We recently identified Homo sapiens systemic RNA interference defective-1 transmembrane family member 1 (SIDT1) to be a transmembrane protein that facilitates intercellular small RNA transfer, leading to non-cell-autonomous RNA interference (RNAi). SIDT1 mediates key interactions between pancreatic adenocarcinoma and stellate cells, which drive tumour desmoplasia. We sought to identify functionally important SIDT1 protein interacting partners with a view to disrupting SIDT1 function, potentially targeting desmoplasia in this cancer.
Methods
A panel of human cells was transfected with a SIDT1-turbo-green fluorescent (tGFP) fusion construct, or a tGFP control construct. A phage display library was screened under high stringency conditions using intact SIDT1 to identify potential ectodomain protein interactors in vitro. Counter-selection bio-panning rounds using control tGFP were carried out. High throughput Ion TorrentTMnext-generation sequencing methods were applied.
Results
Sequence deconvolution identified a shortlist of candidate interacting peptides. A novel 12-mer peptide ectodomain ligand was selected for further evaluation. Peptide-BLAST analysis demonstrated sequence homology with human Growth Arrest-Specific 6 (GAS6). Peptide-SIDT1 co-localisation was confirmed using fluorophore conjugated peptide fragments and corroborated using immunopreciptation, following biotinylated peptide exposure, with streptavidin-mediated detection. Disruption of SIDT1-dependent uptake of Cy3-labelled small RNA by this novel peptide was demonstrated in human adenocarcinoma cells.
Conclusions
The challenges of functionally characterising transmembrane proteins remain significant. However, we demonstrate feasibility of a high throughput phage display bio-panning approach to identify lead peptides that have formed the basis of pre-clinical anti-cancer therapeutic development designed to disrupt the pro-desmoplastic pancreatic adenocarcinoma-stromal cell interaction.