bims-ershed 2022-02-27 papers

Life Sci Alliance. 2022 May;pii: e202201379. [Epub ahead of print]5(5):

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

Alexandra Papaioannou, Federica Centonze, Alice Metais, Marion Maurel, Luc Negroni, Matías Gonzalez-Quiroz, Sayyed Jalil Mahdizadeh, Gabriella Svensson, Ensieh Zare, Alice Blondel, Albert C Koong, Claudio Hetz, Rémy Pedeux, Michel L Tremblay, Leif A Eriksson, Eric Chevet.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

DOI: https://doi.org/10.26508/lsa.202201379

EMBO Mol Med. 2022 Feb 22. e15344

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Zehra Yildirim, Sabyasachi Baboo, Syed M Hamid, Asli E Dogan, Ozlem Tufanli, Sabrina Robichaud, Christina Emerton, Jolene K Diedrich, Hasan Vatandaslar, Fotis Nikolos, Yanghong Gu, Takao Iwawaki, Elizabeth Tarling, Mireille Ouimet, David L Nelson, John R Yates, Peter Walter, Ebru Erbay.

Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Keywords: ER stress; atherosclerosis; cholesterol homeostasis; efferocytosis; translational regulation

DOI: https://doi.org/10.15252/emmm.202115344

Int J Mol Sci. 2022 Feb 10. pii: 1974. [Epub ahead of print]23(4):

Caffeine and MDMA (Ecstasy) Exacerbate ER Stress Triggered by Hyperthermia.

Kathleen A Trychta, Brandon K Harvey.

Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion, which causes ER exodosis, the secretion of ER-resident proteins. In rodent models, club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. In human studies, MDMA stimulated an acute, dose-dependent increase in core body temperature, but an examination of caffeine and MDMA in combination remains a topic for clinical research. Here we examine the secretion of ER-resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER-resident proteins, and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter the canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased BiP/GRP78 mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis, contributing to cellular toxicity.

Keywords: ER exodosis; KDEL receptor; MDMA; caffeine; hyperthermia

DOI: https://doi.org/10.3390/ijms23041974

Discov Oncol. 2021 Nov 30. 12(1): 57

IRE1α-XBP1 but not PERK inhibition exerts anti-tumor activity in osteosarcoma.

Keita Sasa, Tsuyoshi Saito, Taisei Kurihara, Nobuhiko Hasegawa, Kei Sano, Daisuke Kubota, Keisuke Akaike, Taketo Okubo, Takuo Hayashi, Tatsuya Takagi, Takashi Yao, Muneaki Ishijima, Yoshiyuki Suehara.

Osteosarcoma (OS) is the most common primary malignant bone tumor. However, the therapeutic results of the advanced cases at the first visit were still extremely poor. Therefore, more effective therapeutic options based on molecular profiling of OS are needed. In this study, we investigated the functions of endoplasmic reticulum (ER) stress activities in OS and elucidated whether ER stress inhibitors could exert antitumor effects. The expression of 84 key genes associated with unfolded protein response (UPR) was assessed in four OS cells (143B, MG63, U2OS and KHOS) by RT2 Profiler PCR Arrays. Based on results, we performed both siRNA and inhibitor assays focusing on IRE1α-XBP1 and PERK pathways. All OS cell lines showed resistance to PERK inhibitors. Furthermore, ATF4 and EIF2A inhibition by siRNA did not affect the survival of OS cell lines. On the other hand, IRE1α-XBP1 inhibition by toyocamycin suppressed OS cell growth (IC50: < 0.075 μM) and cell viability was suppressed in all OS cell lines by silencing XBP1 expression. The expression of XBP1s and XBP1u in OS cell lines and OS surgical samples were confirmed using qPCR. In MG63 and U2OS, toyocamycin decreased the expression level of XBP1s induced by tunicamycin. On the other hand, in 143B and KHOS, stimulation by toyocamycin did not clearly change the expression level of XBP1s induced by tunicamycin. However, morphological apoptotic changes and caspase activation were observed in these two cell lines. Inhibition of the IRE1α-XBP1s pathway is expected to be a promising new target for OS.

Keywords: ER stress; IRE1α-XBP1 pathway; Osteosarcoma

DOI: https://doi.org/10.1007/s12672-021-00453-2

STAR Protoc. 2022 Mar 18. 3(1): 101169

Protocol for glial Ca2+ imaging in C. elegans following chemical, mechanical, or optogenetic stimulation.

Hankui Cheng, Umar Al-Sheikh, Du Chen, Duo Duan, Lijun Kang.

Caenorhabditis elegans is an exceptionally transparent model to analyze calcium (Ca2+) signals, but available protocols for neuronal Ca2+ imaging may not be suitable for studying glial cells. Here, we present a detailed protocol for glial Ca2+ imaging in C. elegans following three different approaches including chemical, mechanical, and optogenetic stimulation. We also provide the details for imaging analysis using Image-J. For complete details on the use and execution of this protocol, please refer to Duan et al. (2020).

Keywords: Behavior; Microscopy; Model Organisms; Neuroscience

DOI: https://doi.org/10.1016/j.xpro.2022.101169