Unfolded Protein Response Signaling Branches Research Articles

The unfolded protein response to PI*Z alpha-1 antitrypsin in human hepatocellular and murine models.

Alpha‐1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full‐length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R‐like ER kinase and inositol‐requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro‐apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.

The PERK/ATF4/CHOP signaling branch of the unfolded protein response mediates cisplatin-induced ototoxicity in hair cells

Cisplatin is a widely used chemotherapeutic agent. However, its clinical application remains limited due to the high incidence of severe ototoxicity. It has been reported that the unfolded protein response (UPR) is involved in cisplatin-induced ototoxicity. However, the specific mechanism underlying its effect remains unclear. Therefore, the present study aimed to explore the sequential changes in the key UPR signaling branch and its potential pro-apoptotic role in cisplatin-induced ototoxicity. The hair cell-like OC-1 cells were treated with cisplatin for different periods and then the expression levels of the UPR- and apoptosis-related proteins were determined. The results showed that the apoptotic rate of cells was gradually increased with prolonged cisplatin treatment. Furthermore, the sequential changes in three UPR signaling branches were evaluated. The expression levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were gradually increased with up to 12 h of cisplatin treatment. The aforementioned expression profile was consistent with that observed for the apoptosis-related proteins. Subsequently, the proportion of apoptotic cells was notably decreased in CHOP-silenced hair cell-like OC-1 cells following treatment with cisplatin. Moreover, we found significant hair cells loss and a higher level of CHOP in cisplatin-treated cochlear explants in a time-dependent manner. Overall, the present study demonstrated that the protein kinase RNA‑like endoplasmic reticulum kinase (PERK)/ATF4/CHOP signaling branch could play an important role in cisplatin-induced cell apoptosis. Furthermore, the current study suggested that CHOP may be considered as a promising therapeutic target for cisplatin-induced ototoxicity.

Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy.

Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

The PERK-Dependent Molecular Mechanisms as a Novel Therapeutic Target for Neurodegenerative Diseases.

Higher prevalence of neurodegenerative diseases is strictly connected with progressive aging of the world population. Interestingly, a broad range of age-related, neurodegenerative diseases is characterized by a common pathological mechanism—accumulation of misfolded and unfolded proteins within the cells. Under certain circumstances, such protein aggregates may evoke endoplasmic reticulum (ER) stress conditions and subsequent activation of the unfolded protein response (UPR) signaling pathways via the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent manner. Under mild to moderate ER stress, UPR has a pro-adaptive role. However, severe or long-termed ER stress conditions directly evoke shift of the UPR toward its pro-apoptotic branch, which is considered to be a possible cause of neurodegeneration. To this day, there is no effective cure for Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), or prion disease. Currently available treatment approaches for these diseases are only symptomatic and cannot affect the disease progression. Treatment strategies, currently under detailed research, include inhibition of the PERK-dependent UPR signaling branches. The newest data have reported that the use of small-molecule inhibitors of the PERK-mediated signaling branches may contribute to the development of a novel, ground-breaking therapeutic approach for neurodegeneration. In this review, we critically describe all the aspects associated with such targeted therapy against neurodegenerative proteopathies.

Glucose-regulated protein 78 is essential for cardiac myocyte survival

Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.

N-RasG12D-Mediated Dysregulation of IRE1alpha-Xbp1s Signaling Promotes Pre-Leukemic Hematopoietic Stem Cell Expansion

Protein homeostasis is tightly regulated to ensure cell integrity. Upon stimulation by nutrition deprivation, increased reactive oxygen species (ROS), or high protein synthesis demand, increased ER stress triggers unfolded protein response (UPR) pathways to induce either adaptation, including reduced protein synthesis and increased expression of chaperones to promote protein refolding, or apoptosis after severe or prolonged damage. The fate of the cell under ER stress therefore depends on the balance between adaptive and pro-apoptotic signalings. Within the hematopoietic populations, HSCs have the lowest protein synthesis rate. In addition, HSCs are more sensitive to ER-stress-induced apoptosis compared to progenitors. Recent studies have shown that constitutive activation of ER-stress leads to depletion of HSCs. Promotion of adaptation to ER-stress by overexpressing co-chaperones such as ERDJ4, increase the HSC repopulation capacity. Taken together, these studies suggest that protein homeostasis plays an important role in hematopoietic stem cell (HSC) regulation.Genetic lesions that activate oncogenes or inactivate tumor suppressor genes transform HSCs and progenitors first to pre-leukemic stem cells (pre-LSCs) which often exhibit increased proliferation and therefore increased protein synthesis demand. Employing an Mx-Cre+; LSL-NrasG12D/+ conditional knock-in mouse model, our previous studies have shown that N-RasG12Dincreases both self-renewal and proliferation of HSCs. To determine whether UPR pathways contribute to N-RasG12D-mediated pre-leukemic stem cell expansion, we first assessed the response of Nras mutant HSCs to ER stress either by exposure to tunicamycin(TM), which blocks synthesis of N-linked glycoproteins, or thapsigargin(TG), which disrupts Ca2+ homeostasis in the ER. Consistent with recent reports, both TM and TG treatment induced apoptosis in murine SLAM HSCs (CD150+CD48-Lin-sca1+ckit+). Nras mutant HSCs, however, exhibited a significant reduction of apoptosis as measured by Annexin V staining and caspase activation, and their colonogenic potential was unaffected. These effects were mitigated by inhibition of MEK/ERK signaling but not STAT5, and were specific to hyperactive Nras mutant HSCs, since FLT3-ITD leukemic mutation had no effect on HSC survival under ER stress.To identify the mechanism underlying the N-RasG12D-mediated HSC protection, we examined the major signaling branches of UPR: IRE1alpha/XBP1s, ATF6 and PERK/eIF2alpha. We found that N-RasG12D activated the adaptive branch of the UPR, Inositol-requiring enzyme 1 (IRE-1)/XBP1s signaling, as increased level of phospho-IRE1alpha and XBP1s mRNA were observed. Furthermore, inhibition of this pathway by either a small molecular inhibitor specific to the RNase activity of IRE1alpha, or overexpression of a dominant negative mutant of XBP1s, reduced the adaptation to ER-stress and competitive advantage in transplant recipients of Nras mutantHSCs. Moreover, heterozygous knockout of IRE1alpha blocked N-RasG12D-mediated splenomegaly and myeloid expansion in Mx1-cre+; Ire1alphafl/+; LSL-NrasG12D/+ mice. These results indicate that N-RasG12D-mediated activation of IRE1alpha/XBP1s signaling protects HSCs from ER stress and promotes pre-LSC clonal expansion.To identify the downstream targets of IRE1alpha/XBP1s, we examined the potential transcription targets of XBP1s and found that the co-chaperone dnajb8 is significantly upregulated in Nras mutant HSCs. Overexpression of dnajb8 also significantly enhanced the long term reconstitution capacity of HSCs in transplant recipients, suggesting that N-RasG12D-mediated activation of IRE1alpha/XBP1s promotes protein refolding. Consistent with this, in vivo level of protein aggregation was much lower in Nras mutant HSCs as compared to wild type HSCs. Interestingly, unlike Runx1 knockout, which confers resistance to ER stress by inhibiting ribosome biogenesis and protein synthesis, we observed increased protein synthesis in NrasG12D HSCs. Taken together, we conclude that N-RasG12D dysregulates IRE1alpha-Xbp1s signaling to promote adaptation to ER-stress, re-balancing protein homeostasis, and maintaining the integrity of the HSC pool to promote pre-leukemic hematopoietic stem cell expansion. DisclosuresNo relevant conflicts of interest to declare.

Drugging the unfolded protein response in acute leukemias.

The unfolded protein response (UPR), an endoplasmic reticulum (ER) stress-induced signaling cascade, is mediated by three major stress sensors IRE-1α, PERK, and ATF6α. Studies described the UPR as a critical network in selection, adaptation, and survival of cancer cells. While previous reviews focused mainly on solid cancer cells, in this review, we summarize the recent findings focusing on acute leukemias. We take into account the impact of the underlying genetic alterations of acute leukemia cells, the leukemia stem cell pool, and provide an outline on the current genetic, clinical, and therapeutic findings. Furthermore, we shed light on the important oncogene-specific regulation of individual UPR signaling branches and the therapeutic relevance of this information to answer the question if the UPR could be an attractive novel target in acute leukemias.

Memory loss in Alzheimer's disease: are the alterations in the UPR network involved in the cognitive impairment?

Memory loss in Alzheimer's disease: are the alterations in the UPR network involved in the cognitive impairment?

Limitation of individual folding resources in the ER leads to outcomes distinct from the unfolded protein response

ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.

Attenuating the endoplasmic reticulum stress response improves functional recovery after spinal cord injury

Activation of the unfolded protein response (UPR) is involved in the pathogenesis of numerous CNS myelin abnormalities; yet, its direct role in traumatic spinal cord injury (SCI)-induced demyelination is not known. The UPR is an evolutionarily conserved cell defense mechanism initiated to restore endoplasmic reticulum homeostasis in response to various cellular stresses including infection, trauma, and oxidative damage. However, if uncompensated, the UPR triggers apoptotic cell death. We demonstrate that the three signaling branches of UPR including the PERK, ATF6, and IRE1α are rapidly initiated in a mouse model of contusive SCI specifically at the injury epicenter. Immunohistochemical analyses of the various UPR markers revealed that in neurons, the UPR appeared at 6 and 24-h post-SCI. In contrast, in oligodendrocytes and astroglia, UPR persisted at least for up to 3 days post-SCI. The UPR-associated proapoptotic transcriptional regulator CHOP was among the UPR markers upregulated in neurons and oligodendrocytes, but not in astrocytes, of traumatized mouse spinal cords. To directly analyze its role in SCI, WT and CHOP null mice received a moderate T9 contusive injury. Deletion of CHOP led to an overall attenuation of the UPR after contusive SCI. Furthermore, analyses of hindlimb locomotion demonstrated a significant functional recovery that correlated with an increase in white-matter sparing, transcript levels of myelin basic protein, and Claudin 11 and decreased oligodendrocyte apoptosis in CHOP null mice in contrast to WT animals. Thus, our study provides evidence that the UPR contributes to oligodendrocyte loss after traumatic SCI.

GTPase-Mediated Regulation of the Unfolded Protein Response in Caenorhabditis elegans Is Dependent on the AAA+ ATPase CDC-48

When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA+ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.

B- and T-cell Development Both Involve Activity of the Unfolded Protein Response Pathway

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

Intrinsic Capacities of Molecular Sensors of the Unfolded Protein Response to Sense Alternate Forms of Endoplasmic Reticulum Stress

The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings.