Regulation of mammalian IRE1α: Co-chaperones and their importance

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.Medical Research Counci

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

Funder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: European Molecular Biology Organization; FundRef: http://dx.doi.org/10.13039/100004410Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1’s stress-sensing luminal domain (IRE1LD) that favours the latter’s monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling

Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR

Coupling of endoplasmic reticulum stress to dimerisation‑dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP‑induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling

A J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR

ROS and cGMP signaling modulate persistent escape from hypoxia in Caenorhabditis elegans

The ability to detect and respond to acute oxygen (O2) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O2. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O2 closely resemble those evoked by 21% O2, they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O2 due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O2. Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O2. Ca2+ imaging reveals that a 7% to 1% O2 stimulus evokes a Ca2+ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O2 without affecting responses to 21% O2. Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans

Heightened ISR in <i>EIF2B4</i>^<i>A392D</i> cells.

<p>(A) Flow cytometry analysis of <i>CHOP</i>::<i>GFP</i> and <i>XBP1</i>::<i>Turquoise</i> dual reporter-containing parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the <i>CHOP</i>::<i>GFP</i> ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking <i>EIF2B4</i>^<i>A392D</i> mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting <i>EIF2B4</i> and a wildtype or <i>EIF2B4</i>^<i>A392D</i> mutant repair template marked by a silent <i>Spe</i>I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of <i>CHOP</i>::<i>GFP</i> expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of <i>EIF2B4</i> was amplified by PCR and digested with <i>Spe</i>I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with <i>Spe</i>I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of <i>Spe</i>I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the <i>EIF2B4</i>^<i>A392D</i> mutant repair template in the ISR^High bin.</p

Severe VWM mutant cells are unable to tolerate a second <i>EIF2S1</i>^<i>S51A</i> mutation.

<p>(A) Experimental design for tracking <i>EIF2S1</i>^<i>S51A</i> mutant cells. A fluorescent-tagged sgRNA/Cas9 plasmid targeting <i>EIF2S1</i> was co-transfected alongside wild type (WT) or <i>EIF2S1</i>^<i>S51A</i> (Mut) templates into CHO-S21 dual reporter cells. Following FACS selection for the transfected cells they were treated with 250 nM thapsigargin (Tg) for 24 hours and reporter expression was analyzed. (B) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) CHO-S21 cells from the experiment outlined in “A”. Note the emergence of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated cells in the pool offered an <i>EIF2S1</i>^<i>S51A</i> repair template. (C) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) parental CHO-S21 or indicated VWM mutant cells following targeting of the <i>EIF2S1</i> locus with an <i>EIF2S1</i>^<i>S51A</i> repair template (as described in “A”). Note the lack of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>A392D</i> or <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>R484W</i> double mutant cells (lower right panel). (D) Percentage of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i> mutant cells in the indicated population from experiments as in “C”. Shown are means ± S.D. N = 6 (Parent), 5 (<i>EIF2B4</i>^<i>A392D</i>), and 3 (<i>EIF2B4</i>^<i>R484W</i> and <i>EIF2B4</i>^<i>R468W</i>). *** P<0.001, ** P<0.01, n.s. not significant, One way ANOVA followed by Dunnett’s multiple comparisons test.</p

Stress-resistance of wildtype and VWM cells.

<p>(A) Schema of experiments to compare the effect of thapsigargin in parental CHO-S21 and VWM mutant cells. Cells were treated with thapsigargin (Tg; 250 nM) for the indicated time, washed free of compounds and allowed to recover before assay. W = WST-1 assay, P = Puromycin labeling. (B) Immunoblot of puromycinylated proteins following a brief pulse of puromycin, reporting on levels of translation under the indicated experimental conditions. Shown is a representative experiment reproduced four times. P and E indicate parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells, respectively. (C) Quantification of “B”. Signal intensities of puromycinylated proteins were normalized by eIF2α. Shown are means ± SEM of four independent experiments. (D) Cell viability measured by the WST-1 assay in the experiment described in “A”. Shown are the mean ± SEM of four replicates of a representative experiment repeated three (R484W, R468W) to six (A392D) times.</p