Abstract
Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR). This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6). Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity. The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR. Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.
Highlights
The endoplasmic reticulum (ER) is the site of protein folding and post-translational modification of secreted and transmembrane proteins[1]
Spliced transcripts (XBP1s) are translated into the protein XBP1S, a transcription factor that upregulates the expression of proteins involved in ER protein folding, ER associated degradation (ERAD) and lipid biogenesis as part of a concerted effort to increase the capacity of the ER to cope with unfolded proteins
To study the activation of IRE1α RNase activity, we assessed the cleavage of XBP1u mRNA before and after induction of human X-box binding protein 1 (XBP1) expression, using an RT-PCR assay[21]
Summary
The endoplasmic reticulum (ER) is the site of protein folding and post-translational modification of secreted and transmembrane proteins[1]. The mammalian UPR is formed from three distinct but overlapping signalling branches, each governed by an initial effector protein localised to the ER membrane These proteins are activating transcription factor 6 (ATF6), protein kinase-like ER kinase (PERK) and inositol requiring enzyme α (IRE1α), and are activated in the presence of a build-up of incorrectly folded proteins[5]. The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR) This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1
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