Unfolded Protein Response Transducers Research Articles

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

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. 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. 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. 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.

Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.

IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma.

Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1β) with the luminal domain of IRE1β deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1β/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1β/α chimera. In vitro, AGR2 actively de-stabilised the IRE1β luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1β-AGR2 couple suggest that active repression of IRE1β by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

Abstract A033: Exploring synthetic lethality of targeting miR346-unfolded protein response dependent DNA damage response mechanisms in treatment-resistant prostate cancer

Abstract Background: The Unfolded Protein Response (UPR) is a key homeostatic mechanism that is activated during endoplasmic reticulum (ER) stress caused by both intrinsic (genomic instability, metabolic demand) and extrinsic (chemo/radiotherapy, nutrient deprivation, hypoxia) factors. IRE1 is the main UPR transducer, signalling the transcription factor XBP1, and IRE1 Regulated Dependent Decay (RIDD) to resolve ER stress by controlling the expression of mRNA and miRNAs. IRE1 also regulates aspects of the DNA damage response (DDR) and IRE1-binding drugs have clinical effects as monotherapies or as adjuvants to chemotherapeutics. One miRNA involved in both the DDR and IRE1 biology is miR-346, which we have shown to cause widespread DNA damage in prostate cancer (PCa), sensitising PCa to DNA damaging drugs and associating with improved PCa outcomes. Rationale: In RNAseq data datasets we have shown that miR-346 causes a widespread transcriptional downregulation of DDR markers whilst we observed the opposite in IRE1-CRISPR-KO PCa cells. We have also developed novel IRE1 modulators that sensitise cancers to chemotherapy in vivo. These findings offer a strong rationale to explore co-targeting of miR-346 and IRE1 in the presence of DNA damaging agents to treat aggressive forms of PCa. Consequently, we want to elucidate the exact biology underpinning the miR346-IRE1/XBP1/RIDD-DDR axis to prognosticate treatment groups with gene signatures as proxies of PARPi/chemotherapeutic susceptibility. Impact: If miR-346 is a downstream effector of IRE1 activity, then an IRE1 prognostic signature can act as proxy for stratification and selection of patients for miR-346 treatment as sensitiser to DNA damaging agents whilst if miR-346 and IRE1 work in parallel, we could achieve clinically significant results by exploiting synthetic lethality. By stratifying large cohorts through IRE1/miR-346/DDR signatures (personalised precision medicine) we can increase the pool of patients for whom DNA damaging agents are effective by inducing a BRCAness phenotype, improving treatment responses and quality of life. Citation Format: Dimitrios Doultsinos, Ian Mills, Claire Fletcher. Exploring synthetic lethality of targeting miR346-unfolded protein response dependent DNA damage response mechanisms in treatment-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A033.

Deletion of the Unfolded Protein Response Transducer IRE1α Is Detrimental to Aging Photoreceptors and to ER Stress-Mediated Retinal Degeneration.

The unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration. We used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H. OCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation. These data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration.

Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells

The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.

The IRE1α pathway in glomerular diseases: The unfolded protein response and beyond.

Endoplasmic reticulum (ER) function is vital for protein homeostasis ("proteostasis"). Protein misfolding in the ER of podocytes (glomerular visceral epithelial cells) is an important contributor to the pathogenesis of human glomerular diseases. ER protein misfolding causes ER stress and activates a compensatory signaling network called the unfolded protein response (UPR). Disruption of the UPR, in particular deletion of the UPR transducer, inositol-requiring enzyme 1α (IRE1α) in mouse podocytes leads to podocyte injury and albuminuria in aging, and exacerbates injury in glomerulonephritis. The UPR may interact in a coordinated manner with autophagy to relieve protein misfolding and its consequences. Recent studies have identified novel downstream targets of IRE1α, which provide new mechanistic insights into proteostatic pathways. Novel pathways of IRE1α signaling involve reticulophagy, mitochondria, metabolism, vesicular trafficking, microRNAs, and others. Mechanism-based therapies for glomerulopathies are limited, and development of non-invasive ER stress biomarkers, as well as targeting ER stress with pharmacological compounds may represent a therapeutic opportunity for preventing or attenuating progression of chronic kidney disease.

IRE1-mediated cytoplasmic splicing and regulated IRE1-dependent decay of mRNA in the liverwort Marchantia polymorpha.

The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response is a homeostatic cellular response conserved in eukaryotes to alleviate the accumulation of unfolded proteins in the ER. In the present study, we characterized the UPR in the liverwort Marchantia polymorpha to obtain insights into the conservation and divergence of the UPR in the land plants. We demonstrate that the most conserved UPR transducer in eukaryotes, IRE1, is conserved in M. polymorpha, which harbors a single gene encoding IRE1. We showed that MpIRE1 mediates cytoplasmic splicing of mRNA encoding MpbZIP7, a M. polymorpha homolog of bZIP60 in flowering plants, and upregulation of ER chaperone genes in response to the ER stress inducer tunicamycin. We further showed that MpIRE1 also mediates downregulation of genes encoding secretory and membrane proteins in response to ER stress, indicating the conservation of regulated IRE1-dependent decay of mRNA. Consistent with their roles in the UPR, Mpire1 ge and Mpbzip7 ge mutants exhibited higher sensitivity to ER stress. Furthermore, an Mpire1 ge mutant also exhibited retarded growth even without ER stress inducers, indicating the importance of MpIRE1 for vegetative growth in addition to alleviation of ER stress. The present study provides insights into the evolution of the UPR in land plants.

The unfolded protein response transducer IRE1α promotes reticulophagy in podocytes

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.

HBXIP is a novel regulator of the unfolded protein response that sustains tamoxifen resistance in ER+ breast cancer

Endocrine-therapy-resistant estrogen receptor–positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifen-induced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation, enhances UPR-element reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, tamoxifen treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase and activating transcription factor 6α. Clinical investigations and Kaplan–Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression is associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a regulator of EnR homeostasis and a potential target for TamR breast cancer therapy.

Regulation of the unfolded protein response transducer IRE1α by SERPINH1 aggravates periodontitis with diabetes mellitus via prolonged ER stress

The hyperglycemic microenvironment induced by diabetes mellitus aggravates the inflammatory response, in which the IRE1α signal transduction pathway of the unfolded protein response (UPR) participates. However, the mechanism by which hyperglycemia regulates the IRE1α signaling pathway and affects endoplasmic reticulum (ER) homeostasis in human gingival epithelium in periodontitis with diabetes mellitus remains unknown. Our current data provide evidence that diabetes mellitus causes a hyperinflammatory response in the gingival epithelium, which accelerates periodontal inflammation. Next, we assessed UPR-IRE1α signaling in periodontitis with diabetes mellitus by examining human clinical gingival epithelium samples from healthy subjects, subjects with periodontitis and subjects with periodontitis with diabetes mellitus and by in vitro challenge of human epithelial cells with a hyperglycemic microenvironment. The results showed that a hyperglycemic microenvironment inhibited the IRE1α/XBP1 axis, decreased the expression of a UPR target gene (GRP78), and ultimately impaired the UPR, causing ER stress to be prolonged or more severe in human gingival epithelium. Subsequently, RNA sequencing (RNA-seq) data was analyzed to investigate the expression of ER-related genes in human gingival epithelium. Experiments verified that the mechanism by which periodontitis is aggravated in individuals with diabetes mellitus may involve decreased SERPINH1 expression. Furthermore, experiments in SERPINH1-knockdown and SERPINH1-overexpression models established in vitro indicated that SERPINH1 might act as an activator of IRE1α, maintaining human gingival epithelium homeostasis and reducing proinflammatory cytokine expression by preventing prolonged ER stress induced by high-glucose conditions. In conclusion, regulation of the UPR transducer IRE1α by SERPINH1 alleviates periodontitis with diabetes mellitus by mitigating prolonged ER stress. This finding provides evidence for the further study of periodontitis with diabetes mellitus.

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 asregulated 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.

The interplay of GTP-binding protein AGB1 with ER stress sensors IRE1a and IRE1b modulates Arabidopsis unfolded protein response and bacterial immunity

ABSTRACT In eukaryotic cells, the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in ER stress that induces a cascade of reactions called the unfolded protein response (UPR). In Arabidopsis, the most conserved UPR sensor, Inositol-requiring enzyme 1 (IRE1), responds to both abiotic- and biotic-induced ER stress. Guanine nucleotide-binding proteins (G proteins) constitute another universal and conserved family of signal transducers that have been extensively investigated due to their ubiquitous presence and diverse nature of action. Arabidopsis GTP-binding protein β1 (AGB1) is the only G-protein β-subunit encoded by the Arabidopsis genome that is involved in numerous signaling pathways. Mounting evidence suggests the existence of a crosstalk between IRE1 and G protein signaling during ER stress. AGB1 has previously been shown to control a distinct UPR pathway independently of IRE1 when treated with an ER stress inducer tunicamycin. Our results obtained with combinatorial knockout mutants support the hypothesis that both IRE1 and AGB1 synergistically contribute to ER stress responses chemically induced by dithiothreitol (DTT) as well as to the immune responses against a phytopathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000. Our study highlights the crosstalk between the plant UPR transducers under abiotic and biotic stress.

Recombinant human growth hormone improves the immune status of rats with septic encephalopathy: The role of VEGFR2 in the prevalence of endoplasmic reticulum stress repair module

Septic encephalopathy results from the intense reaction of the immune system to infection. The role of growth hormone (GH) signaling in maintaining brain function is well established; however, the involvement of the vascular endothelial growth factor receptor-2 (VEGFR2) in the potential modulatory effect of GH on septic encephalopathy-associated endoplasmic reticulum stress (ERS) and blood–brain barrier (BBB) permeability is not well-understood. Therefore, after the induction of mid-grade sepsis by cecal ligation/perforation, rats were subcutaneously injected with recombinant human GH (rhGH)/somatropin alone or preceded by the VEGFR2 antagonist WAG-4S for 7 days. rhGH/somatropin reduced bodyweight loss and plasma endotoxin, maintained the hyperthermic state, and improved motor/memory functions. Additionally, rhGH/somatropin increased the junctional E-cadherin and β-catenin pool in the cerebral cortex to enhance the BBB competency, effects that were abolished by VEGFR2 blockade. Also, it activated cortical VEGFR2/mammalian target of the Rapamycin (mTOR) axis to mitigate ERS. The latter was reflected by the deactivation of the inositol-requiring enzyme-1α (IRE1α)/spliced X-box binding protein-1 (XBP1s) trajectory and the reduction in the protein levels of the death markers, C/EBP homologous protein (CHOP)/growth arrest and DNA damage-153 (GADD153), c-jun-N-terminal kinase (JNK), and caspase-3 with the simultaneous augmentation of expression of the unfolded protein response transducer proteinkinaseR-like ERkinase (PERK). Furthermore, rhGH/somatropin suppressed the phosphorylation of eukaryotic initiation factor-2α (eIF2α), upregulated the gene expression of activating transcription factor-4 (ATF4), GADD34, and glucose-regulated protein-78/binding immunoglobulin (GRP78/Bip). Moreover, it increased the glutathione level and reduced lipid peroxidation in the cerebral cortex. The VEGFR2 antagonist reversed the effect of rhGH/somatropin on PERK and IRE1α and boosted the apoptotic markers but neither affected p-eIF2α nor GADD34. Hence, we conclude that VEGFR2 activation by rhGH/somatropin plays a crucial role in assembling the BBB adherens junctions via its antioxidant capacity, ERS relief, and reducing endotoxemia in septic encephalopathy.

The Unfolded Protein Response as a Guardian of the Secretory Pathway.

The endoplasmic reticulum (ER) is the major site of membrane biogenesis in most eukaryotic cells. As the entry point to the secretory pathway, it handles more than 10,000 different secretory and membrane proteins. The insertion of proteins into the membrane, their folding, and ER exit are affected by the lipid composition of the ER membrane and its collective membrane stiffness. The ER is also a hotspot of lipid biosynthesis including sterols, glycerophospholipids, ceramides and neural storage lipids. The unfolded protein response (UPR) bears an evolutionary conserved, dual sensitivity to both protein-folding imbalances in the ER lumen and aberrant compositions of the ER membrane, referred to as lipid bilayer stress (LBS). Through transcriptional and non-transcriptional mechanisms, the UPR upregulates the protein folding capacity of the ER and balances the production of proteins and lipids to maintain a functional secretory pathway. In this review, we discuss how UPR transducers sense unfolded proteins and LBS with a particular focus on their role as guardians of the secretory pathway.

Abstract 1934: Modulation of protein homeostasis networks upon Hsp70 inhibition in cancer cells

Abstract Cellular protein homeostasis is tightly regulated by balancing protein synthesis, folding, and degradation. Cancer cells constantly modulate protein folding and degradation efficiency to survive and proliferate without accumulating toxic misfolded proteins under acute stress conditions, such as low oxygen and energy, and exposure to chemotherapeutic agents. Many cancers rely on the unfolded protein response (UPR) to cope with stress, which increases the expression of molecular chaperones and mitigates the accumulation of misfolded proteins. Thus, it is unsurprising that the Hsp70 molecular chaperone, which serves as a master regulator of proteostasis, is upregulated in many cancers. Increased Hsp70 correlates with metastasis formation and poor patient prognosis in different cancers, like breast cancer. To define the role of Hsp70 in cancer survival and identify how cancer cells compensate for chaperone-mediated proteotoxicity, we investigated the response to a specific Hsp70 inhibitor, MAL3-101, in breast cancer cells. We discovered that breast cancer cells bin into distinct groups when subjected to Hsp70 inhibition based on their sensitivity to the compound. Moreover, we demonstrate that resistant cells have higher autophagy levels compared to more sensitive lines. Autophagy was further induced by MAL3-101 in resistant breast cancer cells, as evidenced by the accumulation of both autophagy related genes and proteins, as well as by autophagic-like structures detected by confocal microscopy. These data suggest that resistance to Hsp70 inhibition arises from autophagy induction. We then discovered that Hsp70 inhibition induces the UPR pathway, triggering apoptosis in sensitive cells. In particular, the activation of the UPR transducer, PERK, is required to induce apoptosis in sensitive cells when Hsp70 was inhibited. Overall, our work positions autophagy as a critical compensatory mechanism when molecular chaperone function is overwhelmed and misfolded proteins accumulate. Furthermore, our findings delineate a distinct role of PERK in apoptosis induction upon Hsp70 inhibition, highlighting the interplay between the PERK, Hsp70, and autophagy. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Modulation of protein homeostasis networks upon Hsp70 inhibition in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1934.

Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1.

The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.

Heat shock protein 47 confers chemoresistance on pancreatic cancer cells by interacting with calreticulin and IRE1α.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase 2 and interaction of IRE1α with inositol 1,4,5‐triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase‐induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+/caspases axis. Ablation of HSP47 promotes gemcitabine‐induced suppression of tumor growth in PDAC cell‐bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.

Calcineurin Activity Is Increased in Charcot-Marie-Tooth 1B Demyelinating Neuropathy.

Schwann cells produce a considerable amount of lipids and proteins to form myelin in the PNS. For this reason, the quality control of myelin proteins is crucial to ensure proper myelin synthesis. Deletion of serine 63 from P0 (P0S63del) protein in myelin forming Schwann cells causes Charcot-Marie-Tooth type 1B neuropathy in humans and mice. Misfolded P0S63del accumulates in the ER of Schwann cells where it elicits the unfolded protein response (UPR). PERK is the UPR transducer that attenuates global translation and reduces ER stress by phosphorylating the translation initiation factor eIF2alpha. Paradoxically, Perk ablation in P0S63del Schwann cells (S63del/PerkSCKO ) reduced the level of P-eIF2alpha, leaving UPR markers upregulated, yet unexpectedly improved S63del myelin defects in vivo We therefore investigated the hypothesis that PERK may interfere with signals outside of the UPR and specifically with calcineurin/NFATc4 pro-myelinating pathway. Using mouse genetics including females and males in our experimental setting, we show that PERK and calcineurin interact in P0S63del nerves and that calcineurin activity and NFATc4 nuclear localization are increased in S63del Schwann cells, without altering EGR2/KROX20 expression. Moreover, genetic manipulation of the calcineurin subunits appears to be either protective or toxic in S63del in a context-dependent manner, suggesting that Schwann cells are highly sensitive to alterations of calcineurin activity.SIGNIFICANCE STATEMENT Our work shows a novel activity and function for calcineurin in Schwann cells in the context of ER stress. Schwann cells expressing the S63del mutation in P0 protein induce the unfolded protein response and upregulate calcineurin activity. Calcineurin interacts with the ER stress transducer PERK, but the relationship between the UPR and calcineurin in Schwann cells is unclear. Here we propose a protective role for calcineurin in S63del neuropathy, although Schwann cells appear to be very sensitive to its regulation. The paper uncovers a new important role for calcineurin in a demyelinating diseases.