Abstract 5272: Multimodality interrogation of systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) identifies axon guidance protein interactions.
Authors: Elhassan Mohamed O. , Christie Jennifer , Molendijk Marlieke , Duxbury Mark 
Published: August 2014

Introduction

SIDT1 is an 11-transmembrane domain channel protein that conveys small double stranded RNA (dsRNA) molecules between cells. SIDT1 is highly expressed in human pancreatic stellate cells and disruption of SIDT1 function attenuates synthesis of components of the desmoplastic response that is typical of pancreatic cancer. We sought to identify the intra- and extracellular protein interaction partners of SIDT1 using an unbiased multimodality screening approach.

Methods

SIDT1 was used as ‘bait’ in a membrane-directed split ubiquitin yeast 2-hybrid (Y2H) screen. Candidate interacting partners identified in multiple biological replicates using a stringent selection approach were validated using immunoprecipiation and the functional effects of disrupting candidate interacting partner protein expression were determined using lentiviral short hairpin RNA-based RNA interference. Gene ontology (GO) term analysis was performed. A high-throughput flow cytometry-based assay was used to quantify the effects of depleting SIDT1 interacting partners on fluorescent dsRNA transfer mediated by SIDT1.

Potential SIDT1 ectodomain interactors were probed using cell surface phage display. SIDT1-tGFP transfected HEK293 cells were probed, with tGFP transfectants serving as a counter-selection control population. Phage display hits were deconvoluted using BLASTP. Fluorescently labeled peptides were synthesized and interactions assessed by epifluorescence microscopy using human pancreatic ductal adenocarcinoma cells. Enzymatic quantification of biotinylated synthetic peptide colocalisation was performed. Immunohistochemical and clincopathological correlation using resected pancreatic adenocarcinoma specimens was undertaken.

Results

The Y2H screen identified a shortlist of consistent SIDT1 interacting partners in which axon guidance proteins were significantly overrepresented, based on GO-term analysis. Disruption of Reticulon 4 expression resulted in impairment of SIDT1 intercellular small dsRNA transfer function.

Phage display identified a SIDT1-binding 12-mer with sequence homology to human Growth arrest-specific 6 (GAS6). 12-mer colocalisation with full length SIDT1 and the SIDT1 ectodomain was confirmed using controlled epifluorescence microscopy. Levels of SIDT1 protein expression correlate with the presence of perineural invasion in human resection specimens (P&lt;0.001).

Discussion

Using a multimodality screening approach we have identified novel SIDT1 binding partners. The role of SIDT1 and its interacting partners in heterotypic cellular interactions, e.g. with peritumoral neurons, warrants further investigation.

Citation Format: Mohamed O. Elhassan, Jennifer Christie, Marlieke Molendijk, Mark Duxbury. Multimodality interrogation of systemic RNA interference-defective-1 transmembrane family member 1 (SIDT1) identifies axon guidance protein interactions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5272. doi:10.1158/1538-7445.AM2013-5272