Abstract
BackgroundIRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them.MethodsA multiplexed isoform-specific XBP1 array utilising Biochip array technology (BAT™) was assessed for specificity and suitability when using cell protein lysates. The array was applied to RIPA protein lysates from several relevant pre-clinical models with an aim to quantify XBP1 isoforms in comparison with RT-PCR or immunoblot reference methods.ResultsA novel reliable, specific and sensitive XBP1 biochip was successfully utilised in pre-clinical research. Application of this biochip to detect XBP1 splicing at the protein level in relevant breast cancer models, under basal conditions as well as pharmacological inhibition and paclitaxel induction, confirmed the findings of previous studies. The biochip was also applied to non-adherent cells and used to quantify changes in the XBP1 isoforms upon activation of the NLRP3 inflammasome.ConclusionsThe XBP1 biochip enables isoform specific quantification of protein level changes upon activation and inhibition of IRE1α RNase activity, using a routine clinical methodology. As such it provides a research tool and potential clinical tool with a quantified, simultaneous, rapid output that is not available from any other published method.
Highlights
The unfolded protein response (UPR) is a set of eukaryotic pathways activated by endoplasmic reticulum (ER) stress, which is defined by an accumulation of unfolded or misfolded proteins in the ER [1]
Biochip Array Technology (BAT) multiplexed XBP1s and XBP1u assays are specific to their respective antigen and produce antigen dependent signal To assess the XBP1 biochip’s suitability and performance as a sandwich immunoassay in a multiplexed format the specificity and sensitivity of each assay was assessed
BAT multiplexing of the two XBP1 isoforms requires the spotting of the two identified isoform specific capture antibodies into distinct Discrete Test Region (DTR) on the ceramic biochip surface
Summary
The unfolded protein response (UPR) is a set of eukaryotic pathways activated by endoplasmic reticulum (ER) stress, which is defined by an accumulation of unfolded or misfolded proteins in the ER [1]. Translation of XBP1s mRNA (the result of unconventional IRE1α mediated XBP1 splicing) produces a potent transcription factor of 261 amino acids and ~ 55 kDa (Additional file 1: Figure S1A). IRE1α-mediated unconventional splicing of XBP1 is emerging as a biomarker in several disease states and is indicative of activation of the unfolded protein response sensor IRE1. Splicing of XBP1 mRNA results in the translation of two distinct XBP1 protein isoforms (XBP1s and XBP1u) which, due to post-translational regulation, do not correlate with mRNA levels. As both XBP1 isoforms are implicated in pathogenic or disease progression mechanisms there is a need for a reliable, clinically applicable method to detect them
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