Chemical genetic dissection of efferent IRE1α signalling

Abstract

The Endoplasmic Reticulum is the cellular organelle primarily responsible for producing proteins on the secretory pathway, a pathway important in the production of biopharmaceuticals. One of the requirements for the successful production of a functional protein is correct folding of the polypeptide sequence. During conditions such as viral infection, mutant protein expression and cell differentiation the endoplasmic reticulum is placed under conditions of stress. IRE1 is a protein kinase and endoribonuclease, which along with PERK and ATF6, forms part of the Unfolded Protein Response, the system by which the cell deals with the stress caused by a high protein load. IRE1 is capable of increasing the protein folding capacity of the ER, by upregulating chaperone proteins and reducing the load by attenuating translation, (protective response). This action is mediated by splicing of the mRNA coding for the bZIP transcription factor XBP-1. IRE1 is also capable of causing apoptotic responses via TRAF2 (cell injuring response) resulting in the activation of JNK and NFκB. In this study, using site directed mutagenesis a panel of IRE1 mutants was produced and screened for alterations to the protective and cell injuring responses. Of these the D711A mutant was shown in mouse embryonic fibroblasts to retain endoribonuclease activity, and to display an attenuated cell injuring response. When this mutant was applied to an industrial CHO cell line it appeared to exhibit an increase in biopharmaceutical productivity over the wild type IRE1, indicating its potential for use in the biopharmaceutical cell lines.