Sensing Endoplasmic Reticulum Stress Research Articles

FicD sensitizes cellular response to glucose fluctuations in mouse embryonic fibroblasts

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.

IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells.

Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer. The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction. Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription. We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.

Protein disulfide isomerase-9 interacts with the lumenal region of the transmembrane endoplasmic reticulum stress sensor kinase, IRE1, to modulate the unfolded protein response in Arabidopsis.

Environmental stressors disrupt secretory protein folding and proteostasis in the endoplasmic reticulum (ER), leading to ER stress. The unfolded protein response (UPR) senses ER stress and restores proteostasis by increasing the expression of ER-resident protein folding chaperones, such as protein disulfide isomerases (PDIs). In plants, the transmembrane ER stress sensor kinase, IRE1, activates the UPR by unconventionally splicing the mRNA encoding the bZIP60 transcription factor, triggering UPR gene transcription. The induced PDIs catalyze disulfide-based polypeptide folding to restore the folding capacity in the ER; however, the substrates with which PDIs interact are largely unknown. Here, we demonstrate that the Arabidopsis PDI-M subfamily member, PDI9, modulates the UPR through interaction with IRE1. This PDI9-IRE1 interaction was largely dependent on Cys63 in the first dithiol redox active domain of PDI9, and Cys233 and Cys107 in the ER lumenal domain of IRE1A and IRE1B, respectively. In vitro and in vivo, PDI9 coimmunoprecipitated with IRE1A and IRE1B. Moreover, the PDI9:RFP and Green Fluorescence Protein (GFP):IRE1 fusions exhibited strong interactions as measured by fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) when coexpressed in mesophyll protoplasts. The UPR-responsive PDI9 promoter:mCherry reporter and the UPR-dependent splicing of the bZIP60 intron from the mRNA of the 35S::bZIP60-intron:GFP reporter were both significantly induced in the pdi9 mutants, indicating a derepression and hyperactivation of UPR. The inductions of both reporters were substantially attenuated in the ire1a-ire1b mutant. We propose a model in which PDI9 modulates the UPR through two competing activities: secretory protein folding and via interaction with IRE1 to maintain proteostasis in plants.

The role of the ER stress sensor IRE1 in cardiovascular diseases.

Despite enormous advances in the treatment of cardiovascular diseases, including I/R injury and heart failure, heart diseases remain a leading cause of mortality worldwide. Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor endoplasmic reticulum (ER) transmembrane protein that senses ER stress. It manages ER stress induced by the accumulation of unfolded/misfolded proteins via the unfolded protein response (UPR). However, if the stress still persists, the UPR pathways are activated and induce cell death. Emerging evidence shows that, beyond the UPR, IRE1 participates in the progression of cardiovascular diseases by regulating inflammation levels, immunity, and lipid metabolism. Here, we summarize the recent findings and discuss the potential therapeutic effects of IRE1 in the treatment of cardiovascular diseases.

Fundamental and Applicative Aspects of the Unfolded Protein Response in Yeasts.

Upon the dysfunction or functional shortage of the endoplasmic reticulum (ER), namely, ER stress, eukaryotic cells commonly provoke a protective gene expression program called the unfolded protein response (UPR). The molecular mechanism of UPR has been uncovered through frontier genetic studies using Saccharomyces cerevisiae as a model organism. Ire1 is an ER-located transmembrane protein that directly senses ER stress and is activated as an RNase. During ER stress, Ire1 promotes the splicing of HAC1 mRNA, which is then translated into a transcription factor that induces the expression of various genes, including those encoding ER-located molecular chaperones and protein modification enzymes. While this mainstream intracellular UPR signaling pathway was elucidated in the 1990s, new intriguing insights have been gained up to now. For instance, various additional factors allow UPR evocation strictly in response to ER stress. The UPR machineries in other yeasts and fungi, including pathogenic species, are another important research topic. Moreover, industrially beneficial yeast strains carrying an enforced and enlarged ER have been produced through the artificial and constitutive induction of the UPR. In this article, we review canonical and up-to-date insights concerning the yeast UPR, mainly from the viewpoint of the functions and regulation of Ire1 and HAC1.

ER Stress and the Unfolded Protein Response: Homeostatic Regulation Coordinate Plant Survival and Growth

The endoplasmic reticulum (ER), a eukaryotic organelle, is the major site of protein biosynthesis. The disturbance of ER function by biotic or abiotic stress triggers the accumulation of misfolded or unfolded proteins in the ER. The unfolded protein response (UPR) is the best-studied ER stress response. This transcriptional regulatory system senses ER stress, activates downstream genes that function to mitigate stress, and restores homeostasis. In addition to its conventional role in stress responses, recent reports indicate that the UPR is involved in plant growth and development. In this review, we summarize the current knowledge of ER stress sensing and the activation and downstream regulation of the UPR. We also describe how the UPR modulates both plant growth and stress tolerance by maintaining ER homeostasis. Lastly, we propose that the UPR is a major component of the machinery that balances the trade-off between plant growth and survival in a dynamic environment.

Role of systems biology and multi-omics analyses in delineating spatial interconnectivity and temporal dynamicity of ER stress mediated cellular responses.

The endoplasmic reticulum (ER) is a membranous organelle involved in calcium storage, lipid biosynthesis, protein folding and processing. Many patho-physiological conditions and pharmacological agents are known to perturb normal ER function and can lead to ER stress, which severely compromise protein folding mechanism and hence poses high risk of proteotoxicity. Upon sensing ER stress, the different stress signaling pathways interconnect with each other and work together to preserve cellular homeostasis. ER stress response is a part of the integrative stress response (ISR) and might play an important role in the pathogenesis of chronic neurodegenerative diseases, where misfolded protein accumulation and cell death are common. The initiation, manifestation and progression of ER stress mediated unfolded protein response (UPR) is a complex procedure involving multiple proteins, pathways and cellular organelles. To understand the cause and consequences of such complex processes, implementation of an integrative holistic approach is required to identify novel players and regulators of ER stress. As multi-omics data-based systems analyses have shown potential to unravel the underneath molecular mechanism of complex biological systems, it is important to emphasize the utility of this approach in understanding the ER stress biology. In this review we first discuss the ER stress signaling pathways and regulatory players, along with their inter-connectivity. We next highlight the importance of systems and network biology approaches using multi-omics data in understanding ER stress mediated cellular responses. This report would help advance our current understanding of the multivariate spatial interconnectivity and temporal dynamicity of ER stress.

Endoplasmic Reticulum Stress and Unfolded Protein Response Signaling in Plants.

Plants are sensitive to a variety of stresses that cause various diseases throughout their life cycle. However, they have the ability to cope with these stresses using different defense mechanisms. The endoplasmic reticulum (ER) is an important subcellular organelle, primarily recognized as a checkpoint for protein folding. It plays an essential role in ensuring the proper folding and maturation of newly secreted and transmembrane proteins. Different processes are activated when around one-third of newly synthesized proteins enter the ER in the eukaryote cells, such as glycosylation, folding, and/or the assembling of these proteins into protein complexes. However, protein folding in the ER is an error-prone process whereby various stresses easily interfere, leading to the accumulation of unfolded/misfolded proteins and causing ER stress. The unfolded protein response (UPR) is a process that involves sensing ER stress. Many strategies have been developed to reduce ER stress, such as UPR, ER-associated degradation (ERAD), and autophagy. Here, we discuss the ER, ER stress, UPR signaling and various strategies for reducing ER stress in plants. In addition, the UPR signaling in plant development and different stresses have been discussed.

The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER‐resident protein, inositol‐requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1‐mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence‐related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin‐induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1‐mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

AtIRE1C, an unconventional isoform of the UPR master regulator AtIRE1, is functionally associated with AtIRE1B in Arabidopsis gametogenesis.

The unfolded protein response (UPR), a highly conserved set of eukaryotic intracellular signaling cascades, controls the homeostasis of the endoplasmic reticulum (ER) in normal physiological growth and situations causing accumulation of potentially toxic levels of misfolded proteins in the ER, a condition known as ER stress. During evolution, eukaryotic lineages have acquired multiple UPR effectors, which have increased the pliability of cytoprotective responses to physiological and environmental stresses. The ER‐associated protein kinase and ribonuclease IRE1 is a UPR effector that is conserved from yeast to metazoans and plants. IRE1 assumes dispensable roles in growth in yeast but it is essential in mammals and plants. The Arabidopsis genome encodes two isoforms of IRE1, IRE1A and IRE1B, whose protein functional domains are conserved across eukaryotes. Here, we describe the identification of a third Arabidopsis IRE1 isoform, IRE1C. This protein lacks the ER lumenal domain that has been implicated in sensing ER stress in the IRE1 isoforms known to date. Through functional analyses, we demonstrate that IRE1C is not essential in growth and stress responses when deleted from the genome singularly or in combination with an IRE1A knockout allele. However, we found that IRE1C exerts an essential role in gametogenesis when IRE1B is also depleted. Our results identify a novel, plant‐specific IRE1 isoform and highlight that at least the control of gametogenesis in Arabidopsis requires an unexpected functional coordination of IRE1C and IRE1B. More broadly, our findings support the existence of a functional form of IRE1 that is required for development despite the remarkable absence of a protein domain that is critical for the function of other known IRE1 isoforms.

Clustering of IRE1α depends on sensing ER stress but not on its RNase activity.

The sensors of the unfolded protein response react to endoplasmic reticulum (ER) stress by transient activation of their enzymatic activities, which initiate various signaling cascades. In addition, the sensor IRE1α exhibits stress-induced clustering in a transient time frame similar to activation of its endoRNase activity. Previous work had suggested that the clustering response and RNase activity of IRE1α are functionally linked, but here we show that they are independent of each other and have different behaviors and modes of activation. Although both clustering and the RNase activity are responsive to luminal stress conditions and to depletion of the ER chaperone binding protein, RNase-inactive IRE1α still clusters and, conversely, full RNase activity can be accomplished without clustering. The clusters formed by RNase-inactive IRE1α are much larger and persist longer than those induced by ER stress. Clustering requires autophosphorylation, and an IRE1α mutant whose RNase domain is responsive to ligands that bind the kinase domain forms yet a third type of stress-independent cluster, with distinct physical properties and half-lives. These data suggest that IRE1α clustering can follow distinct pathways upon activation of the sensor.-Ricci, D., Marrocco, I., Blumenthal, D., Dibos, M., Eletto, D., Vargas, J., Boyle, S., Iwamoto, Y., Chomistek, S., Paton, J. C., Paton, A. W., Argon, Y. Clustering of IRE1α depends on sensing ER stress but not on its RNase activity.

4-Phenylbutyrate suppresses the unfolded protein response without restoring protein folding in Saccharomyces cerevisiae.

Accumulation of unfolded secretory proteins in the endoplasmic reticulum (ER), namely ER stress, is hazardous to eukaryotic cells and promotes the unfolded protein response (UPR). Ire1 is an ER-located transmembrane protein that senses ER stress and triggers the UPR. According to previous in vitro experiments, 4-phenylbutyrate (4-PBA) works as a chemical molecular chaperone. Since 4-PBA attenuates the UPR in mammalian tissue cultures, this chemical may have clinical potential for restoring ER-stressing conditions. In this study, we investigated 4-PBA's mode of action using the yeast Saccharomyces cerevisiae as a model organism. Although 4-PBA blocked a dithiothreitol (DTT)-induced UPR, it did not appear to restore impairment of ER protein folding that was caused by DTT. Moreover, even under non-stress conditions, 4-PBA attenuated UPR that was induced by an Ire1 mutant that exhibits a substantial activity without sensing ER accumulation of unfolded proteins. We also found that 4-PBA drastically promotes the degradation of Ire1. These observations indicate that at least in the case of yeast cells, 4-PBA suppresses the UPR not through restoration of the ER function to correctly fold proteins. Instead, the accelerated degradation of Ire1 possibly explains the reason why the UPR is attenuated by 4-PBA.

Ratiometric sensing of BiP-client versus BiP levels by the unfolded protein response determines its signaling amplitude.

Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µs), is well-tolerated in HeLa cells. Upon µs expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µs model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.

Abstract 257: Endogenous Activating Transcription Factor 6 Preserves Heart Structure and Function in a Mouse Model of Myocardial Infarction-induced Heart Failure

Rationale: The ER stress response is activated by the accumulation of misfolded, toxic proteins in the endoplasmic reticulum (ER), and upregulates proteins that restore ER protein-folding capacity. The ER-transmembrane protein, activating transcription factor 6 (ATF6) senses ER stress and responds by transcriptionally inducing many of these genes and is thus a key component of the adaptive ER stress response. We previously showed that in the heart, ischemia activates ATF6. Furthermore, transgenic mouse hearts expressing a conditionally activated form of ATF6, and subjected to ex vivo ischemia/reperfusion, exhibited preserved heart function and smaller infarcts. Our lab also showed that by serving as a novel inducer of a global anti-oxidant gene program, endogenous ATF6 limits cardiac damage caused by reactive oxygen species during reperfusion. However, the effect of endogenous ATF6 in the failing heart is not known. Given that acute ischemia caused by occlusion of the coronary arteries is the cause of myocardial infarction (MI), we hypothesized that endogenous ATF6 limits infarct size and preserves heart function during MI. Additionally, since deleterious cardiac remodeling and heart failure can be long-term consequences of MI, we hypothesized that ATF6 can mitigate these effects. Objective/Methods: To examine the role of endogenous ATF6 in heart failure, in vivo, we used a mouse model of MI-induced heart failure in mice with a global deletion of the ATF6 gene (ATF6 KO). Infarct size was measured by TTC staining and heart function was observed via longitudinal echocardiogram. Results: We found that following infarction, ATF6 KO mouse hearts had larger infarcts compared to control. Thus, ischemic cardiac tissue in the peri-infarct region requires ATF6 to limit cardiac myocyte death. Interestingly, ejection fraction following MI decreased more over 13 weeks in ATF6 KO mice relative to control. While control and ATF6 KO mouse hearts hypertrophied to a similar degree, KO mice showed greater cardiac dilation. Conclusions: Together these findings show for the first time that endogenous ATF6 acts to preserve heart structure and function in an MI model of heart failure, suggesting that ATF6 may be a viable therapeutic target for treatment of this disease.

Calnexin Controls the STAT3-Mediated Transcriptional Response to EGF

Calnexin is a well-characterized transmembrane chaperone involved in the folding of newly synthesized glycoproteins in the lumen of the endoplasmic reticulum (ER). Here, we reveal a previously unrecognized function of calnexin in regulating the transcriptional response downstream of epidermal growth factor receptor (EGF), the product of a well-known human oncogene. We find that cell stimulation with EGF leads to the caspase-8-dependent cleavage of the calnexin cytoplasmic domain, preferentially at ER-mitochondria interaction sites. The released fragment translocates into the nucleus, binds to PIAS3--a natural inhibitor of activated STAT3--and, thus, acts as an enhancer of the STAT3-mediated transcriptional response to EGF. Also, we reveal the unsuspected capacity of calnexin to sense ER stress and, in response, prevent the EGF-induced processing of its cytosolic domain. Thus, cells integrate the health status of the ER to determine the amplitude of their response to EGF.

Introduction: the unfolded protein response’s role in disease pathophysiology

Introduction: the unfolded protein response’s role in disease pathophysiology

Luminal Domain of ATF6 Alone Is Sufficient for Sensing Endoplasmic Reticulum Stress and Subsequent Transport to the Golgi Apparatus

The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.

Mechanism of ER Stress-Induced Brain Damage by IP 3 Receptor

Deranged Ca(2+) signaling and an accumulation of aberrant proteins cause endoplasmic reticulum (ER) stress, which is a hallmark of cell death implicated in many neurodegenerative diseases. However, the underlying mechanisms are elusive. Here, we report that dysfunction of an ER-resident Ca(2+) channel, inositol 1,4,5-trisphosphate receptor (IP(3)R), promotes cell death during ER stress. Heterozygous knockout of brain-dominant type1 IP(3)R (IP(3)R1) resulted in neuronal vulnerability to ER stress invivo, and IP(3)R1 knockdown enhanced ER stress-induced apoptosis via mitochondria in cultured cells. The IP(3)R1 tetrameric assembly was positively regulated by the ER chaperone GRP78 in an energy-dependent manner. ER stress induced IP(3)R1 dysfunction through an impaired IP(3)R1-GRP78 interaction, which has also been observed in the brain of Huntington's disease model mice. These results suggest that IP(3)R1 senses ER stress through GRP78 to alter the Ca(2+) signal to promote neuronal cell death implicated in neurodegenerative diseases.

IRE1α controls cyclin A1 expression and promotes cell proliferation through XBP-1

IRE1 is a conserved dual endoribonuclease/protein kinase that is indispensable for directing the endoplasmic reticulum (ER) stress response in yeast, flies, and worms. In mammalian systems, however, the precise biological activities carried out by IRE1alpha are unclear. Here, molecular and chemical genetic approaches were used to control IRE1 activity in a number of prostate cancer cell lines and the resulting impact on gene transcription, cell survival, and proliferation was examined. Modulating IRE1alpha activity had no transcriptional effect on the induction of genes classically associated with the ER stress response (Grp78 and CHOP) or cell survival when confronted with ER stress agents. Rather, IRE1alpha activity was positively correlated to proliferation. Since Xbp-1 mRNA is the sole known substrate for IRE1 endoribonuclease activity, the role of this transcription factor in mediating proliferation was examined. Repressing total Xbp-1 levels by siRNA techniques effectively slowed proliferation. In an effort to identify IRE1/XBP-1 targets responsible for the cell cycle response, genome-wide differential mRNA expression analysis was performed. Consistent with its ability to sense ER stress, IRE1alpha induction led to an enrichment of ER-Golgi, plasma membrane, and secretory gene products. An increase in cyclin A1 expression was the only differentially expressed cell cycle regulatory gene found. Greater cyclin A protein levels were consistently observed in cells with active IRE1alpha and were dependent on XBP-1. We conclude that IRE1alpha activity controls a subset of the ER stress response and mediates proliferation through tight control of Xbp-1 splicing.

Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Abstract 551Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures:No relevant conflicts of interest to declare.