relevance to mosquito control in areas where people are administered with ivermectin to treat parasitic diseases.

We have demonstrated genetic changes in telomere length and oxidative stress, which may be relevant to cataractogenesis. Our study shows that in control cells telomere length increases as passage increases. We have also demonstrated that telomere length increases at higher doses of 1.0 Gy and 2.0 Gy. However, telomerase activity decreases dose dependently and as passages increase. These results are not conclusive and further studies ex vivo measuring lens opacity and telomere length in the model would be beneficial in a bigger cohort, hence confirming a link between telomere length, cataractogenesis and genetic factors.

The broad substrate capacity of the intestinal oligopeptide transporter, PepT1, has made it a key target of research into drug delivery. Whilst the substrate capacity of this transporter is broad, studies have largely been limited to small peptides and peptide-like drugs. Here, we demonstrate for the first time that a diverse range of drugs can be targeted towards transport by PepT1 using a hydrolysis resistant carrier. Eleven prodrugs were synthesized by conjugating modified dipeptides containing a thioamide bond to the approved drugs ibuprofen, gabapentin, propofol, aspirin, acyclovir, nabumetone, atenolol, zanamivir, baclofen and mycophenolate. Except for the aspirin and acyclovir prodrugs, which were unstable in the assay conditions and were not further studied, the prodrugs were tested for affinity and transport by PepT1 expressed in Xenopus laevis oocytes: binding affinities ranged from approximately 0.1 to 2 mM. Compounds which showed robust transport in an oocyte trans-stimulation assay were then tested for transcellular transport in Caco-2 cell monolayers: all five tested prodrugs showed significant PepT1-mediated transcellular uptake. Finally, the ibuprofen and propofol prodrugs were tested for absorption in rats: following oral dosing the intact prodrugs and free ibuprofen were measured in the plasma. This provides proof-of-concept for the idea of targeting poorly bioavailable drugs towards PepT1 transport as a general means of improving oral permeability.