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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. Cell...
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... (IPs) were analyzed using two-dimensional liquid chromatography along with tandem mass spectrometry followed by bioinformatic analysis. Comparison of three independent experiments led us to identify ten candidate pro- teins that differentially interact with IRE1a, in addition to the known regulator BiP (Table S1). These proteins are involved in different cellular functions including mitochondrial biology, collagen biosynthesis, cytoskeleton, and cell signaling, and display different subcellular distributions (Table S1). ...
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... of three independent experiments led us to identify ten candidate pro- teins that differentially interact with IRE1a, in addition to the known regulator BiP (Table S1). These proteins are involved in different cellular functions including mitochondrial biology, collagen biosynthesis, cytoskeleton, and cell signaling, and display different subcellular distributions (Table S1). The majority of the candidates identified in IRE1a-containing complexes were present in both basal and ER stress conditions, whereas only three proteins were exclusively detected at resting conditions and two upon 6 hr of Tm treatment ( Figure 1A). ...
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... incubated RED-labeled IRE1a-NLD protein with increasing concentrations of purified HSP47 ( Figure 4I). Calculation of the dissociation constant (KD) revealed a high affinity between HSP47 and IRE1a-NLD in the nanomolar range (Table 1). These data were further confirmed using surface plasmon resonance to deter- mine that the biophysical parameters of the interaction were in the nanomolar range (Table 1). ...
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... of the dissociation constant (KD) revealed a high affinity between HSP47 and IRE1a-NLD in the nanomolar range (Table 1). These data were further confirmed using surface plasmon resonance to deter- mine that the biophysical parameters of the interaction were in the nanomolar range (Table 1). We then compared the strength of the interaction between HSP47 and IRE1a-NLD with its affinity for BiP or PDIA6. ...
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... this experimental setting, recombinant BiP associated with the luminal domain of IRE1a with a KD value of 39.7 nM, in the same range as recombinant HSP47. In contrast, PDIA6 displayed a lower binding affinity to IRE1a-NLD under the same experimental conditions (Table 1). As control for the specificity of this binding, we tested the asso- ciation of the ER luminal domain of PERK to recombinant Hsp47. ...
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Background:
Defects in protein homeostasis are sufficient to provoke cardiac remodeling and dysfunction. Although posttranslational modifications by ubiquitin and ubiquitin-like proteins are emerging as an important regulatory mechanism of protein function, the role of Ufm1 (ubiquitin-fold modifier 1)-a novel ubiquitin-like protein-has not been ex...
Citations
... We subsequently investigated the mechanism by which HSP47 enhances the expression of type I collagen in human foreskin fibroblasts. It has been reported that HSP47 binds to the α isoform of IRE1 (IRE1α), leading to its activation [37]. Therefore, we examined whether IRE1α activation is involved in the HSP47-dependent induction of type I collagen. ...
... We subsequently investigated the mechanism by which HSP47 enhances the e sion of type I collagen in human foreskin fibroblasts. It has been reported that HSP47 to the α isoform of IRE1 (IRE1α), leading to its activation [37]. Therefore, we exa whether IRE1α activation is involved in the HSP47-dependent induction of type gen. ...
... This interaction displaces the negative regulator BiP from the complex, facilitating IRE1α oligomerization. Consequently, HSP47 expression enhances IRE1α activation, amplifying downstream signaling and effectively mitigating ER stress [6,37]. Given this information, we hypothesized that IRE1α might play a role in the upregulation of type I collagen mediated by HSP47. ...
Heat shock protein 47 (HSP47), also known as SERPINH1, functions as a collagen-specific molecular chaperone protein essential for the formation and stabilization of the collagen triple helix. Here, we delved into the regulatory pathways governed by HSP47, shedding light on collagen homeostasis. Our investigation revealed a significant reduction in HSP47 mRNA levels in the skin tissue of older mice as compared to their younger counterparts. The augmented expression of HSP47 employing lentivirus infection in fibroblasts resulted in an increased secretion of type I collagen. Intriguingly, the elevated expression of HSP47 in fibroblasts correlated with increased protein and mRNA levels of type I collagen. The exposure of fibroblasts to IRE1α RNase inhibitors resulted in the reduced manifestation of HSP47-induced type I collagen secretion and expression. Notably, HSP47-overexpressing fibroblasts exhibited increased XBP1 mRNA splicing. The overexpression of HSP47 or spliced XBP1 facilitated the nuclear translocation of β-catenin and transactivated a reporter harboring TCF binding sites on the promoter. Furthermore, the overexpression of HSP47 or spliced XBP1 or the augmentation of nuclear β-catenin through Wnt3a induced the expression of type I collagen. Our findings substantiate that HSP47 enhances type I collagen expression and secretion in fibroblasts by orchestrating a mechanism that involves an increase in nuclear β-catenin through IRE1α activation and XBP1 splicing. This study therefore presents potential avenues for an anti-skin-aging strategy targeting HSP47-mediated processes.
... CBNPs can enter the central nervous system through blood circulation and olfactory nerves, affecting brain development and increasing neurological disease susceptibility [3]. In the present study, we investigated the neurotoxicity of CBNPs in the brains of mice exposed to the CBNP Printex90, which is often used as a model particle in toxicological research of particulate air pollution [18]. ...
... Activated IRE1 further oligomerizes, trans-autophosphorylates, and causes the activation of its cytosolic kinase domain. Phosphorylation of IRE1 occurs at Ser724, Ser729, and Ser726 positions [22]. This phosphorylation is important for the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2), which activates apoptosis signal-regulating kinase 1 (ASK1), thus activating c-Jun N-terminal kinase (JNK) pathway signaling [13]. ...
Endoplasmic reticulum (ER) is the site for synthesis and folding of secreted and trans-membrane proteins. Disturbance in the functioning of ER leads to the accumulation of unfolded and misfolded proteins, which finally activate the unfolded protein response (UPR) signaling. The three branches of UPR-IRE1 (Inositol requiring enzyme 1), PERK (Protein kinase RNA-activated (PKR)-like ER kinase), and ATF6 (Activating transcription factor 6)-modulate the gene expression pattern through increased expression of chaperones and restore ER homeostasis by enhancing ER protein folding capacity. The liver is a central organ which performs a variety of functions which help in maintaining the overall well-being of our body. The liver plays many roles in cellular physiology , blood homeostasis, and detoxification, and is the main site at which protein synthesis occurs. Disturbance in ER homeostasis is triggered by calcium level imbalance, change in redox status, viral infection, and so on. ER dysfunction and subsequent UPR signaling participate in various hepatic disorders like metabolic (dysfunction) associated fatty liver disease, liver cancer, viral hepatitis, and cholestasis. The exact role of ER stress and UPR signaling in various liver diseases is not fully understood and needs further investigation. Targeting UPR signaling with drugs is the subject of intensive research for therapeutic use in liver diseases. The present review summarizes the role of UPR signaling in liver disorders and describes why UPR regulators are promising therapeutic targets.
... As a major intracellular Ca 2+ store, the ER is essential for protein folding, secretion and Ca 2+ homeostasis [1]. Environmental stressors, such as disruption of Ca 2+ homeostasis, viral infection, and redox homoeostasis, lead to the accumulation of misfolded proteins in the ER, eliciting ER stress [2,3]. ...
... IRE1α activation has been reported to enhance the susceptibility of cells to apoptosis. In view of the pro-survival properties of XBP1 splicing [1], we set out to test whether IRE1α activation induced by TMCO1 deficiency could promote cell survival. WT and KD cells were treated with KIRA6 for 24 h and then the early apoptotic, late apoptotic, and dead cells were quantified by flow cytometry using Annexin V/PI. ...
... To test the effect of TMCO1 on IRE1α signaling in vivo, Tmco1 +/+ and Tmco1 −/− mice at 2 months of age were intraperitoneally injected with a single dose of TM to trigger a strong UPR reaction in livers [1,[64][65][66][67][68]. ...
Background:
Maintaining homeostasis of Ca2+ stores in the endoplasmic reticulum (ER) is crucial for proper Ca2+ signaling and key cellular functions. Although Ca2+ depletion has been known to cause ER stress which in turn activates the unfolded protein response (UPR), how UPR sensors/transducers respond to excess Ca2+ when ER stores are overloaded remain largely unclear.
Results:
Here, we report for the first time that overloading of ER Ca2+ can directly sensitize the IRE1α-XBP1 axis. The overloaded ER Ca2+ in TMCO1-deficient cells can cause BiP dissociation from IRE1α, promote the dimerization and stability of the IRE1α protein, and boost IRE1α activation. Intriguingly, attenuation of the over-activated IRE1α-XBP1s signaling by a IRE1α inhibitor can cause a significant cell death in TMCO1-deficient cells.
Conclusions:
Our data establish a causal link between excess Ca2+ in ER stores and the selective activation of IRE1α-XBP1 axis, underscoring an unexpected role of overload of ER Ca2+ in IRE1α activation and in preventing cell death.
... Thus, cells need to adjust their protein production capacity at multiple levels, including translation, folding, maturation, quality control, degradation, and trafficking. The ER operates as a sensing unit to monitor the efficiency of protein production, mediated by the activation of the unfolded protein response (URP), an adaptive signaling pathway that engages multiple mechanisms to cope with protein misfolding and restore proteostasis [19,20]. ...
... The relative amounts of mRNAs were calculated from the values of comparative threshold cycle by using Actin mRNA as control. Xbp1 mRNA splicing assay have been previously described [20,95,96]. Semiquantitative PCR was performed using specific primer pair (Supplementary Materials and Methods). ...
... No reuse allowed without permission. [48] of Ern1 were bred with Mx-Cre ([41] and deletion was carried out with 150 μg intraperitoneal poly I:C (invivogen) injection thrice after every two days [20,93]. For induction of acute liver toxicity, single doses of 1.6 g/Kg of CCl4/olive oil (1:1 ratio) were injected intraperitoneally 2, 6, 12 and 24 h before the mice were sacrificed, and livers were harvested and immediately weighed. ...
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 (CCl4) 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.
... The protein assists in the transport of triple-helix procollagen from the ER lumen to the Golgi [88,89]. HSP47 associates with the ER luminal domain of IRE1α promoting IRE1α dimerization/oligomerization and activation of IRE1α-mediated UPR (Fig. 2) [90]. HSP47 does not alter PERK or ATF6-mediated UPR signaling [90]. ...
... HSP47 associates with the ER luminal domain of IRE1α promoting IRE1α dimerization/oligomerization and activation of IRE1α-mediated UPR (Fig. 2) [90]. HSP47 does not alter PERK or ATF6-mediated UPR signaling [90]. HSP47-dependent activation of IRE1α may play a role in the pathogenesis of cardiac fibrosis [91]. ...
Cellular homeostasis is crucial for the healthy functioning of the organism. Disruption of cellular homeostasis activates endoplasmic reticulum (ER) stress coping responses including the unfolded protein response (UPR). There are three ER resident stress sensors responsible for UPR activation - IRE1α, PERK and ATF6. Ca2+ signaling plays an important role in stress responses including the UPR and the ER is the main Ca2+ storage organelle and a source of Ca2+ for cell signaling. The ER contains many proteins involved in Ca2+ import/export/ storage, Ca2+ movement between different cellular organelles and ER Ca2+ stores refilling. Here we focus on selected aspects of ER Ca2+ homeostasis and its role in activation of the ER stress coping responses.
... Frontiers in Cell and Developmental Biology frontiersin.org 2012; Sepulveda et al., 2018;Oka et al., 2019;Yu et al., 2020;Wiseman et al., 2022). The three UPR pathways work together with the aim to reestablish ER proteostasis and functions. ...
Endoplasmic reticulum (ER) is the largest membrane-bound compartment in all cells and functions as a key regulator in protein biosynthesis, lipid metabolism, and calcium balance. Mammalian endoplasmic reticulum has evolved with an orchestrated protein quality control system to handle defective proteins and ensure endoplasmic reticulum homeostasis. Nevertheless, the accumulation and aggregation of misfolded proteins in the endoplasmic reticulum may occur during pathological conditions. The inability of endoplasmic reticulum quality control system to clear faulty proteins and aggregates from the endoplasmic reticulum results in the development of many human disorders. The efforts to comprehensively understand endoplasmic reticulum quality control network and protein aggregation will benefit the diagnostics and therapeutics of endoplasmic reticulum storage diseases. Herein, we overview recent advances in mammalian endoplasmic reticulum protein quality control system, describe protein phase transition model, and summarize the approaches to monitor protein aggregation. Moreover, we discuss the therapeutic applications of enhancing endoplasmic reticulum protein quality control pathways in endoplasmic reticulum storage diseases.
... Correctly folded and assembled proteins will be transported to the cell membrane or released into the extracellular space through the endomembrane system [1]. Protein folding in ER is exquisitely sensitive to internal and external adverse stresses, which lead to the interruption of normal protein folding process, accumulation of unfolded or misfolded proteins and entry into a special condition termed endoplasmic reticulum stress (ER stress) [2][3][4][5]. The induction of ER stress starts from the liberation of three ER stress sensors (ATF6, PERK or IRE1α) away from GRP78 (BIP) and could separately trigger downstream signaling events [6] by reprogramming cellular protein translation and gene expression to rescue back to normal ER homeostasis, which is termed unfolded protein response (UPR) [7,8]. ...
Background
Endoplasmic reticulum stress (ER stress) may destroy endoplasmic reticulum homeostasis (ER homeostasis) and leads to programmable cell death. Unfolded protein response (UPR) originally stimulated by ER stress is critical for the survival of tumor cells through trying to re-establish ER homeostasis as an adaption to harsh microenvironment. However, mechanisms involving key regulators in modulating UPR remain underexplored.
Methods
The expression of LINP1 in cutaneous squamous cell carcinoma (cSCC) tissues and cell lines was assessed. Subsequently, LINP1 was knocked out, knocked down or overexpressed in cSCC cells. CCK-8 assays, colony forming assays, transwell migration assays and invasiveness measurement by matrigel-coated transwell were performed to examine the role of LINP1 in cSCC development through gain-of-function and loss-of-function experiments. Transcriptomic sequencing (RNA-Seq) was conducted and indicated the key downstream signaling events regulated by LINP1 including UPR and apoptosis signaling. Furthermore, the direct interaction between LINP1 and eIF2α to modulate UPR and apoptosis was confirmed by RNA pulldown, RNA immunoprecipitation (RIP), ChIP-qPCR and in vitro phosphorylation assays.
Results
In this study, LncRNA in non-homologous end joining pathway 1 (LINP1) was identified to be one of the top ten highest-expressed LncRNAs in cSCC, the second most common cancer in the world. Functional studies using in vitro and in vivo models revealed that LINP1 functions as an oncogene to promote cell proliferation, colony formation, migration and invasiveness while inhibiting cell apoptosis in cSCC. Transcriptomic sequencing after knockdown of LINP1 indicated LINP1 negatively regulates UPR-related pathways involving key effectors for activating UPR and the apoptosis following the prolonged UPR. Mechanistic study showed LINP1 physically interacts with eIF2α to inhibit its phosphorylation for avoiding unmitigated UPR. Loss of LINP1 followed by enhanced eIF2α phosphorylation led to overactivated UPR and induced DDIT3 expression, contributing to ER stress-induced apoptosis and suppression of cSCC development.
Conclusions
Our findings demonstrate a novel regulatory hierarchy of UPR by demonstrating LINP1 as a critical modulator for eIF2α phosphorylation and a suppressor of UPR-mediated apoptosis, which suggests a novel therapeutic target for cSCC treatment.
... The inositolase-1α pathway is a transmembrane protein that is present in the UPRs and has kinase and ribonucleic acid endonuclease as its cytoplasmic structural domains (65). In a healthy state, inositolase-1 binds to immunoglobulins and is inactive. ...
The majority of new cases and fatalities from hepatocellular carcinoma (HCC) occur in China; however, the overall morbidity and mortality rates are decreasing. A major risk factor due to the evolving epidemiology is improper lipid metabolism. Although investigations on aberrant lipid metabolism are numerous, there are only a limited number of studies available on proteasomal degradation processes. The degradation process is mainly involved in endoplasmic reticulum stabilization, the balance of lipid metabolism, and physiological functions of Golgi apparatus, endoplasmic reticulum, lysosomes and other organelles, however, this process has been little studied in the development of tumorigenesis. In order to provide some theoretical support for future research on ubiquitin regulatory X domain‑containing protein 3B (UBXN3B), the present review focuses on the role of UBXN3B, which is involved in the stabilization of the endoplasmic reticulum and the maintenance of lipid homeostasis, as well as in the promotion and development of non‑alcoholic fatty liver disease and HCC.
... Inositol-requiring enzyme-1α (IRE-1α, also called ERN1) is a sensor of the unfolded protein response (UPR), widely involved in various diseases [15]. After IRE-1α is activated, the endoribonuclease activity of IRE-1α induces unconventional X-box binding protein 1 (XBP1) splicing. ...
Necroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD), respectively. However, the crosstalk between them remains largely unknown. Here, we disclose that inositol-requiring enzyme-1α (IRE-1α) is a potential modulator of both necroptosis and pyroptosis, particularly in liver pathology. In vivo, we found that pharmacological suppression of IRE-1α resulted in serious acute liver failure, which may be attributable to the downregulation of GSDMD and caspase-8 while remarkable upregulation of necroptosis markers receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. However, by using thapsigargin (THP) to moderately restore the IRE-1α level, liver failure was distinctly alleviated. Conversely, ER stressor tunicamycin (TM) promoted IRE-1α activity, which initiated liver pyroptosis by increasing GSDMD and NLRP3. But maintaining the IRE-1α balance by moderate inhibition of IRE-1α effectively improved mouse survival. In vitro, we demonstrated that inhibition of IRE-1α led to distinct necroptosis accompanied by the reduction of GSDMD in LO-2 cells and mouse primary hepatocytes. Nevertheless, by using lipopolysaccharide (LPS) to specifically inspire the GSDMD level, necroptosis was obvious ameliorated. In addition, overexpression of IRE-1α in LO-2 cells obviously increased pyroptotic markers, such as GSDMD and NLRP3, but downregulated the necroptosis markers p-MLKL and p-RIPK3. In conclusion, enhanced expression of IRE-1α triggers hepatic pyroptosis, while IRE-1α deficiency activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD in mice. So, IRE-1α may be a promising therapeutic target in tissue injuries.
