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IRE1 is a transmembrane signalling protein that activates the unfolded protein response under endoplasmic reticulum stress. IRE1 is endowed with kinase and endoribonuclease activities. The ribonuclease activity of IRE1 can switch substrate specificities to carry out atypical splicing of Xbp1 mRNA or trigger the degradation of specific mRNAs. The mechanisms regulating the distinct ribonuclease activities of IRE1 have yet to be fully understood. Here, we report the Bcl-2 family protein Bid as a novel recruit of the IRE1 complex, which directly interacts with the cytoplasmic domain of IRE1. Bid binding to IRE1 leads to a decrease in IRE1 phosphorylation in a way that it can only perform Xbp1 splicing while mRNA degradation activity is repressed. The RNase outputs of IRE1 have been found to regulate the homeostatic-apoptotic switch. This study, thus, provides insight into IRE1-mediated cell survival.

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BackgroundAquilegia nivalis Flax Jackson, also called Aquilegia vulgaris sub sp. nivalis (Bak.) Brühl or columbine, locally known as “Zoe-neel”, is a wild edible plant traditionally used as an anti-inflammatory medicine by the local nomadic tribes inhabiting the Himalayas of Jammu and Kashmir. The plant has been used as herbal medicine since middle ages in treating ailments that include chronic rhinitis and various infectious diseases. The extracts from the plant possess antioxidant properties and have been reported to be hepatoprotective in rats. Our preliminary studies, however, pointed to hitherto unexplored anti-apoptotic potential of the plant which lead us to carry the in-depth study using breast cancer cell lines to validate its anti-cancerous properties and explore the affected pathways.MethodsMTT assay was used to draw the dose response curve and evaluate the effect of increasing concentrations of the extract on cell lines to determine the appropriate dosage to be used for further experimentation. DNA fragmentation analysis was followed through gel electrophoresis and DAPI staining was pursued by phase contrast microscopy to study apoptosis. Quantitative PCR was used to study the expression of UPR signaling and RIDD markers at the level of mRNA. Western blot analysis was used in studying the expression of the various markers of the signaling pathways. The cell cycle analysis was carried out using flow cytometry.ResultsMTT assay revealed that the methanolic extract of the plant (ANME) was selectively cytotoxic to various cancer cell lines as revealed by lower IC50 values relative to normal cell lines. The results of cell cycle analysis were similar as ANME caused Sub G1 arrest of the cell cycle. DNA fragmentation analysis, DAPI staining and western blot analysis for PARP and caspases revealed that the extract selectively induced apoptosis in cancerous cell lines. UPR markers p-Ire1α and Xbp1 splicing were consistently alleviated in a dose dependent manner, the rate of phosphorylation of eIF2a and ATF4 also decreased with increasing concentration of ANME. The RT PCR results of the RIDD marker, Blos1S1 revealed a similar dose dependent association. The methanolic extract was especially chosen for it could be easily internalized by the cells and any resultant potential bioactive compounds could gain access to the cells because of their hydrophobic nature.ConclusionOur results suggest that ANME causes deactivation of UPR signaling pathway facilitating apoptosis selectively in cancerous cells, paving the way forward for a novel approach in cancer therapeutics.

ER is facilitated with a dynamic cellular pathway namely Unfolded Protein Response (UPR): an adaptive signalling mechanism that maintains proteostasis in response to ER stress. IRE1 is one of the three transmembrane sensors of UPR with dual protein kinase and ribonuclease activities. IRE1 acts as a central molecule of UPR, which associates with a number of proteins that either regulate its activity or connect it to other pathways. Here, we report sMEK1 and Akt as novel interacting partners of IRE1 which associate to orchestrate the IRE1 and Akt signalling networks. Our study revealed that ER stress negatively regulates Akt through IRE1 protein. We found that IRE1/sMEK1/Akt form a ternary complex, which results in the dephosphorylation of Akt by protein phosphotase sMEK1 in presence of activated IRE1. Together, this study highlights the UPR/Akt link by delineating the molecular mechanism along with giving insights into the overall impact of this interaction.