bims-ershed Biomed News
on ER Stress in Health and Diseases
Issue of 2021‒05‒23
twenty-two papers selected by
Matías Eduardo González Quiroz
Worker’s Hospital
  1. An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation.
  2. Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.
  3. Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.
  4. TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis.
  5. Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.
  6. Retrograde signaling mediates an adaptive survival response to endoplasmic reticulum stress.
  7. The Intricacies of NRF2 Regulation in Cancer.
  8. An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.
  9. Links between the unfolded protein response and the DNA damage response in hypoxia: a systematic review.
  10. Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells.
  11. In vivo assay and modelling of protein and mitochondrial turnover during aging.
  12. A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.
  13. PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.
  14. Inhibition of DNA-PK with AZD7648 sensitizes tumor cells to radiotherapy and induces type I interferon-dependent durable tumor control.
  15. Global in situ profiling of RNA-RNA spatial interactions with RIC-seq.
  16. USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response.
  17. PKD2 deficiency suppresses amino acid biosynthesis in ADPKD by impairing the PERK-TBL2-eIF2ɑ-ATF4 pathway.
  18. Hawaii's surprise volcanic eruption: Lessons from Kilauea 2018.
  19. Mitochondrial DNA instability in mammalian cells.
  20. Nucleic Acid-Based Therapeutics in Orphan Neurological Disorders: Recent Developments.
  21. ER membrane protein complex 1 interacts with STIM1 and regulates store-operated Ca2+ entry.
  22. The state of neuro-oncology during the COVID-19 pandemic: a worldwide assessment.
  1. J Biol Chem. 2021 May 14. pii: S0021-9258(21)00574-3. [Epub ahead of print] 100781
    An interdomain helix in IRE1α mediates the conformational change required for the sensor's activation.
    Daniela Ricci, Stephen Tutton, Ilaria Marrocco, Mingjie Ying, Daniel Blumenthal, Daniela Eletto, Jade Vargas, Sarah Boyle, Hossein Fazelinia, Lei Qian, Krishna Suresh, Deanne Taylor, James C Paton, Adrienne W Paton, Chih-Hang Anthony Tang, Chih-Chi Andrew Hu, Ravi Radhakrishnan, Tali Gidalevitz, Yair Argon.
      The unfolded protein response (UPR) plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1α is a major transducer that conveys endoplasmic reticulum (ER) stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its ER luminal domain, IRE1α dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1α, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix αK, located between the kinase and RNase domains of IRE1α, in conveying this critical conformational change. Using constructs containing mutations within this inter-domain helix, we show that distinct substitutions affect oligomerization, kinase activity and the RNase activity of IRE1α differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Importantly, an RNase-inactive mutant, L827P, can still dimerize with wild type monomers, but this mutation inactivates the wild type molecule and renders leukemic cells more susceptible to stress. We surmise that helix αK is a conduit for the activation of IRE1α in response to stress.
    Keywords:  IRE1 oligomerization; Kinase RNase interdomain helix; RNase activity; conformational change; differential autophosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2021.100781
  2. J Cell Sci. 2020 Jan 01. pii: jcs.244855. [Epub ahead of print]
    Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.
    Andria A Lytridou, Anthi Demetriadou, Melina Christou, Louiza Potamiti, Nikolas P Mastroyiannopoulos, Kyriacos Kyriacou, Leonidas A Phylactou, Anthi Drousiotou, Petros P Petrou.
      Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionary conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing, ER structures. We demonstrate that, the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process which does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.
    Keywords:  Apoptosis; ER stress; Glycogen; Glycogen synthase; Glycogenin; UPR
    DOI:  https://doi.org/10.1242/jcs.244855
  3. J Biol Chem. 2021 May 14. pii: S0021-9258(21)00572-X. [Epub ahead of print] 100779
    Calcium as a reliable marker for the quantitative assessment of endoplasmic reticulum stress in live cells.
    Paul F Lebeau, Khrystyna Platko, Jae Hyun Byun, Richard C Austin.
      Calcium (Ca2+) is an essential mineral of endoplasmic reticulum (ER) luminal biochemistry due to the Ca2+-dependence of ER-resident chaperones charged with folding de novo proteins that transit this cellular compartment. ER Ca2+ depletion reduces the ability of chaperones to properly fold the proteins entering the ER, thus leading to an accumulation of misfolded proteins and the onset of a state known as ER stress. However, not all conditions that cause ER stress do so in a manner dependent on ER Ca2+ depletion. Agents like tunicamycin (TM) inhibit the glycosylation of de novo polypeptides, a key step in the maturation process of newly synthesized proteins. Despite this established effect of TM, our understanding of how such conditions modulate ER Ca2+ levels is still limited. In the present study, we report that a variety of ER stress-inducing agents that have not been known to directly alter ER Ca2+ homeostasis can also cause a marked reduction in ER Ca2+ levels. Consistent with these observations, protecting against ER stress using small chemical chaperones, such as 4PBA and TUDCA, also attenuated ER Ca2+ depletion caused by these agents. We also describe a novel high-throughput and low-cost assay for the rapid quantification of ER stress using ER Ca2+ levels as a surrogate marker. This report builds on our understanding of ER Ca2+ levels in the context of ER stress, and also provides the scientific community with a new, reliable tool to study this important cellular process in vitro.
    Keywords:  Calcium; Mag-Fluo-4; endoplasmic reticulum stress (ER stress); unfolded protein response (UPR)
    DOI:  https://doi.org/10.1016/j.jbc.2021.100779
  4. Cell Metab. 2021 May 17. pii: S1550-4131(21)00223-0. [Epub ahead of print]
    TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis.
    Dong Huang, Bolin Xu, Lu Liu, Lingzhi Wu, Yuangang Zhu, Alireza Ghanbarpour, Yawei Wang, Feng-Jung Chen, Jia Lyu, Yating Hu, Yunlu Kang, Wenjing Zhou, Xiao Wang, Wanqiu Ding, Xin Li, Zhaodi Jiang, Jizheng Chen, Xu Zhang, Hongwen Zhou, John Zhong Li, Chunguang Guo, Wen Zheng, Xiuqin Zhang, Peng Li, Thomas Melia, Karin Reinisch, Xiao-Wei Chen.
      How amphipathic phospholipids are shuttled between the membrane bilayer remains an essential but elusive process, particularly at the endoplasmic reticulum (ER). One prominent phospholipid shuttling process concerns the biogenesis of APOB-containing lipoproteins within the ER lumen, which may require bulk trans-bilayer movement of phospholipids from the cytoplasmic leaflet of the ER bilayer. Here, we show that TMEM41B, present in the lipoprotein export machinery, encodes a previously conceptualized ER lipid scramblase mediating trans-bilayer shuttling of bulk phospholipids. Loss of hepatic TMEM41B eliminates plasma lipids, due to complete absence of mature lipoproteins within the ER, but paradoxically also activates lipid production. Mechanistically, scramblase deficiency triggers unique ER morphological changes and unsuppressed activation of SREBPs, which potently promotes lipid synthesis despite stalled secretion. Together, this response induces full-blown nonalcoholic hepatosteatosis in the TMEM41B-deficient mice within weeks. Collectively, our data uncovered a fundamental mechanism safe-guarding ER function and integrity, dysfunction of which disrupts lipid homeostasis.
    Keywords:  SREBP; endoplasmic reticulum; fatty liver disease; lipid scramblase; lipoprotein metabolism
    DOI:  https://doi.org/10.1016/j.cmet.2021.05.006
  5. J Vis Exp. 2021 Apr 28.
    Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging.
    Julia O'Sullivan, Sofiane Y Mersaoui, Guy Poirier, Jean-Yves Masson.
      The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.
    DOI:  https://doi.org/10.3791/62553
  6. J Cell Sci. 2020 Jan 01. pii: jcs.241539. [Epub ahead of print]
    Retrograde signaling mediates an adaptive survival response to endoplasmic reticulum stress.
    Imadeddin Hijazi, Jeffrey Knupp, Amy Chang.
      One major cause of endoplasmic reticulum (ER) stress is homeostatic imbalance between biosynthetic protein folding and protein folding capacity. Cells utilize mechanisms such as the unfolded protein response (UPR) to cope with ER stress. Nevertheless, when ER stress is prolonged or severe, cell death may occur, accompanied by production of mitochondrial reactive oxygen species (ROS). Using a yeast model, we describe an innate, adaptive response to ER stress to increase select mitochondrial proteins, O2 consumption, and cell survival. The mitochondrial response allows cells to resist additional ER stress. ER stress-induced mitochondrial response is mediated by activation of retrograde (RTG) signaling to enhance anapleurotic reactions of the TCA cycle. Mitochondrial response to ER stress is accompanied by inactivation of the conserved TORC1 pathway, and activation of Snf1/AMPK, the conserved energy sensor and regulator of metabolism. Our results provide new insight into the role of respiration in cell survival in the face of ER stress, and should help in developing therapeutic strategies to limit cell death in disorders linked to ER stress.
    Keywords:  ER stress; Endoplasmic reticulum; Mitochondria; Yeast
    DOI:  https://doi.org/10.1242/jcs.241539
  7. Semin Cancer Biol. 2021 May 18. pii: S1044-579X(21)00146-2. [Epub ahead of print]
    The Intricacies of NRF2 Regulation in Cancer.
    Cody J Schmidlin, Aryatara Shakya, Matthew Dodson, Eli Chapman, Donna D Zhang.
      The complex role of NRF2 in the context of cancer continues to evolve. As a transcription factor, NRF2 regulates various genes involved in redox homeostasis, protein degradation, DNA repair, and xenobiotic metabolism. As such, NRF2 is critical in preserving cell function and viability, particularly during stress. Importantly, NRF2 itself is regulated via a variety of mechanisms, and the mode of NRF2 activation often dictates the duration of NRF2 signaling and its role in either preventing cancer initiation or promoting cancer progression. Herein, different modes of NRF2 regulation, including oxidative stress, autophagy dysfunction, protein-protein interactions, and epigenetics, as well as pharmacological modulators targeting this cascade in cancer, are explored. Specifically, how the timing and duration of these different mechanisms of NRF2 induction affect tumor initiation, progression, and metastasis are discussed. Additionally, progress in the discovery and development of NRF2 inhibitors for the treatment of NRF2-addicted cancers is highlighted, including modulators that inhibit specific NRF2 downstream targets. Overall, a better understanding of the intricate nature of NRF2 regulation in specific cancer contexts should facilitate the generation of novel therapeutics designed to not only prevent tumor initiation, but also halt progression and ultimately improve patient wellbeing and survival.
    Keywords:  KEAP1; NRF2; carcinogenesis; chemoprevention; chemoresistance
    DOI:  https://doi.org/10.1016/j.semcancer.2021.05.016
  8. STAR Protoc. 2021 Jun 18. 2(2): 100504
    An optimized two-step chromatin immunoprecipitation protocol to quantify the associations of two separate proteins and their common target DNA.
    Lingli He, Wentao Yu, Wenxiang Zhang, Lei Zhang.
      Sequential chromatin immunoprecipitation (ChIP) is commonly used to investigate DNA-protein and protein-protein interactions to a specific genomic region. However, it can be tricky to achieve a robust and reproducible signal with sequential ChIP. Here, we provide an optimized two-step ChIP protocol to quantify the in vivo associates of multiple proteins with the same DNA regulatory element. For complete details on the use and execution of this protocol, please refer to He et al. (2020).
    Keywords:  Chromatin immunoprecipitation (ChIP); Molecular Biology
    DOI:  https://doi.org/10.1016/j.xpro.2021.100504
  9. Biochem Soc Trans. 2021 May 18. pii: BST20200861. [Epub ahead of print]
    Links between the unfolded protein response and the DNA damage response in hypoxia: a systematic review.
    Hannah Bolland, Tiffany S Ma, Syafiq Ramlee, Kristijan Ramadan, Ester M Hammond.
      Hypoxia is a feature of most solid tumours and predicts for poor prognosis. In radiobiological hypoxia (<0.1% O2) cells become up to three times more resistant to radiation. The biological response to radiobiological hypoxia is one of few physiologically relevant stresses that activates both the unfolded protein and DNA damage responses (UPR and DDR). Links between these pathways have been identified in studies carried out in normoxia. Based in part on these previous studies and recent work from our laboratory, we hypothesised that the biological response to hypoxia likely includes overlap between the DDR and UPR. While inhibition of the DDR is a recognised strategy for improving radiation response, the possibility of achieving this through targeting the UPR has not been realised. We carried out a systematic review to identify links between the DDR and UPR, in human cell lines exposed to <2% O2. Following PRISMA guidance, literature from January 2010 to October 2020 were retrieved via Ovid MEDLINE and evaluated. A total of 202 studies were included. LAMP3, ULK1, TRIB3, CHOP, NOXA, NORAD, SIAH1/2, DYRK2, HIPK2, CREB, NUPR1, JMJD2B, NRF2, GSK-3B, GADD45a, GADD45b, STAU1, C-SRC, HK2, CAV1, CypB, CLU, IGFBP-3 and SP1 were highlighted as potential links between the hypoxic DDR and UPR. Overall, we identified very few studies which demonstrate a molecular link between the DDR and UPR in hypoxia, however, it is clear that many of the molecules highlighted warrant further investigation under radiobiological hypoxia as these may include novel therapeutic targets to improve radiotherapy response.
    Keywords:  DDR; ER stress; UPR; hypoxia; radiation; replication stress
    DOI:  https://doi.org/10.1042/BST20200861
  10. J Cell Sci. 2020 Jan 01. pii: jcs.243709. [Epub ahead of print]
    Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells.
    R San Gil, D Cox, L McAlary, T Berg, A K Walker, J J Yerbury, L Ooi, H Ecroyd.
      Protein aggregates that result in inclusions formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyQ-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR may contribute to the development of inclusion pathology in neurodegenerative diseases.
    Keywords:  HSF1; Heat shock response; Inclusions; Neurodegenerative disorders; Protein aggregation
    DOI:  https://doi.org/10.1242/jcs.243709
  11. Fly (Austin). 2021 Dec;15(1): 60-72
    In vivo assay and modelling of protein and mitochondrial turnover during aging.
    Hans S Bell, John Tower.
      To maintain homoeostasis, cells must degrade damaged or misfolded proteins and synthesize functional replacements. Maintaining a balance between these processes, known as protein turnover, is necessary for stress response and cellular adaptation to a changing environment. Damaged mitochondria must also be removed and replaced. Changes in protein and mitochondrial turnover are associated with aging and neurodegenerative disease, making it important to understand how these processes occur and are regulated in cells. To achieve this, reliable assays of turnover must be developed. Several methods exist, including pulse-labelling with radioactive or stable isotopes and strategies making use of fluorescent proteins, each with their own advantages and limitations. Both cell culture and live animals have been used for these studies, in systems ranging from yeast to mammals. In vivo assays are especially useful for connecting turnover to aging and disease. With its short life cycle, suitability for fluorescent imaging, and availability of genetic tools, Drosophila melanogaster is particularly well suited for this kind of analysis.
    Keywords:  Drosophila; aging; fluorescence microscopy; isotope labelling; mitophagy; protein turnover; video tracking
    DOI:  https://doi.org/10.1080/19336934.2021.1911286
  12. J Cell Sci. 2020 Jan 01. pii: jcs.239236. [Epub ahead of print]
    A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.
    Nadine Sen Nkwe, Salima Daou, Maxime Uriarte, Jessica Gagnon, Nicholas Victor Iannantuono, Haithem Barbour, Helen Yu, Louis Masclef, Erlinda Fernández, Natalia Zamorano Cuervo, Nazar Mashtalir, Loïc Binan, Mikhail Sergeev, François Bélanger, Elliot Drobetsky, Eric Milot, Hugo Wurtele, Santiago Costantino, El Bachir Affar.
      USP16/UBP-M has emerged as a histone H2AK119 deubiquitinase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also observed in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.
    Keywords:  Cell proliferation; DNA double stand break repair; Deubiquitinase; H2AK119; Mitosis; Nuclear export; UBP-M; USP16; Ubiquitin
    DOI:  https://doi.org/10.1242/jcs.239236
  13. Cancer Sci. 2021 May 03.
    PP1 regulatory subunit NIPP1 regulates transcription of E2F1 target genes following DNA damage.
    Shunsuke Hanaki, Makoto Habara, Takahiro Masaki, Keisuke Maeda, Yuki Sato, Makoto Nakanishi, Midori Shimada.
      DNA damage induces transcriptional repression of E2F1 target genes and a reduction in histone H3-Thr11 phosphorylation (H3-pThr11 ) at E2F1 target gene promoters. Dephosphorylation of H3-pThr11 is partly mediated by Chk1 kinase and protein phosphatase 1γ (PP1γ) phosphatase. Here, we isolated NIPP1 as a regulator of PP1γ-mediated H3-pThr11 by surveying nearly 200 PP1 interactor proteins. We found that NIPP1 inhibits PP1γ-mediated dephosphorylation of H3-pThr11 both in vivo and in vitro. By generating NIPP1-depleted cells, we showed that NIPP1 is required for cell proliferation and the expression of E2F1 target genes. Upon DNA damage, activated protein kinase A (PKA) phosphorylated the NIPP1-Ser199 residue, adjacent to the PP1 binding motif (RVxF), and triggered the dissociation of NIPP1 from PP1γ, leading to the activation of PP1γ. Furthermore, the inhibition of PKA activity led to the activation of E2F target genes. Statistical analysis confirmed that the expression of NIPP1 was positively correlated with E2F target genes. Taken together, these findings demonstrate that the PP1 regulatory subunit NIPP1 modulates E2F1 target genes by linking PKA and PP1γ during DNA damage.
    Keywords:  DNA damage; E2F1; histone phosphorylation; protein phosphatase; transcription
    DOI:  https://doi.org/10.1111/cas.14924
  14. Clin Cancer Res. 2021 May 19. pii: clincanres.CCR-20-3701-A.2020. [Epub ahead of print]
    Inhibition of DNA-PK with AZD7648 sensitizes tumor cells to radiotherapy and induces type I interferon-dependent durable tumor control.
    Kyoko Nakamura, Ankur Karmokar, Paul M Farrington, Neil H James, Antonio Ramos-Montoya, Susan J Bickerton, Gareth D Hughes, Timothy M Illidge, Elaine B Cadogan, Barry R Davies, Simon J Dovedi, Viia Valge-Archer.
      PURPOSE: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA dependent protein kinase (DNA-PK) plays an important role in DNA double strand break repair via the non-homologous end joining (NHEJ) pathway. We hypothesised that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anti-cancer immune response.EXPERIMENTAL DESIGN: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26 and B16-F10 models. Immunological consequences were analysed by gene expression and flow-cytometric analysis.
    RESULTS: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The anti-tumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T cell PD-1 expression, increased NK cell granzyme B expression, and elevated type I interferon (IFN) signaling in mice treated with the combination when compared to RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunological memory capable of supressing tumor growth following rechallenge.
    CONCLUSIONS: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-3701
  15. Nat Protoc. 2021 May 21.
    Global in situ profiling of RNA-RNA spatial interactions with RIC-seq.
    Changchang Cao, Zhaokui Cai, Rong Ye, Ruibao Su, Naijing Hu, Hailian Zhao, Yuanchao Xue.
      Emerging evidence has demonstrated that RNA-RNA interactions are vital in controlling diverse biological processes, including transcription, RNA splicing and protein translation. RNA in situ conformation sequencing (RIC-seq) is a technique for capturing protein-mediated RNA-RNA proximal interactions globally in living cells at single-base resolution. Cells are first treated with formaldehyde to fix all the protein-mediated RNA-RNA interactions in situ. After cell permeabilization and micrococcal nuclease digestion, the proximally interacting RNAs are 3' end-labeled with pCp-biotin and subsequently ligated using T4 RNA ligase. The chimeric RNAs are then enriched and converted into libraries for paired-end sequencing. After deep sequencing, computational analysis yields interaction strength scores for every base on proximally interacting RNAs in the starting populations. The whole experimental procedure is designed to be completed within 6 d, followed by an additional 8 d for computational analysis. RIC-seq technology can unbiasedly detect intra- and intermolecular RNA-RNA interactions, thereby rendering it useful for reconstructing RNA higher-order structures and identifying direct noncoding RNA targets.
    DOI:  https://doi.org/10.1038/s41596-021-00524-2
  16. Oncogene. 2021 May 18.
    USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response.
    Evangelos Prokakis, Anna Dyas, Regina Grün, Sonja Fritzsche, Upasana Bedi, Zahra B Kazerouni, Robyn L Kosinsky, Steven A Johnsen, Florian Wegwitz.
      The Ubiquitin-Specific Protease 22 (USP22) is a deubiquitinating subunit of the mammalian SAGA transcriptional co-activating complex. USP22 was identified as a member of the so-called "death-from-cancer" signature predicting therapy failure in cancer patients. However, the importance and functional role of USP22 in different types and subtypes of cancer remain largely unknown. In the present study, we leveraged human cell lines and genetic mouse models to investigate the role of USP22 in HER2-driven breast cancer (HER2+-BC) and demonstrate for the first time that USP22 is required for the tumorigenic properties in murine and human HER2+-BC models. To get insight into the underlying mechanisms, we performed transcriptome-wide gene expression analyses and identified the Unfolded Protein Response (UPR) as a pathway deregulated upon USP22 loss. The UPR is normally induced upon extrinsic or intrinsic stresses that can promote cell survival and recovery if shortly activated or programmed cell death if activated for an extended period. Strikingly, we found that USP22 actively suppresses UPR induction in HER2+-BC cells by stabilizing the major endoplasmic reticulum (ER) chaperone HSPA5. Consistently, loss of USP22 renders tumor cells more sensitive to apoptosis and significantly increases the efficiency of therapies targeting the ER folding capacity. Together, our data suggest that therapeutic strategies targeting USP22 activity may sensitize tumor cells to UPR induction and could provide a novel, effective approach to treat HER2+-BC.
    DOI:  https://doi.org/10.1038/s41388-021-01814-5
  17. Biochem Biophys Res Commun. 2021 May 17. pii: S0006-291X(21)00782-8. [Epub ahead of print]561 73-79
    PKD2 deficiency suppresses amino acid biosynthesis in ADPKD by impairing the PERK-TBL2-eIF2ɑ-ATF4 pathway.
    Xingquan Zhou, Hui Xiong, Yi Lu, Meijuan Geng, Yumei Lu, Lirong Zhang, Xu Zhu.
      Metabolic reprogramming is emerging as a key pathological contributor to the progression of autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanisms underlying dysregulated cellular metabolism remain elusive. Here we report that amino acid biosynthesis is reprogrammed in Pkd2-knockout mouse kidneys via a defective PERK-eIF2ɑ-ATF4 pathway. Transcriptomic analysis revealed that the amino acid biosynthesis pathways such as serine, arginine and cysteine were impaired, and associated critical enzymes were downregulated in Pkd2-knockout mouse kidneys. ATF4 and CHOP, transcription factors downstream of the endoplasmic reticulum (ER) stress sensor PERK, were identified as master regulators of these enzymes' expression. PKD2 deficiency impaired the expression of ATF4 and amino acid synthesis enzymes in RCTEC cells under ER stress. Mechanistically, as an ER-resident protein, PKD2 interacts with TBL2, which functions as an adaptor bridging eIF2ɑ to PERK. PKD2 depletion impaired the recruitment of eIF2ɑ to TBL2, thus impeding activation of the PERK-eIF2ɑ-ATF4 pathway and downstream amino acid biosynthesis. These findings illuminate a molecular mechanism linking the PKD2-mediated PERK-eIF2ɑ-ATF4 pathway and amino acid metabolic reprogramming in ADPKD.
    Keywords:  ADPKD; ATF4; Amino acid biosynthesis; PKD2; TBL2
    DOI:  https://doi.org/10.1016/j.bbrc.2021.05.012
  18. Nature. 2021 May 19.
    Hawaii's surprise volcanic eruption: Lessons from Kilauea 2018.
    Charlotte Stoddart.
      
    Keywords:  Volcanology
    DOI:  https://doi.org/10.1038/d41586-021-01363-7
  19. Antioxid Redox Signal. 2021 May 20.
    Mitochondrial DNA instability in mammalian cells.
    Gustavo Carvalho, Bruno Marçal Repolês, Isabela Mendes, Paulina H Wanrooij.
      SIGNIFICANCE: The small, multicopy mitochondrial genome (mtDNA) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress and an insufficient nucleotide supply. Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides and strand breaks. We also discuss the newly-discovered link between mtDNA instability and activation of the innate immune response.CRITICAL ISSUES: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how?
    FUTURE DIRECTIONS: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease.
    DOI:  https://doi.org/10.1089/ars.2021.0091
  20. Front Mol Biosci. 2021 ;8 643681
    Nucleic Acid-Based Therapeutics in Orphan Neurological Disorders: Recent Developments.
    Olga Khorkova, Jane Hsiao, Claes Wahlestedt.
      The possibility of rational design and the resulting faster and more cost-efficient development cycles of nucleic acid-based therapeutics (NBTs), such as antisense oligonucleotides, siRNAs, and gene therapy vectors, have fueled increased activity in developing therapies for orphan diseases. Despite the difficulty of delivering NBTs beyond the blood-brain barrier, neurological diseases are significantly represented among the first targets for NBTs. As orphan disease NBTs are now entering the clinical stage, substantial efforts are required to develop the scientific background and infrastructure for NBT design and mechanistic studies, genetic testing, understanding natural history of orphan disorders, data sharing, NBT manufacturing, and regulatory support. The outcomes of these efforts will also benefit patients with "common" diseases by improving diagnostics, developing the widely applicable NBT technology platforms, and promoting deeper understanding of biological mechanisms that underlie disease pathogenesis. Furthermore, with successes in genetic research, a growing proportion of "common" disease cases can now be attributed to mutations in particular genes, essentially extending the orphan disease field. Together, the developments occurring in orphan diseases are building the foundation for the future of personalized medicine. In this review, we will focus on recent achievements in developing therapies for orphan neurological disorders.
    Keywords:  antisense oligonucleotide; gene therapy; neurological disorder; noncoding RNA; orphan disorder; siRNA
    DOI:  https://doi.org/10.3389/fmolb.2021.643681
  21. J Biochem. 2021 May 20. pii: mvab063. [Epub ahead of print]
    ER membrane protein complex 1 interacts with STIM1 and regulates store-operated Ca2+ entry.
    Kazuhiko Kawata, Akemi Baba, Masayuki Shiota, Hideki Wanibuchi, Yoshihiro Baba.
      Store-operated calcium entry (SOCE) is the process by which the emptying of endoplasmic reticulum (ER) Ca2+ stores causes an influx of Ca2+ across the plasma membrane. It is the major Ca2+ influx pathway in non-excitable cells and has a wide array of physiological functions. Upon store depletion, stromal interaction molecule 1 (STIM1), an ER calcium sensor relocates into discrete puncta at the ER-plasma membrane junction region, which results in the coupling of Ca2+ channels to initiate SOCE. However, the mechanism regulating STIM1 activity remains poorly understood. Here, we performed affinity purification of STIM1 and uncovered ER membrane protein complex 1 (EMC1) as a STIM1 binding partner. We showed that this interaction occurred in the ER through the intraluminal region of STIM1. After store depletion, EMC1 does not cluster adjacent to the plasma membrane, which suggests that it is distributed differently from STIM1. EMC1 knockdown with small interfering RNA resulted in a marked decrease in SOCE. Thus, these findings suggest that EMC1 functions as a positive regulator of SOCE.
    Keywords:  Calcium; Endoplasmic Reticulum; Signal Transduction; Store-operated calcium entry
    DOI:  https://doi.org/10.1093/jb/mvab063
  22. Neurooncol Adv. 2021 Jan-Dec;3(1):3(1): vdab035
    The state of neuro-oncology during the COVID-19 pandemic: a worldwide assessment.
    Maciej M Mrugala, Quinn T Ostrom, Shelley M Pressley, Jennie W Taylor, Alissa A Thomas, Jeffrey S Wefel, Scott L Coven, Alvina A Acquaye, Chas Haynes, Sameer Agnihotri, Michael Lim, Katherine B Peters, Erik P Sulman, Joanne T Salcido, Nicholas A Butowski, Shawn Hervey-Jumper, Alireza Mansouri, Kathy R Oliver, Alyx B Porter, Farshad Nassiri, David Schiff, Erin M Dunbar, Monika E Hegi, Terri S Armstrong, Martin J van den Bent, Susan M Chang, Gelareh Zadeh, Milan G Chheda.
      Background: It remains unknown how the COVID-19 pandemic has changed neuro-oncology clinical practice, training, and research efforts.Methods: We performed an international survey of practitioners, scientists, and trainees from 21 neuro-oncology organizations across 6 continents, April 24-May 17, 2020. We assessed clinical practice and research environments, institutional preparedness and support, and perceived impact on patients.
    Results: Of 582 respondents, 258 (45%) were US-based and 314 (55%) international. Ninety-four percent of participants reported changes in their clinical practice. Ninety-five percent of respondents converted at least some practice to telemedicine. Ten percent of practitioners felt the need to see patients in person, specifically because of billing concerns and pressure from their institutions. Sixty-seven percent of practitioners suspended enrollment for at least one clinical trial, including 62% suspending phase III trial enrollments. More than 50% believed neuro-oncology patients were at increased risk for COVID-19. Seventy-one percent of clinicians feared for their own personal safety or that of their families, specifically because of their clinical duties; 20% had inadequate personal protective equipment. While 69% reported increased stress, 44% received no psychosocial support from their institutions. Thirty-seven percent had salary reductions and 63% of researchers temporarily closed their laboratories. However, the pandemic created positive changes in perceived patient satisfaction, communication quality, and technology use to deliver care and mediate interactions with other practitioners.
    Conclusions: The pandemic has changed treatment schedules and limited investigational treatment options. Institutional lack of support created clinician and researcher anxiety. Communication with patients was satisfactory. We make recommendations to guide clinical and scientific infrastructure moving forward and address the personal challenges of providers and researchers.
    Keywords:  COVID-19; clinical trial enrollment; neuro-oncology outcomes
    DOI:  https://doi.org/10.1093/noajnl/vdab035
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