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• W2783306773 abstract "•Proteomic screening identified the ER chaperone Hsp47 as an adjustor of the UPR•Hsp47 is a selective regulator of the ER stress transducer IRE1α•Hsp47 displaces BiP from the IRE1 UPRosome to promote its oligomerization•The regulation of the UPR by Hsp47 is conserved in evolution Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. The endoplasmic reticulum (ER) is the main subcellular compartment involved in protein folding and secretion in addition to operating as a central site for calcium storage and lipid synthesis. Multiple conditions favor the accumulation of misfolded proteins in the ER lumen leading to a cellular state known as ER stress (Walter and Ron, 2011Walter P. Ron D. The unfolded protein response: from stress pathway to homeostatic regulation.Science. 2011; 334: 1081-1086Crossref PubMed Scopus (3881) Google Scholar). Chronic ER stress is emerging as a relevant factor contributing to various pathological conditions, highlighting cancer, diabetes, inflammatory diseases, and neurodegeneration (Oakes and Papa, 2015Oakes S.A. Papa F.R. The role of endoplasmic reticulum stress in human pathology.Annu. Rev. Pathol. 2015; 10: 173-194Crossref PubMed Scopus (673) Google Scholar, Wang and Kaufman, 2016Wang M. Kaufman R.J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease.Nature. 2016; 529: 326-335Crossref PubMed Scopus (879) Google Scholar). ER stress engages the unfolded protein response (UPR), an adaptive mechanism to cope with protein misfolding and restore proteostasis (Walter and Ron, 2011Walter P. Ron D. The unfolded protein response: from stress pathway to homeostatic regulation.Science. 2011; 334: 1081-1086Crossref PubMed Scopus (3881) Google Scholar). Inositol-requiring enzyme 1 alpha (IRE1α) is a type I ER transmembrane protein with a serine and threonine protein kinase and endoribonuclease activity, representing the most conserved ER stress signal transducer. Upon activation, IRE1α catalyzes the unconventional splicing of the mRNA encoding X-box binding protein 1 (XBP1), leading to the expression of a potent and stable transcription factor termed XBP1s (Walter and Ron, 2011Walter P. Ron D. The unfolded protein response: from stress pathway to homeostatic regulation.Science. 2011; 334: 1081-1086Crossref PubMed Scopus (3881) Google Scholar). XBP1s transactivates a cluster of genes involved in different aspects of the secretory pathway, including protein folding, ER-associated degradation (ERAD), protein quality control, and phospholipid synthesis (Acosta-Alvear et al., 2007Acosta-Alvear D. Zhou Y. Blais A. Tsikitis M. Lents N.H. Arias C. Lennon C.J. Kluger Y. Dynlacht B.D. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks.Mol. Cell. 2007; 27: 53-66Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, Lee et al., 2003Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1617) Google Scholar). The RNase activity of IRE1α also degrades selected mRNAs and microRNAs through a process known as regulated IRE1-dependent decay (RIDD), contributing to cell death, inflammation, and other biological processes (Maurel et al., 2014Maurel M. Chevet E. Tavernier J. Gerlo S. Getting RIDD of RNA: IRE1 in cell fate regulation.Trends Biochem. Sci. 2014; 39: 245-254Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). Although irreversible ER stress triggers an apoptosis program involving a network of interconnected pathways (Hetz et al., 2015Hetz C. Chevet E. Oakes S.A. Proteostasis control by the unfolded protein response.Nat. Cell Biol. 2015; 17: 829-838Crossref PubMed Scopus (457) Google Scholar), the mechanisms determining the transition from prosurvival to a terminal UPR phase remain poorly defined. Sustained ER stress attenuates IRE1α signaling, which ablates the adaptive activity of XBP1s, eventually sensitizing cells to cell death (Lin et al., 2007Lin J.H. Li H. Yasumura D. Cohen H.R. Zhang C. Panning B. Shokat K.M. Lavail M.M. Walter P. IRE1 signaling affects cell fate during the unfolded protein response.Science. 2007; 318: 944-949Crossref PubMed Scopus (1054) Google Scholar, Lisbona et al., 2009Lisbona F. Rojas-Rivera D. Thielen P. Zamorano S. Todd D. Martinon F. Glavic A. Kress C. Lin J.H. Walter P. et al.BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1alpha.Mol. Cell. 2009; 33: 679-691Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). However, in certain experimental systems, hyperactivation of IRE1α results in apoptosis, involving changes in its oligomerization state and RIDD (Ghosh et al., 2014Ghosh R. Wang L. Wang E.S. Perera B.G. Igbaria A. Morita S. Prado K. Thamsen M. Caswell D. Macias H. et al.Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.Cell. 2014; 158: 534-548Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, Han et al., 2009Han D. Lerner A.G. Vande Walle L. Upton J.P. Xu W. Hagen A. Backes B.J. Oakes S.A. Papa F.R. IRE1alpha kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates.Cell. 2009; 138: 562-575Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). Therefore, IRE1α acts as a central adjustor of cell fate under ER stress by integrating information about the intensity and duration of the injury. The molecular events underlying ER stress sensing are not completely defined. The binding of the ER chaperone BiP to the luminal domain of IRE1α has been proposed to maintain its monomeric inactive state. Under ER stress, BiP preferentially associates with improperly folded proteins, allowing the dimerization and auto-transphosphorylation of IRE1α in order to activate its RNase domain (Bertolotti et al., 2000Bertolotti A. Zhang Y. Hendershot L.M. Harding H.P. Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.Nat. Cell Biol. 2000; 2: 326-332Crossref PubMed Scopus (2095) Google Scholar, Carrara et al., 2015Carrara M. Prischi F. Nowak P.R. Kopp M.C. Ali M.M. Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling.eLife. 2015; 4https://doi.org/10.7554/eLife.03522Crossref PubMed Scopus (106) Google Scholar). In this model, BiP is the actual sensor because of its ability to directly detect misfolded proteins, whereas IRE1α operates as a signal transducer. In contrast, in yeast cells IRE1p may directly bind to misfolded proteins (Gardner and Walter, 2011Gardner B.M. Walter P. Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response.Science. 2011; 333: 1891-1894Crossref PubMed Scopus (484) Google Scholar) through a structure analogous to the peptide binding pocket of MHC-I (Credle et al., 2005Credle J.J. Finer-Moore J.S. Papa F.R. Stroud R.M. Walter P. On the mechanism of sensing unfolded protein in the endoplasmic reticulum.Proc. Natl. Acad. Sci. USA. 2005; 102: 18773-18784Crossref PubMed Scopus (404) Google Scholar). In vitro evidence suggests that the direct recognition model may not operate in mammals (Kimata and Kohno, 2011Kimata Y. Kohno K. Endoplasmic reticulum stress-sensing mechanisms in yeast and mammalian cells.Curr. Opin. Cell Biol. 2011; 23: 135-142Crossref PubMed Scopus (150) Google Scholar). However, a recent report indicated that unfolded proteins may bind to IRE1α, inducing allosteric changes that trigger its oligomerization (Karagöz et al., 2017Karagöz G.E. Acosta-Alvear D. Nguyen H.T. Lee C.P. Chu F. Walter P. An unfolded protein-induced conformational switch activates mammalian IRE1.eLife. 2017; 6: e30700Crossref PubMed Scopus (119) Google Scholar). Thus, in mammals, ER stress sensing may involve BiP in addition to the binding of misfolded proteins to engage an optimal UPR reaction. Besides, other studies identified the disulfide isomerase PDIA6 as an additional ER foldase that binds to the luminal IRE1α and adjusts its signaling behavior (Eletto et al., 2014Eletto D. Eletto D. Dersh D. Gidalevitz T. Argon Y. Protein disulfide isomerase A6 controls the decay of IRE1α signaling via disulfide-dependent association.Mol. Cell. 2014; 53: 562-576Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Groenendyk et al., 2014Groenendyk J. Peng Z. Dudek E. Fan X. Mizianty M.J. Dufey E. Urra H. Sepulveda D. Rojas-Rivera D. Lim Y. et al.Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis.Sci. Signal. 2014; 7: ra54Crossref PubMed Scopus (59) Google Scholar). Overall, despite the central role of IRE1α as a master regulator of ER proteostasis, the mechanisms connecting fluctuation in protein folding efficiency and the engagement of the UPR remain poorly understood. The amplitude and kinetics of ER stress signaling are regulated by the binding of different cofactors to the main UPR transducers, their post-translational modifications, and proteosomal degradation (Hetz et al., 2015Hetz C. Chevet E. Oakes S.A. Proteostasis control by the unfolded protein response.Nat. Cell Biol. 2015; 17: 829-838Crossref PubMed Scopus (457) Google Scholar). IRE1α can be viewed as a scaffold into which many components assemble to modulate its activity and instigate specific signaling outputs, a platform referred to as the UPRosome (Hetz and Glimcher, 2009Hetz C. Glimcher L.H. Fine-tuning of the unfolded protein response: assembling the IRE1alpha interactome.Mol. Cell. 2009; 35: 551-561Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). For example, several adaptor proteins bind to IRE1α and mediate the crosstalk with other alarm pathways including c-JNK (Urano et al., 2000Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000; 287: 664-666Crossref PubMed Scopus (2311) Google Scholar) and NF-κB (Hu et al., 2006Hu P. Han Z. Couvillon A.D. Kaufman R.J. Exton J.H. Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression.Mol. Cell. Biol. 2006; 26: 3071-3084Crossref PubMed Scopus (588) Google Scholar), among others (Hetz, 2012Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond.Nat. Rev. Mol. Cell Biol. 2012; 13: 89-102Crossref PubMed Scopus (2472) Google Scholar). The levels of IRE1α phosphorylation are modulated by several phosphatases and kinases (Lu et al., 2013Lu G. Ota A. Ren S. Franklin S. Rau C.D. Ping P. Lane T.F. Zhou Z.H. Reue K. Lusis A.J. et al.PPM1l encodes an inositol requiring-protein 1 (IRE1) specific phosphatase that regulates the functional outcome of the ER stress response.Mol. Metab. 2013; 2: 405-416Crossref PubMed Scopus (33) Google Scholar, Mao et al., 2011Mao T. Shao M. Qiu Y. Huang J. Zhang Y. Song B. Wang Q. Jiang L. Liu Y. Han J.D. et al.PKA phosphorylation couples hepatic inositol-requiring enzyme 1alpha to glucagon signaling in glucose metabolism.Proc. Natl. Acad. Sci. USA. 2011; 108: 15852-15857Crossref PubMed Scopus (65) Google Scholar, Qiu et al., 2010Qiu Y. Mao T. Zhang Y. Shao M. You J. Ding Q. Chen Y. Wu D. Xie D. Lin X. et al.A crucial role for RACK1 in the regulation of glucose-stimulated IRE1alpha activation in pancreatic beta cells.Sci. Signal. 2010; 3: ra7Crossref PubMed Scopus (122) Google Scholar), whereas IRE1α oligomerization into large clusters is assisted by non-muscle myosin II (NMII) and the actin cytoskeleton (He et al., 2012He Y. Beatty A. Han X. Ji Y. Ma X. Adelstein R.S. Yates 3rd, J.R. Kemphues K. Qi L. Nonmuscle myosin IIB links cytoskeleton to IRE1α signaling during ER stress.Dev. Cell. 2012; 23: 1141-1152Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). IRE1α signaling is also controlled by the targeting of the XBP1 mRNA to the ER membrane followed by the ligation of Xbp1 mRNA by the RtcB ligase (reviewed in Hetz et al., 2015Hetz C. Chevet E. Oakes S.A. Proteostasis control by the unfolded protein response.Nat. Cell Biol. 2015; 17: 829-838Crossref PubMed Scopus (457) Google Scholar). Under prolonged ER stress, IRE1α is turned off, involving the physical interaction with several factors previously linked to the regulation of apoptosis (Lisbona et al., 2009Lisbona F. Rojas-Rivera D. Thielen P. Zamorano S. Todd D. Martinon F. Glavic A. Kress C. Lin J.H. Walter P. et al.BAX inhibitor-1 is a negative regulator of the ER stress sensor IRE1alpha.Mol. Cell. 2009; 33: 679-691Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, Rodriguez et al., 2012Rodriguez D.A. Zamorano S. Lisbona F. Rojas-Rivera D. Urra H. Cubillos-Ruiz J.R. Armisen R. Henriquez D.R. Cheng E.H. Letek M. et al.BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α.EMBO J. 2012; 31: 2322-2335Crossref PubMed Scopus (92) Google Scholar). Finally, the selectivity of the IRE1α RNase activity for RIDD substrates and the Xbp1 mRNA may depend on its oligomerization status (Ghosh et al., 2014Ghosh R. Wang L. Wang E.S. Perera B.G. Igbaria A. Morita S. Prado K. Thamsen M. Caswell D. Macias H. et al.Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.Cell. 2014; 158: 534-548Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, Tam et al., 2014Tam A.B. Koong A.C. Niwa M. Ire1 has distinct catalytic mechanisms for XBP1/HAC1 splicing and RIDD.Cell Rep. 2014; 9: 850-858Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Thus, IRE1α signaling represents a highly regulated process involving multiple checkpoints, setting up the threshold to induce an adaptive UPR or transit into a terminal proapoptotic program when ER proteostasis is irreversibly damaged. The nature of the IRE1α interactome is not well defined, but it is essential to understanding the significance of the UPR to cell physiology. To uncover regulatory elements of the UPR, we identified IRE1α binding partners using proteomics followed by a functional genomics validation. This study allowed the identification of several interactors, highlighting heat shock protein 47 (Hsp47) as a major regulator of the UPR. Hsp47 is an ER chaperone specialized in the maturation and trafficking of collagen, the most abundant ER client in mammals (Nagata, 2003Nagata K. HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development.Semin. Cell Dev. Biol. 2003; 14: 275-282Crossref PubMed Scopus (105) Google Scholar). We provide evidence indicating that Hsp47 expression enhances IRE1α activation and potentiates its downstream signaling, resulting in efficient ER stress mitigation. Using complementary approaches, we demonstrate that Hsp47 binds to the ER luminal domain of IRE1α and reduces the association of BiP to the complex, a phenomenon that may enhance its oligomerization for optimal signaling. Hsp47 deficiency in flies and mice attenuates IRE1α signaling and sensitizes cells to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. Overall, our results unravel a previously unanticipated biological function of Hsp47 as an adjustor of the UPR, setting up the threshold for IRE1α activation. To identify potential new components of the UPRosome, we immunoprecipitated HA-tagged IRE1α expressed in IRE1 (Ern1) null mouse embryonic fibroblasts (MEFs). This reconstitution strategy led to physiological levels of IRE1α expression and fully restored Xbp1 mRNA splicing and RIDD activity (Figures S1A–S1D). IRE1α interacting complexes were isolated at basal conditions or in cells undergoing ER stress induced by the treatment with the N-glycosylation inhibitor tunicamycin (Tm). Immunoprecipitates (IPs) were analyzed using two-dimensional liquid chromatography along with tandem mass spectrometry followed by bioinformatic analysis. Comparison of three independent experiments led us to identify ten candidate proteins that differentially interact with IRE1α, in addition to the known regulator BiP (Table S1). These proteins are involved in different cellular functions including mitochondrial biology, collagen biosynthesis, cytoskeleton, and cell signaling, and display different subcellular distributions (Table S1). The majority of the candidates identified in IRE1α-containing complexes were present in both basal and ER stress conditions, whereas only three proteins were exclusively detected at resting conditions and two upon 6 hr of Tm treatment (Figure 1A). To assess the functional contribution of the putative IRE1α interactors, we knocked down their expression by the stable delivery of shRNAs (Figure S1E). To avoid saturation of the Xbp1 mRNA splicing assay, we used low doses of Tm. Analysis of Xbp1 mRNA splicing by RT-PCR in shRNA cells treated with Tm revealed that a subgroup of genes modulated IRE1α signaling to different extents (Figure 1B). To monitor the adaptive capacity of the cell to ER stress, we measured cell viability after Tm treatment in dose-response curves (Figure 1C). Of note, the Tm doses used to monitor UPR signaling did not result in significant apoptosis in control cells. However, at higher concentrations it was possible to detect an enhancement of ER stress-induced cell death when Atp5h, Hsp47 or Tubα1a were knocked down (Figure 1C). To define possible genes that enhance IRE1α signaling when they are downregulated, we performed kinetic analysis to measure Xbp1 mRNA splicing, allowing the identification of Tubα1a as an additional regulator of the pathway (Figure 1D). The global analysis of these experiments led us to select four candidates for further functional validation, three putative activators, the phosphatase Ppa2, the ER chaperone Hsp47 and the mitochondrial ATPase Atp5h, in addition to one putative inhibitor of the pathway, Tubα1a. Secondary validation was performed with newly generated knockdown cells to monitor Xbp1 mRNA splicing, which recapitulated the phenotypes identified in the primary screening (Figure 1E). To determine the specificity over IRE1α regulation, we measured the levels of two UPR-target genes controlled by the PERK and ATF6 branches. From this analysis, targeting Pp2a, Atp5h or Hsp47 did not affect the upregulation of Bip and Chop. In contrast, knockdown of Tubα1a increased the expression of these genes suggesting global alterations to ER physiology, resulting on ER stress (Figure 1F). To further validate the four selected IRE1α interacting partners, we performed gain-of-function experiments and assessed UPR signaling using different approaches. We stably expressed V5-tagged versions of human HSP47, TUBα1a, PPA2 and ATP5h in MEFs using retroviral transduction (Figure S2A). Next, we treated these cells with Tm and monitored Xbp1 mRNA splicing in time-course experiments (Figure 2A). In agreement with our previous results, overexpression of PPA2, ATP5H and HSP47 enhanced the levels of Xbp1 mRNA splicing (Figure 2A, right panel). Unexpectedly, TUBα1a overexpression also resulted in sustained Xbp1 mRNA splicing after Tm treatment (Figure 2A). We confirmed these results using an alternative Xbp1 mRNA splicing assay based on PstI digestion of RT-PCR products (Figure S2B). To determine the activity of XBP1s in cells overexpressing IRE1α regulators, we measured the mRNA levels of Edem1 and Sec61, two classical XBP1s-target genes (Lee et al., 2003Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1617) Google Scholar). Real time PCR analysis revealed that the overexpression of the four candidates significantly enhanced the induction of Edem1 and Sec61 mRNA under ER stress (Figure 2B), whereas the upregulation of Bip and Chop was not altered in these cells (Figure 2C). We also measured RIDD activity as an additional output of the pathway. Surprisingly, analysis of two canonical RIDD substrates, Sparc and Blocs1 (Hollien et al., 2009Hollien J. Lin J.H. Li H. Stevens N. Walter P. Weissman J.S. Regulated Ire1-dependent decay of messenger RNAs in mammalian cells.J. Cell Biol. 2009; 186: 323-331Crossref PubMed Scopus (698) Google Scholar), indicated that the overexpression of HSP47-V5 or ATP5-V5 enhanced the decay of these two mRNAs, whereas the expression of PP2A-V5 or TUBα1a-V5 did not affect RIDD levels under the same conditions (Figure 2D) despite augmented Xbp1 mRNA splicing. Based on the global analysis of our results, we decided to focus our further studies on assessing the contribution of Hsp47 to the regulation of IRE1α because (i) it is an ER-located chaperone, (ii) its modulation generated a strongest effect on cell survival, and (iii) was the greater modifier of Xbp1 mRNA splicing and RIDD in our analysis. To further explore the effects of Hsp47 on IRE1α signaling we complemented our previous analysis with a promoter reporter assay using luciferase controlled by a UPR element (UPRE) sequence (Lee et al., 2003Lee A.H. Iwakoshi N.N. Glimcher L.H. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response.Mol. Cell. Biol. 2003; 23: 7448-7459Crossref PubMed Scopus (1617) Google Scholar). Expression of HSP47-V5 in MEFs led to an enhancement in the activity of the UPRE reporter after Tm treatment (Figure 3A). Similar results were observed when cells were stimulated with other ER stressors, including thapsigargin and brefeldin A (Figure 3B). This assay was highly dependent on IRE1α expression since no signal was observed when experiments were performed in IRE1α null MEFs (Figure S2C). We confirmed these results using real time PCR to measure Xbp1s levels in MEFs with manipulated levels of Hsp47, observing that its expression enhanced IRE1α signaling (Figures 3C and S2D). IRE1α activation involves its dimerization and auto-transphosphorylation, leading to a conformational change in the cytosolic region that engages the RNase domain. We used a Phostag assay to assess the phosphorylation status of IRE1α in MEFs. Treatment of Hsp47 silenced cells with Tm revealed a delay in the appearance of the electrophoretic shift associated with its activation (Figure 3D, upper panel). In agreement with these results, overexpression of HSP47-V5 accelerated the activation process, reflected by an almost complete phosphorylation of IRE1α that was sustained over time (Figure 3D, bottom panel). Importantly, total IRE1α levels were not altered when the expression of Hsp47 was modulated in MEFs (Figures S3A and S3B). IRE1α phosphorylation leads to its oligomerization into large clusters. We took advantage of a stable TREX293 cell line overexpressing mammalian IRE1α tagged with GFP controlled by doxycycline to visualize IRE1α oligomerization (Li et al., 2010Li H. Korennykh A.V. Behrman S.L. Walter P. Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering.Proc. Natl. Acad. Sci. USA. 2010; 107: 16113-16118Crossref PubMed Scopus (247) Google Scholar). We knocked down HSP47 in TREX293 cells using siRNA and monitored the generation of GFP-positive foci. Downregulation of HSP47 reduced the number of IRE1α clusters per cell, leading to a faster attenuation (Figure 3E). In agreement with this, increased number of IRE1α-GFP puncta was observed in HSP47-V5 overexpressing cells after Tm treatment (Figure 3F, see controls in Figures S4A and S4B). We then further explored the specificity of Hsp47 over the regulation of IRE1α signaling. The overexpression or the knockdown of Hsp47 in MEFs did not alter the upregulation of ATF4 and CHOP under ER stress, as determined using western blot analysis (Figure 3G). Thus, Hsp47 expression specifically enhances molecular events related to IRE1α activation. Our initial IP-mass spectrometry analysis suggested that Hsp47 associates with IRE1α-containing protein complexes. To validate this interaction, we first determined the subcellular distribution of IRE1α and Hsp47 using immunofluorescence and then used a sensitive method based on a confined displacement analysis algorithm to calculate colocalization coefficients (Ramírez et al., 2010Ramírez O. García A. Rojas R. Couve A. Härtel S. Confined displacement algorithm determines true and random colocalization in fluorescence microscopy.J. Microsc. 2010; 239: 173-183Crossref PubMed Scopus (45) Google Scholar). Quantification of Manders coefficients M1 and M2 indicated an average 0.32 and 0.35 index of colocalization, respectively, under basal conditions (Figure 4A, right). Stimulation of ER stress for 6 hr slightly reduced the colocalization index, consistent with our proteomic data (Figure 4A, right). We moved forward to validate this interaction in living cells using an in situ proximal ligation assay (Duolink), a method that is predicted to have sensitivity for protein complexes in the range of 40 nm. As control, we knocked down Hsp47 with an shRNA construct (Figure S4C). This experiment corroborated a close proximity between Hsp47 and IRE1α (Figure 4B). We then performed co-IP assays by analyzing the interaction of endogenous Hsp47 with IRE1α-HA expressed at physiological levels in an Ern1-null background (Figure 4C and Figure S1). We also detected a reduced interaction in cells treated with Tm for 4 hr (Figure 4C). Similarly, induction of ER stress with brefeldin A, DTT, and thapsigargin resulted in the release of Hsp47 from the IRE1α complex after prolonged treatment (Figure 4D). Since Hsp47 expression enhances IRE1α signaling and the activation of the pathway occurs very fast after stimulation of ER stress, we then monitored the interaction of Hsp47 with IRE1α during shorter time points. Treatment of cells with Tm revealed a fast and progressive release of BiP from the complex, starting at 1 hr after stimulation (Figure 4E). Interestingly, the binding of endogenous Hsp47 to IRE1α-HA was enhanced at 1 hr after ER stress induction, correlating with the release of BiP from IRE1α (Figure 4E). We also confirmed the formation of a protein complex between the endogenous proteins that was enhanced by ER stress (Figure 4F). Based on the subcellular distribution of Hsp47, we expressed an IRE1α-deletion mutant containing only the N-terminal luminal domain (NLD). Again, our co-IP experiments supported the establishment of a complex between Hsp47 and IRE1α, restricting this association to the ER lumen (Figure 4G). In addition, we also detected the interaction by pull-down assays in COS-1 cells transfected with vectors encoding the NLD of IRE1α fused with a polyhistidine sequence (His-IRE1α-NLD) (Figure 4H). To determine if Hsp47 directly binds to IRE1α, we used two sensitive in vitro assays. MicroScale Thermophoresis (MST) was performed using recombinant HSP47 and IRE1α-NLD produced in E. coli and COS-1 cells, respectively. We incubated RED-labeled IRE1α-NLD protein with increasing concentrations of purified HSP47 (Figure 4I). Calculation of the dissociation constant (KD) revealed a high affinity between HSP47 and IRE1α-NLD in the nanomolar range (Table 1). These data were further confirmed using surface plasmon resonance to determine that the biophysical parameters of the interaction were in the nanomolar range (Table 1). We then compared the strength of the interaction between HSP47 and IRE1α-NLD with its affinity for BiP or PDIA6. In this experimental setting, recombinant BiP associated with the luminal domain of IRE1α with a KD value of 39.7 nM, in the same range as" @default.
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• W2783306773 date "2018-01-01" @default.
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• W2783306773 title "Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α" @default.
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