Mutation in protein disulfide isomerase A3 causes neurodevelopmental defects by disturbing endoplasmic reticulum proteostasis.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α.

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.

IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

The UPRosome - decoding novel biological outputs of IRE1α function.

Different perturbations alter the function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded proteins in its lumen, a condition termed ER stress. To restore ER proteostasis, a highly conserved pathway is engaged, known as the unfolded protein response (UPR), triggering adaptive programs or apoptosis of terminally damaged cells. IRE1α (also known as ERN1), the most conserved UPR sensor, mediates the activation of responses to determine cell fate under ER stress. The complexity of IRE1α regulation and its signaling outputs is mediated in part by the assembly of a dynamic multi-protein complex, named the UPRosome, that regulates IRE1α activity and the crosstalk with other pathways. We discuss several studies identifying components of the UPRosome that have illuminated novel functions in cell death, autophagy, DNA damage, energy metabolism and cytoskeleton dynamics. Here, we provide a theoretical analysis to assess the biological significance of the UPRosome and present the results of a systematic bioinformatics analysis of the available IRE1α interactome data sets followed by functional enrichment clustering. This in silico approach decoded that IRE1α also interacts with proteins involved in the cell cycle, transport, differentiation, response to viral infection and immune response. Thus, defining the spectrum of IRE1α-binding partners will reveal novel signaling outputs and the relevance of the pathway to human diseases.

A novel ER stress-independent function of the UPR in angiogenesis.

Tumors rely on the unfolded protein response (UPR) and angiogenesis to survive the metabolic stress of hypoxia. Karali et al. (2014) revealed that VEGF signaling engages UPR sensors in an unconventional manner that is independent of endoplasmic reticulum (ER) stress, mediated by mTOR signaling to promote endothelial cell survival and angiogenesis.

Homeostatic interplay between FoxO proteins and ER proteostasis in cancer and other diseases.

Cancer cells are exposed to adverse conditions within the tumor microenvironment that challenge cells to adapt and survive. Several of these homeostatic perturbations insults alter the normal function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded proteins. ER stress triggers a conserved signaling pathway known as the unfolded protein response (UPR) to cope with the stress or trigger apoptosis of damaged cells. The UPR has been described as a major driver in the acquisition of malignant characteristics that ultimately lead to cancer progression. Although, several reports describe the relevance of the UPR in tumor growth, the possible crosstalk with other cancer-related pathways is starting to be elucidated. The Forkhead Box O (FoxO) subfamily of proteins has a major role in cancer progression, where chromosomal translocations and deregulated signaling lead to loss-of-function of FoxO proteins, contributing to tumor progression. Here we discuss the homeostatic connection between the UPR and FoxO proteins and its possible implications to tumor progression and the acquisition of several hallmarks of cancer. In addition, studies linking a crosstalk between the UPR and FoxO proteins in other diseases, including neurodegeneration and metabolic disorders is provided.

Synaptotagmin-1 overexpression under inflammatory conditions affects secretion in salivary glands from Sjögren's syndrome patients.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy associated with severe secretory alterations by disruption of the glandular architecture integrity, which is fundamental for a correct function and localization of the secretory machinery. Syt-1, PI(4,5)P2 and Ca2+ are significant factors controlling exocytosis in different secretory cells, the Ca2+ role being the most studied. Salivary acinar cells from SS-patients show a defective agonist-regulated intracellular Ca2+ release together with a decreased IP3R expression level, and this condition may explain a reduced water release. However, there are not reports where Syt-1, PI(4,5)P2 and Ca2+ in acinar cells of SS patients had been studied. In the present study, we analyzed the expression and/or localization of Syt-1 and PI(4,5)P2 in acinar cells of labial salivary gland biopsies from SS-patients and control individuals. Also, we evaluated whether the overexpression of Syt-1 and the loss of cell polarity induced by TNF-α or loss of interaction between acinar cell and basal lamina, alters directionality of the exocytosis process, Ca2+ signaling and α-amylase secretion in a 3D-acini model stimulated with cholinergic or ß-adrenergic agonists. In addition, the correlation between Syt-1 protein levels and clinical parameters was evaluated. The results showed an increase of Syt-1 mRNA and protein levels, and a high number of co-localization points of Syt-1/STX4 and PI(4,5)P2/Ezrin in the acinar basolateral region of LSG from SS-patients. With regard to 3D-acini, Syt-1 overexpression increased exocytosis in the apical pole compared to control acini. TNF-α stimulation increased exocytic events in the basal pole, which was further enhanced by Syt-1 overexpression. Additionally, altered acinar cell polarity affected Ca2+ signaling and amylase secretion. Overexpression of Syt-1 was associated with salivary gland alterations revealing that the secretory dysfunction in SS-patients is linked to altered expression and/or localization of secretory machinery components together with impaired epithelial cell polarity. These findings provide a novel insight on the pathological mechanism implicated in ectopic secretory products to the extracellular matrix of LSG from SS-patients, which might initiate inflammation.

ER proteostasis addiction in cancer biology: Novel concepts.

Endoplasmic reticulum (ER) stress is generated by various physiological and pathological conditions that induce an accumulation of misfolded proteins in its lumen. ER stress activates the unfolded protein response (UPR), an adaptive reaction to cope with protein misfolding to and restore proteostasis. However, chronic ER stress results in apoptosis. In solid tumors, the UPR mediates adaptation to various environmental stressors, including hypoxia, low in pH and low nutrients availability, driving positive selection. Recent findings support the concept that UPR signaling also contributes to other relevant cancer-related event that may not be related to ER stress, including angiogenesis, genomic instability, metastasis and immunomodulation. In this article, we overview novel discoveries highlighting the impact of the UPR to different aspects of cancer biology beyond its known role as a survival factor to the hypoxic environment observed in solid tumors.

BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α.

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

When ER stress reaches a dead end.

Endoplasmic reticulum (ER) stress is a common feature of several physiological and pathological conditions affecting the function of the secretory pathway. To restore ER homeostasis, an orchestrated signaling pathway is engaged that is known as the unfolded protein response (UPR). The UPR has a primary function in stress adaptation and cell survival; however, under irreversible ER stress a switch to pro-apoptotic signaling events induces apoptosis of damaged cells. The mechanisms that initiate ER stress-dependent apoptosis are not fully understood. Several pathways have been described where we highlight the participation of the BCL-2 family of proteins and ER calcium release. In addition, recent findings also suggest that microRNAs and oxidative stress are relevant players on the transition from adaptive to cell death programs. Here we provide a global and integrated overview of the signaling networks that may determine the elimination of a cell under chronic ER stress. This article is part of a Special Section entitled: Cell Death Pathways.

The endoplasmic reticulum stress sensor IRE1 regulates collagen secretion through the enforcement of the proteostasis factor P4HB/PDIA1 contributing to liver damage and fibrosis.

Collagen is one the most abundant proteins and the main cargo of the secretory pathway, contributing to hepatic fibrosis and cirrhosis due to excessive deposition of extracellular matrix. Here we investigated the possible contribution of the unfolded protein response, the main adaptive pathway that monitors and adjusts the protein production capacity at the endoplasmic reticulum, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 reduced liver damage and diminished collagen deposition in models of liver fibrosis triggered by carbon tetrachloride (CCl 4 ) administration or by high fat diet. Proteomic and transcriptomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1), which is known to be critical for collagen maturation, as a major IRE1-induced gene. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER and altered secretion, a phenotype rescued by P4HB overexpression. Taken together, our results collectively establish a role of the IRE1/P4HB axis in the regulation of collagen production and its significance in the pathogenesis of various disease states.

Assays to Study IRE1 Activation and Signaling.

The endoplasmic reticulum (ER) stress sensor IRE1 is a a major player of the unfolded protein response (UPR), the main pathway driving adaptation processes to restore proteostasis.  In addition, overactivation of IRE1 signaling contributes to a variety of pathologies including diabetes, neurodegenerative diseases, and cancer. Under ER stress, IRE1 auto-transphosphorylates and oligomerizes, triggering the activation of its endoribonuclease domain located in the cytosolic region. Active IRE1 catalyzes the splicing of the mRNA encoding for the XBP1 transcription factor, in addition to degrade several RNAs through a process known as regulated IRE1-dependent decay of mRNA (RIDD). Besides its role as an UPR transducer, several posttranslational modifications and protein-protein interactions can regulate IRE1 activity and modulate its signaling in the absence of stress. Thus, investigating the function of IRE1 in physiology and disease requires the use of complementary approaches. Here, we provide detailed protocols to perform four different assays to study IRE1 activation and signaling: (i) Phos-tag gels to evaluate the phosphorylation status of IRE1, (ii) microscopy using TREX-IRE1-GFP cells to measure IRE1 oligomerization, (iii) conventional RT-PCR to assess XBP1 mRNA processing, and (iv) quantitative PCR to determine the levels of canonical UPR target genes and the degradation of several mRNAs that are target of RIDD. We propose to use these experimental strategies as "gold standards" to study IRE1 signaling.

Emerging roles of endoplasmic reticulum proteostasis in brain development.

The development of the central nervous system requires a series of morphogenetic events that shape brain and spinal cord structures. Several brain regions and neural circuits are formed by differential gene expression patterns and cell migration events involving neurons. During neurogenesis and neuritogenesis, increased demand for protein synthesis occurs to express key neuronal proteins to generate axons, dendrites, and synapsis. The endoplasmic reticulum (ER) is a central hub controlling protein homeostasis (proteostasis), impacting a wide range of cellular processes required for brain function. Although most of the field has focused on studying the role of ER stress in neurodegenerative diseases marked by abnormal protein aggregation, accumulating evidence indicates that ER proteostasis contributes to brain development and may impact neurodevelopmental processes such as neuronal migration, differentiation, and function. Here, we review emerging evidence linking neurodevelopment with ER proteostasis and its relevance to human disorders.

Helicobacter pylori-induced loss of the inhibitor-of-apoptosis protein survivin is linked to gastritis and death of human gastric cells.

Helicobacter pylori infects the human stomach and modifies signaling pathways that affect gastric epithelial cell proliferation and viability. Chronic exposure to this pathogen contributes to the onset of gastric atrophy, an early event in the genesis of gastric cancer associated with H. pylori infection. Susceptibility to H. pylori-induced cell death ultimately depends on the presence of protective host cell factors. Although expression of the inhibitor-of-apoptosis protein survivin in adults is frequently linked to the development of cancer, evidence indicating that the protein is present in normal gastric mucosa is also available. Thus, we investigated in human gastric tissue samples and cell lines whether H. pylori infection is linked to loss of survivin and increased cell death. Our results show that infection with H. pylori decreased survivin protein levels in the mucosa of patients with gastritis. Furthermore, survivin down-regulation correlated with apoptosis and loss of cell viability in gastrointestinal cells cocultured with different H. pylori strains. Finally, overexpression of survivin in human gastric cells was sufficient to reduce cell death after infection. Taken together, these findings implicate survivin as an important survival factor in the gastric mucosa of humans.

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1.

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Caveolin-1 suppresses tumor formation through the inhibition of the unfolded protein response.

Caveolin-1 (CAV1), is a broadly expressed, membrane-associated scaffolding protein that acts both, as a tumor suppressor and a promoter of metastasis, depending on the type of cancer and stage. CAV1 is downregulated in human tumors, tumor cell lines and oncogene-transformed cells. The tumor suppressor activity of CAV1 is generally associated with its presence at the plasma membrane, where it participates, together with cavins, in the formation of caveolae and also has been suggested to interact with and inhibit a wide variety of proteins through interactions mediated by the scaffolding domain. However, a pool of CAV1 is also located at the endoplasmic reticulum (ER), modulating the secretory pathway in a manner dependent on serine-80 (S80) phosphorylation. In melanoma cells, CAV1 expression suppresses tumor formation, but the protein is largely absent from the plasma membrane and does not form caveolae. Perturbations to the function of the ER are emerging as a central driver of cancer, highlighting the activation of the unfolded protein response (UPR), a central pathway involved in stress mitigation. Here we provide evidence indicating that the expression of CAV1 represses the activation of the UPR in vitro and in solid tumors, reflected in the attenuation of PERK and IRE1α signaling. These effects correlated with increased susceptibility of cells to ER stress and hypoxia. Interestingly, the tumor suppressor activity of CAV1 was abrogated by site-directed mutagenesis of S80, correlating with a reduced ability to repress the UPR. We conclude that the tumor suppression by CAV1 involves the attenuation of the UPR, and identified S80 as essential in this context. This suggests that intracellular CAV1 regulates cancer through alternative signaling outputs.

Genotoxic stress triggers the activation of IRE1α-dependent RNA decay to modulate the DNA damage response.

The molecular connections between homeostatic systems that maintain both genome integrity and proteostasis are poorly understood. Here we identify the selective activation of the unfolded protein response transducer IRE1α under genotoxic stress to modulate repair programs and sustain cell survival. DNA damage engages IRE1α signaling in the absence of an endoplasmic reticulum (ER) stress signature, leading to the exclusive activation of regulated IRE1α-dependent decay (RIDD) without activating its canonical output mediated by the transcription factor XBP1. IRE1α endoribonuclease activity controls the stability of mRNAs involved in the DNA damage response, impacting DNA repair, cell cycle arrest and apoptosis. The activation of the c-Abl kinase by DNA damage triggers the oligomerization of IRE1α to catalyze RIDD. The protective role of IRE1α under genotoxic stress is conserved in fly and mouse. Altogether, our results uncover an important intersection between the molecular pathways that sustain genome stability and proteostasis.

Emerging Roles of the Endoplasmic Reticulum Associated Unfolded Protein Response in Cancer Cell Migration and Invasion.

Endoplasmic reticulum (ER) proteostasis is often altered in tumor cells due to intrinsic (oncogene expression, aneuploidy) and extrinsic (environmental) challenges. ER stress triggers the activation of an adaptive response named the Unfolded Protein Response (UPR), leading to protein translation repression, and to the improvement of ER protein folding and clearance capacity. The UPR is emerging as a key player in malignant transformation and tumor growth, impacting on most hallmarks of cancer. As such, the UPR can influence cancer cells' migration and invasion properties. In this review, we overview the involvement of the UPR in cancer progression. We discuss its cross-talks with the cell migration and invasion machinery. Specific aspects will be covered including extracellular matrix (ECM) remodeling, modification of cell adhesion, chemo-attraction, epithelial-mesenchymal transition (EMT), modulation of signaling pathways associated with cell mobility, and cytoskeleton remodeling. The therapeutic potential of targeting the UPR to treat cancer will also be considered with specific emphasis in the impact on metastasis and tissue invasion.