bims-ershed Biomed News
on ER Stress in Health and Diseases
Issue of 2021‒07‒11
nine papers selected by
Matías Eduardo González Quiroz
Worker’s Hospital

  1. Curr Opin Hematol. 2021 Jul 06.
      PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) possess the ability to regenerate over a lifetime in the face of extreme cellular proliferation and environmental stress. Yet, mechanisms that control the regenerative properties of HSCs remain elusive. ER stress has emerged as an important signaling event that supports HSC self-renewal and multipotency. The purpose of this review is to summarize the pathways implicating ER stress as cytoprotective in HSCs.RECENT FINDINGS: Recent studies have shown multiple signaling cascades of the unfolded protein response (UPR) are persistently activated in healthy HSCs, suggesting that low-dose ER stress is a feature HSCs. Stress adaptation is a feature ascribed to cytoprotection and longevity of cells as well as organisms, in what is known as hormesis. However, assembling this information into useful knowledge to improve the therapeutic application of HSCs remains challenging and the upstream activators and downstream transcriptional programs induced by ER stress that are required in HSCs remain to be discovered.
    SUMMARY: The maintenance of HSCs requires a dose-dependent simulation of ER stress responses that involves persistent, low-dose UPR. Unraveling the complexity of this signaling node may elucidate mechanisms related to regeneration of HSCs that can be harnessed to expand HSCs for cellular therapeutics ex vivo and transplantation in vivo.
  2. Front Cell Dev Biol. 2021 ;9 695041
      Moderate-intensity exercise can help delay the development of osteoarthritis (OA). Previous studies have shown that the purinergic receptor P2X ligand gated ion channel 7 (P2X7) is involved in OA development and progression. To investigate the effect of exercise on P2X7 activation and downstream signaling in OA, we used the anterior cruciate ligament transection (ACLT)-induced OA rat model and primary chondrocyte culture system. Our in vivo experiments confirmed that treadmill exercise increased P2X7 expression and that this effect was more pronounced at the later time points. Furthermore, P2X7 activation induced endoplasmic reticulum (ER) stress and increased the expression levels of ER stress markers, such as 78 kDa glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor 6 (ATF6). At the early time points, IRE1 and PERK were activated, and mTOR was inhibited. At the later time points, mTOR was activated, mediating PERK to promote ER stress-apoptosis, whereas IRE1 and autophagy were inhibited. To confirm our observations in vitro, we treated primary chondrocytes with the P2X7 agonist benzoylbenzoyl-ATP (Bz-ATP). Our results confirmed that P2X7-mediated Ca2+ influx activated IRE1-mediated autophagic flux and induced PERK-mediated ER stress-apoptosis. To further investigate the role of P2X7 in OA, we injected mTOR antagonist rapamycin or P2X7 antagonist A740003 into the knee joints of ACLT rats. Our results demonstrated that mTOR inhibition induced autophagy, decreased apoptosis, and reduced cartilage loss. However, injection of mTOR agonist MHY1485 or Bz-ATP had the opposite effect. In summary, our results indicated that during the early stages of moderate-intensity exercise, P2X7 was activated and autophagic flux was increased, delaying OA development. At the later stages, P2X7 became over-activated, and the number of apoptotic cells increased, promoting OA development. We propose that the IRE1-mTOR-PERK signaling axis was involved in the regulation of autophagy inhibition and the induction of apoptosis. Our findings provide novel insights into the positive and preventative effects of exercise on OA, suggesting that the intensity and duration of exercise play a critical role. We also demonstrated that on a molecular level, P2X7 and its downstream pathways could be potential therapeutic targets for OA.
    Keywords:  P2X7 receptor; autophagy; endoplasmic reticulum stress; inositol-requiring enzyme-1; mammalian target of rapamycin; osteoarthritis; protein kinase R-like endoplasmic reticulum kinase; treadmill exercise
  3. Int J Med Sci. 2021 ;18(13): 2890-2896
      Background: When an imbalance occurs between the demand and capacity for protein folding, unfolded proteins accumulate in the endoplasmic reticulum (ER) lumen and activate the unfolded protein response (UPR). In addition, unfolded proteins are cleared from the ER lumen for ubiquitination and subsequent cytosolic proteasomal degradation, which is termed as the ER-associated degradation (ERAD) pathway. This study focused on changes in the UPR and ERAD pathways induced by the repeated inhalation anesthetic exposure in Caenorhabditis elegans. Methods: Depending on repeated isoflurane exposure, C. elegans was classified into the control or isoflurane group. To evaluate the expression of a specific gene, RNA was extracted from adult worms in each group and real-time polymerase chain reaction was performed. Ubiquitinated protein levels were measured using western blotting, and behavioral changes were evaluated by chemotaxis assay using various mutant strains. Results: Isoflurane upregulated the expression of ire-1 and pek-1 whereas the expression of atf-6 was unaffected. The expression of both sel-1 and sel-11 was decreased by isoflurane exposure, possibly indicating the inhibition of retro-translocation. The expression of cdc-48.1 and cdc-48.2 was decreased and higher ubiquitinated protein levels were observed in the isoflurane group than in the control, suggesting that deubiquitination and degradation of misfolded proteins were interrupted. The chemotaxis indices of ire-1, pek-1, sel-1, and sel-11 mutants decreased significantly compared to N2, and they were not suppressed further even after the repeated isoflurane exposure. Conclusion: Repeated isoflurane exposure caused significant ER stress in C. elegans. Following the increase in UPR, the ERAD pathway was disrupted by repeated isoflurane exposure and ubiquitinated proteins was accumulated subsequently. UPR and ERAD pathways are potential modifiable neuroprotection targets against anesthesia-induced neurotoxicity.
    Keywords:  Caenorhabditis elegans; endoplasmic reticulum-associated degradation pathway; inhalation anesthetics; unfolded protein response
  4. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00688-4. [Epub ahead of print]36(1): 109312
      Efforts to overcome resistance to immune checkpoint blockade therapy have focused on vaccination strategies using neoepitopes, although they cannot be applied on a large scale due to the "private" nature of cancer mutations. Here, we show that infection of tumor cells with Salmonella induces the opening of membrane hemichannels and the extracellular release of proteasome-generated peptides by the exacerbation of endoplasmic reticulum (ER) stress. Peptides released by cancer cells foster an antitumor response in vivo, both in mice bearing B16F10 melanomas and in dogs suffering from osteosarcoma. Mass spectrometry analysis on the supernatant of human melanoma cells revealed 12 peptides capable of priming healthy-donor CD8+ T cells that recognize and kill human melanoma cells in vitro and when xenotransplanted in vivo. Hence, we identified a class of shared tumor antigens that are generated in ER-stressed cells, such as tumor cells, that do not induce tolerance and are not presented by healthy cells.
    Keywords:  ER-stress response; Salmonella; cancer; immunotherapy; tumor antigens; vaccine
  5. Biochem Biophys Res Commun. 2021 Jun 30. pii: S0006-291X(21)01001-9. [Epub ahead of print]569 17-22
      The pathogenesis of Parkinson's disease (PD) remains elusive, but mitochondrial dysfunction is believed to be one crucial step in its pathogenesis. The mitochondrial unfolded protein response (UPRmt) is an important mitochondrial quality control strategy that maintains mitochondrial function in response to disturbances of mitochondrial protein homeostasis. Activation of the UPRmt and the beneficial effect of rescuing mitochondrial proteostasis have been reported in several genetic models of PD. However, the pathogenic relevance of the UPRmt in idiopathic PD is unknown. The present study examined the link between the UPRmt and mitochondrial dysfunction in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells. Treatment with MPP + induced activation of the UPRmt, reflected by an increase in the expression of UPRmt-related chaperones, proteases, and transcription mediators. UPRmt activation that was induced by overexpressing mutant ornithine transcarbamylase significantly reduced the production of mitochondrial reactive oxygen species (ROS) and improved cell survival in SH-SY5Y cells following MPP+ treatment. Moreover, the overexpression of activating transcription factor 5 (mammalian UPRmt transcription factor) conferred protection against MPP+-induced ROS production and against cell death in SH-SY5Y cells. Overall, our results demonstrate the beneficial effect of UPRmt activation in MPP + -treated cells, shedding new light on the mechanism of mitochondrial dysfunction in the pathogenesis of PD.
    Keywords:  ATF5; MPP(+); Parkinson's disease; UPR(mt)
  6. Curr Protoc. 2021 Jul;1(7): e181
      Circular RNAs (circRNAs) are a class of endogenous noncoding RNAs that have been shown to play a role in normal development, homeostasis, and disease, including cancer. CircRNAs are formed through a process called back-splicing, which results in a covalently closed loop with a nonlinear back-spliced junction (BSJ). In general, circRNA BSJs are predicted in RNA sequencing data using one of numerous circRNA detection algorithms. Selected circRNAs are then typically validated using an orthogonal method such as reverse transcription quantitative PCR (RT-qPCR) with circRNA-specific primers. However, linear transcripts originating from endogenous trans-splicing can lead to false-positive signals both in RNA sequencing and in RT-qPCR experiments. Therefore, it is essential to perform the RT-qPCR validation step only after linear RNAs have been degraded using an exonuclease such as ribonuclease R (RNase R). Several RNase R protocols are available for circRNA detection using RNA sequencing or RT-qPCR. These protocols-which vary in enzyme concentration, RNA input amount, incubation times, and cleanup steps-typically lack a detailed validated standard protocol and fail to provide a range of conditions that deliver accurate results. As such, some protocols use RNase R concentrations that are too high, resulting in partial degradation of the target circRNAs. Here, we describe an optimized workflow for circRNA validation, combining RNase R treatment and RT-qPCR. First, we outline the steps for circRNA primer design and qPCR assay validation. Then, we describe RNase R treatment of total RNA and, importantly, a subsequent essential buffer cleanup step. Lastly, we outline the steps to perform the RT-qPCR and discuss the downstream data analyses. © 2021 Wiley Periodicals LLC. Basic Protocol 1: CircRNA primer design and qPCR assay validation Basic Protocol 2: RNase R treatment, cleanup, and RT-qPCR.
    Keywords:  RT-qPCR; circular RNA; noncoding RNA; primer design; ribonuclease R
  7. EJHaem. 2021 May 17.
      The coronavirus disease 2019 (COVID-19) pandemic has already left an indelible mark in human lives. Despite the havoc it created, this pandemic also saw significant advances in the management of an infectious disease wherein worldwide collaborative efforts from health care professionals have been unprecedented. One of the commonest complications recognised early in the pandemic is the development of coagulopathy. In this review, the lessons learnt from COVID-19 coagulopathy are summarised with some perspectives on future clinical and research strategies. These include how local versus systemic coagulopathy can matter, how we can put D-dimers to effective use, exhort more input into identifying a simple platelet activation marker, rethink the role of fibrinogen, look differently at lupus anticoagulant and heparin-induced thrombocytopenia, bring back disseminated intravascular coagulation into our differential diagnosis slate and most importantly channel more funding into haemostasis and thrombosis research.
    Keywords:  COVID‐19; D‐dimer; coagulopathy; fibrinogen; platelets; thrombocytopenia
  8. Chem Sci. 2021 May 20. 12(24): 8468-8476
      Co-aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual-color fluorogenic thermal shift assay to simultaneously report on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.