Dissection of Prosurvival and Apoptotic Signalling By Human IRE1α

Abstract

The endoplasmic reticulum (ER) is a complex organelle whose primary function is concerned with protein folding and modification, interconnected network of membraneous tubules. Its primary function is concerned with appropriately folding and modifying proteins so as they can progress along the secretory pathway. However, occasionally , if a mutated gene is expressed or if there is a sudden increase in the influx of nascent polypeptide chains, then unfolded proteins accumulate in the ER, causing ER stress. In these cases, t. The unfolded response (UPR) is consequentially activated to try and in an attempt to restore ER homeostasis. IRE1α is the most ancient There are three mammalian UPR signal transducers. IRE1α is the most ancient of these in the mammalian UPR and. It has two functional domains: an RNase and a kinase., both of which have been implicated in several diseases. The RNase domain is often regarded as promoting cell survival via non-conventional splicing of XBP-1 mRNA to generate the XBP-1(s) transcription factor. An effective transcription factor is generated as a result, which is able to upregulate foldases and enhance degradation of proteins which cannot be refolded. This domain has also been implicated in the RIDD pathway where ER-localised mRNAs are degraded. The kinase function domain is generally thought of as being proapoptotic due to its ability tovia TRAF2 interaction and the subsequent activate the JNK MAP kinase cascade.signalling cascade.
However, recent work has highlighted a potential role for IRE1α kinase activity in cell survival. This project focussed on segregating cell fate decisions according to kinase and RNase functions. This was done by comparing MEFs expressing human WT-IRE1α to MEFs expressing a kinase-deficient IRE1α mutant, or a kinase- and RNase-dead form of IRE1α. Western blotting, qPCR analysis and electron microscopy were principally used to identify biochemical, transcriptional and physical differences between these cells. Western blot analysis highlighted a correlation between the loss of IRE1α kinase activity and increased expression of the proapoptotic marker, CHOP, during ER stress. Further strengthening this observation, an increase in mRNA levels of TRB3, which is a downstream target of CHOP was identified by qPCR. The latter technique also suggested that ERAD, the process by which misfolded ER lumenal proteins are degraded, was also compromised. Finally, electron micrographs clearly showed loss of the ability in cells expressing either IRE1α mutants to structurally organise the proliferating ER during stress. To conclude, the results strongly argue that lack of the kinase domain negatively influencesd ER physiology and gene expression when the UPR iswas active. Therefore, this study has broughtbrings to attention the importance of the IRE1α kinase activity as well as its RNase, for cytoprotection which, .in turn, has major implications on the role of the UPR in several different diseases.