bims-unfpre 2019-03-24 papers

bims-unfpre

Biomed News

on Unfolded protein response

Issue of 2019–03–24
sixteen papers selected by
Susan Logue, Apoptosis Research Centre

STING-mediated disruption of calcium homeostasis chronically activates ER stress and primes T cell death.
ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression.
The PKR-like endoplasmic reticulum kinase promotes the dissemination of Myc-induced leukemic cells.
Targeting off-target effects: endoplasmic reticulum stress and autophagy as effective strategies to enhance temozolomide treatment.
The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii.
Molecular Insights Into the Relationship Between Autoimmune Thyroid Diseases and Breast Cancer: A Critical Perspective on Autoimmunity and ER Stress.
Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.
Preservation of circadian rhythms by the protein folding chaperone, BiP.
Dynamic analysis of proteomic alterations in response to N-linked glycosylation inhibition in a drug-resistant ovarian carcinoma cell line.
Cadmium induces cell centrosome amplification via reactive oxygen species as well as endoplasmic reticulum stress pathway.
Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes.
GRASP55 and UPR Control Interleukin-1β Aggregation and Secretion.
Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis.
Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells.
Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response.
Carbon monoxide induces the assembly of stress granule through the integrated stress response.

J Exp Med. 2019 Mar 18. pii: jem.20182192. [Epub ahead of print]

STING-mediated disruption of calcium homeostasis chronically activates ER stress and primes T cell death.

Jianjun Wu, Yu-Ju Chen, Nicole Dobbs, Tomomi Sakai, Jen Liou, Jonathan J Miner, Nan Yan.

STING gain-of-function mutations cause lung disease and T cell cytopenia through unknown mechanisms. Here, we found that these mutants induce chronic activation of ER stress and unfolded protein response (UPR), leading to T cell death by apoptosis in the StingN153S/+ mouse and in human T cells. Mechanistically, STING-N154S disrupts calcium homeostasis in T cells, thus intrinsically primes T cells to become hyperresponsive to T cell receptor signaling-induced ER stress and the UPR, leading to cell death. This intrinsic priming effect is mediated through a novel region of STING that we name "the UPR motif," which is distinct from known domains required for type I IFN signaling. Pharmacological inhibition of ER stress prevented StingN153S/+ T cell death in vivo. By crossing StingN153S/+ to the OT-1 mouse, we fully restored CD8+ T cells and drastically ameliorated STING-associated lung disease. Together, our data uncover a critical IFN-independent function of STING that regulates calcium homeostasis, ER stress, and T cell survival.

DOI: https://doi.org/10.1084/jem.20182192
Nat Commun. 2019 Mar 20. 10(1): 1280

ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression.

Yu Cao, Jimena Trillo-Tinoco, Rosa A Sierra, Carmen Anadon, Wenjie Dai, Eslam Mohamed, Ling Cen, Tara L Costich, Anthony Magliocco, Douglas Marchion, Richard Klar, Sven Michel, Frank Jaschinski, Richard R Reich, Shikhar Mehrotra, Juan R Cubillos-Ruiz, David H Munn, Jose R Conejo-Garcia, Paulo C Rodriguez.

Understanding the intrinsic mediators that render CD8+ T cells dysfunctional in the tumor microenvironment is a requirement to develop more effective cancer immunotherapies. Here, we report that C/EBP homologous protein (Chop), a downstream sensor of severe endoplasmic reticulum (ER) stress, is a major negative regulator of the effector function of tumor-reactive CD8+ T cells. Chop expression is increased in tumor-infiltrating CD8+ T cells, which correlates with poor clinical outcome in ovarian cancer patients. Deletion of Chop in T cells improves spontaneous antitumor CD8+ T cell immunity and boosts the efficacy of T cell-based immunotherapy. Mechanistically, Chop in CD8+ T cells is elevated primarily through the ER stress-associated kinase Perk and a subsequent induction of Atf4; and directly represses the expression of T-bet, a master regulator of effector T cell function. These findings demonstrate the primary role of Chop in tumor-induced CD8+ T cell dysfunction and the therapeutic potential of blocking Chop or ER stress to unleash T cell-mediated antitumor immunity.

DOI: https://doi.org/10.1038/s41467-019-09263-1
Mol Cancer Res. 2019 Mar 22. pii: molcanres.0002.2019. [Epub ahead of print]

The PKR-like endoplasmic reticulum kinase promotes the dissemination of Myc-induced leukemic cells.

Jun Gui, Kanstantsin V Katlinski, Constantinos Koumenis, J Alan Diehl, Serge Y Fuchs.

Hyperactive oncogenic Myc stimulates protein synthesis that induces the unfolded protein response, which requires the function of the eukaryotic translation initiation factor 2-alpha kinase 3, also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Activated PERK acts to limit mRNA translation, enable proper protein folding and restore the homeostasis in the endoplasmic reticulum. Given that Myc activation contributes to many types of lymphoid and myeloid human leukemias, we used a mouse model to examine the importance of PERK in development and progression of Myc-induced leukemias. We found that genetic ablation of Perk does not suppress the generation of the leukemic cells in the bone marrow. However, the cell-autonomous Perk deficiency restricts the dissemination of leukemic cells into peripheral blood, lymph nodes and vital peripheral organs. Whereas the loss of the IFNAR1 chain of type I interferon receptor stimulated leukemia, Perk ablation did not stabilize IFNAR1 suggesting that PERK stimulates the leukemic cells dissemination in an IFNAR1-independent manner. We discuss the rationale for using PERK inhibitors against Myc-driven leukemias. Implications: The role of PERK in dissemination of Myc-induced leukemic cells demonstrated in this study argues for the use of PERK inhibitors against leukemia progression.

DOI: https://doi.org/10.1158/1541-7786.MCR-19-0002
Onco Targets Ther. 2019 ;12 1857-1865

Targeting off-target effects: endoplasmic reticulum stress and autophagy as effective strategies to enhance temozolomide treatment.

Yichun He, Jing Su, Beiwu Lan, Yufei Gao, Jingxia Zhao.

  Glioblastoma multiforme (GBM) is the most common and aggressive adult primary central nervous system tumor. Unfortunately, GBM is resistant to the classic chemotherapy drug, temozolomide (TMZ). As well as its classic DNA-targeting effects, the off-target effects of TMZ can have pro-survival or pro-death roles and regulate GBM chemoradiation sensitivity. Endoplasmic reticulum (ER) stress is one of the most common off-target effects. ER stress and its downstream induction of autophagy, apoptosis, and other events have important roles in regulating TMZ sensitivity. Autophagy is an evolutionarily conserved cellular homeostasis mechanism that is closely associated with ER stress-induced apoptosis. Under ER stress, autophagy cannot only remove misfolded/unfolded proteins and damaged organelles and degrade and inhibit apoptosis-related caspase activation to reduce cell damage, but may also promote apoptosis dependent on ER stress intensity. Although some protein interactions between autophagy and apoptosis and common upstream signaling pathways have been found, the underlying regulatory mechanisms are still not fully understood. This review summarizes the possible mechanisms underlying the current known off-target roles of ER stress and downstream autophagy in the regulation of cell fate and evaluates their role in TMZ treatment and their potential as therapeutic targets.

Keywords:  apoptosis; autophagy; chemotherapy resistance; endoplasmic reticulum stress; glioma; temozolomide

DOI:  https://doi.org/10.2147/OTT.S194770
Plant Cell. 2019 Mar 20. pii: tpc.00723.2018. [Epub ahead of print]

The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii.

Yasuyo Yamaoka, Seungjun Shin, Bae Young Choi, Hanul Kim, Sunghoon Jang, Masataka Kajikawa, Takashi Yamano, Fantao Kong, Bertrand Legeret, Hideya Fukuzawa, Yonghua Li-Beisson, Youngsook Lee.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Here, we identified proteins and lipids that function downstream of the ER stress sensor inositol-requiring enzyme1 (CrIRE1), which contributes to ER stress tolerance in Chlamydomonas reinhardtii. Treatment with the ER stress inducer tunicamycin resulted in the splicing of a 32-nucleotide fragment of a bZIP transcription factor (CrbZIP1) mRNA by CrIRE1, which resulted in the loss of the transmembrane domain in CrbZIP1, and the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed to induce the expression of the unfolded protein response genes and grew poorly under ER stress. Levels of diacylglyceryltrimethylhomo-Ser (DGTS) and pinolenic acid (18:3Δ5,9,12) increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the biosynthesis of DGTS and pinolenic acid. Moreover, two independent alleles of crdes mutant, which failed to synthesize pinolenic acid, were more sensitive to ER stress than were their parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling.

DOI: https://doi.org/10.1105/tpc.18.00723
Front Immunol. 2019 ;10 344

Molecular Insights Into the Relationship Between Autoimmune Thyroid Diseases and Breast Cancer: A Critical Perspective on Autoimmunity and ER Stress.

Safikur Rahman, Ayyagari Archana, Arif Tasleem Jan, Durgashree Dutta, Abhishek Shankar, Jihoe Kim, Rinki Minakshi.

  The etiopathologies behind autoimmune thyroid diseases (AITDs) unravel misbehavior of immune components leading to the corruption of immune homeostasis where thyroid autoantigens turn foe to the self. In AITDs lymphocytic infiltration in the thyroid shows up a deranged immune system charging the follicular cells of the thyroid gland (thyrocytes) leading to the condition of either hyperthyroidism or hypothyroidism. The inflammation in AITDs consistently associate with ER function due to which disturbances in the ER protein homeostasis leads to unfolded protein response (UPR) that promotes pathogenesis of autoimmunity. The roles of ER stress in the instantaneous downregulation of MHC class I molecules on thyrocytes and the relevance of IFN γ in the pathogenesis of AITD has been well-documented. Thyroglobulin being the major target of autoantibodies in most of the AITDs is because of its unusual processing in the ER. Autoimmune disorders display a conglomeration of ER stress-induced UPR activated molecules. Several epidemiological data highlight the preponderance of AITDs in women as well as its concurrence with breast cancer. Both being an active glandular system displaying endocrine activity, thyroid as well as breast tissue show various commonalities in the expression pattern of heterogenous molecules that not only participate in the normal functioning but at the same time share the blame during disease establishment. Studies on the development and progression of breast carcinoma display a deranged and uncontrolled immune response, which is meticulously exploited during tumor metastasis. The molecular crosstalks between AITDs and breast tumor microenvironment rely on active participation of immune cells. The induction of ER stress by Tunicamycin advocates to provide a model for cancer therapy by intervening glycosylation. Therefore, this review attempts to showcase the molecules that are involved in feeding up the relationship between breast carcinoma and AITDs.

Keywords:  Grave's disease; Hashimotos's thyroiditis; autoantibodies; autoantigens; lymphocytic infiltration

DOI:  https://doi.org/10.3389/fimmu.2019.00344
J Immunol. 2019 Mar 20. pii: ji1801233. [Epub ahead of print]

Brucella abortus Cyclic Dinucleotides Trigger STING-Dependent Unfolded Protein Response That Favors Bacterial Replication.

Erika S Guimarães, Marco Túlio R Gomes, Priscila C Campos, Daniel S Mansur, Adara A Dos Santos, Jerome Harms, Gary Splitter, Judith A Smith, Glen N Barber, Sergio C Oliveira.

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-β, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-β is also important for the UPR induction during B. abortus infection. Indeed, IFN-β shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-β favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-β signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.

DOI: https://doi.org/10.4049/jimmunol.1801233
FASEB J. 2019 Mar 19. fj201802366RR

Preservation of circadian rhythms by the protein folding chaperone, BiP.

Adam Pickard, Joan Chang, Nissrin Alachkar, Ben Calverley, Richa Garva, Peter Arvan, Qing-Jun Meng, Karl E Kadler.

  Dysregulation of collagen synthesis is associated with disease progression in cancer and fibrosis. Collagen synthesis is coordinated with the circadian clock, which in cancer cells is, curiously, deregulated by endoplasmic reticulum (ER) stress. We hypothesized interplay between circadian rhythm, collagen synthesis, and ER stress in normal cells. Here we show that fibroblasts with ER stress lack circadian rhythms in gene expression upon clock-synchronizing time cues. Overexpression of binding immunoglobulin protein (BiP) or treatment with chemical chaperones strengthens the oscillation amplitude of circadian rhythms. The significance of these findings was explored in tendon, where we showed that BiP expression is ramped preemptively prior to a surge in collagen synthesis at night, thereby preventing protein misfolding and ER stress. In turn, this forestalls activation of the unfolded protein response in order for circadian rhythms to be maintained. Thus, targeting ER stress could be used to modulate circadian rhythm and restore collagen homeostasis in disease.-Pickard, A., Chang, J., Alachkar, N., Calverley, B., Garva, R., Arvan, P., Meng, Q.-J., Kadler, K. E. Preservation of circadian rhythms by the protein folding chaperone, BiP.

Keywords:  4PBA; ER stress; Per2::luc; UDCA; collagen

DOI:  https://doi.org/10.1096/fj.201802366RR
FEBS J. 2019 Mar 18.

Dynamic analysis of proteomic alterations in response to N-linked glycosylation inhibition in a drug-resistant ovarian carcinoma cell line.

Jian Shu, Liuyi Dang, Dandan Zhang, Punit Shah, Lijun Chen, Hui Zhang, Shisheng Sun.

  Glycosylation inhibition can improve the efficacy of antitumor drugs and enhance the apoptosis of cancer cells, thus holding great potential for cancer treatment. Inhibition of N-glycosylation induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), and eventually triggers ER stress-related apoptosis. Unfortunately, the detailed timeline of these cell responses and protein expression alterations related to N-glycosylation inhibition is not explicit yet, and the pathways involved in different stages of N-glycosylation inhibition still need to be characterized. In this study, the dynamic proteome alterations related to N-glycosylation inhibition were investigated by further analyzing our previously published quantitative proteomics data from tunicamycin-treated ovarian carcinoma (OVCAR-3) cells. The results revealed that N-glycosylation inhibition not only directly affects the expression of glycosylated proteins but also alters an extended scale of proteins. Functional annotation of these altered proteins demonstrated that proteins related to ER stress start changing within 6 h, followed by UPR within 24 h, and eventually ER stress-related apoptosis is triggered after 48 h, indicating the conversion of cellular response from positive to negative. The dynamic proteome data presented here provide important information for better understanding of the significance of N-glycosylation to cell survival and TM-related cancer treatment. This article is protected by copyright. All rights reserved.

Keywords:  Cellular response; ER Stress; Glycosylation; Mass Spectrometry; Proteome; Tunicamycin

DOI:  https://doi.org/10.1111/febs.14811
J Cell Physiol. 2019 Mar 18.

Cadmium induces cell centrosome amplification via reactive oxygen species as well as endoplasmic reticulum stress pathway.

Rui Kai Zhang, Pu Wang, Yu Cheng Lu, Lang Lang, Lan Wang, Shao Chin Lee.

  There is evidence that cadmium can initiate carcinogenesis. However, the underlying mechanisms remain unknown. There is also evidence that moderate centrosome amplification can initiate tumorigenesis. The present study investigated whether cadmium could trigger cell centrosome amplification, and examined the underlying molecular mechanisms. We found that cadmium was able to cause cell centrosome amplification at the subtoxic concentrations, in a dose-dependent manner. It could cause centrosome amplification via the signaling of reactive oxygen species (ROS). Proteomic analysis revealed that cadmium caused differential expressions of three proteins, which included HSPA1A which is associated with endoplasmic reticulum (ER) stress. Western blot analysis confirmed that cadmium upregulated HSPA1A. Further analyses showed that cadmium upregulated Bip and decreased the phosphorylation of ASK1 as well as increased the phosphorylation of MKK7 and c-Jun N-terminal kinases (JNK). Knockdown of JNK2 using small interfering RNA inhibited the cadmium-induced centrosome amplification but not the level of ROS. N-acetylcysteine did not inhibit the cadmium-activated ER stress pathway. In conclusion, our results suggest that cadmium can induce cell centrosome amplification via ROS as well as ER stress through the Bip-TRAF2-ASK1-MKK7-JNK signaling route, in parallel. More studies are required to clarify whether centrosome amplification underlies cadmium-induced carcinogenesis.

Keywords:  ER stress; ROS; cadmium; centrosome amplification

DOI:  https://doi.org/10.1002/jcp.28455
Diabetes. 2019 Mar 18. pii: db180933. [Epub ahead of print]

Ameliorating Methylglyoxal-Induced Progenitor Cell Dysfunction for Tissue Repair in Diabetes.

Hainan Li, Megan O'Meara, Xiang Zhang, Kezhong Zhang, Berhane Seyoum, Zhengping Yi, Randal J Kaufman, Terrence J Monks, Jie-Mei Wang.

Patient-derived progenitor cell (PC) dysfunction is severely impaired in diabetes, but the molecular triggers that contribute to mechanisms of PC dysfunction are not fully understood. Methylglyoxal (MGO) is one of the highly reactive dicarbonyl species formed during hyperglycemia. We hypothesize that the MGO scavenger glyoxalase 1 (GLO1) reverses bone marrow-derived progenitor cell (BMPCs) dysfunction through augmenting the activity of an important endoplasmic reticulum (ER) stress sensor, inositol-requiring enzyme 1α (IRE1α), resulting in improved diabetic wound healing. BMPCs were isolated from adult male db/db type-2 diabetic mice and their healthy corresponding control db/+ mice. MGO at the concentration of 10µM induced immediate and severe BMPC dysfunction, including impaired network formation, migration, proliferation, and increased apoptosis, which were rescued by adenovirus-mediated GLO1 overexpression. IRE1α expression and activation in BMPCs were significantly attenuated by MGO exposure but rescued by GLO1 overexpression. MGO can diminish IRE1α RNase activity by directly binding to IRE1α in vitro In a diabetic mouse cutaneous wound model in vivo, cell therapies using diabetic cells with GLO1 overexpression remarkably accelerated wound closure by enhancing angiogenesis, compared with diabetic control cell therapy. Augmenting tissue GLO1 expression by adenovirus-mediated gene transfer or with the small-molecule inducer trans-resveratrol and hesperetin formulation also improved wound closure and angiogenesis in diabetic mice. In conclusion, our data suggest that GLO1 rescues BMPC dysfunction and facilitates wound healing in diabetic animals, at least partly through preventing MGO-induced impairment of IRE1α expression and activity. Our results provide important knowledge for the development of novel therapeutic approaches targeting MGO to improve PC-mediated angiogenesis and tissue repair in diabetes.

DOI: https://doi.org/10.2337/db18-0933
Dev Cell. 2019 Mar 08. pii: S1534-5807(19)30102-9. [Epub ahead of print]

GRASP55 and UPR Control Interleukin-1β Aggregation and Secretion.

Marioara Chiritoiu, Nathalie Brouwers, Gabriele Turacchio, Marinella Pirozzi, Vivek Malhotra.

  Signal-sequence-lacking interleukin (IL)-1β, is cleaved by caspase-1 to mature mIL-1β, which is secreted, without entering the endoplasmic reticulum. We report that macrophages of GRASP55-/- mice are defective in mIL-1β secretion and retain it as intracellular aggregates. Intriguingly, GRASP55-/- macrophages are defective in the IRE1α branch of the unfolded protein response. This finding fits well with our data that inhibition of IRE1α also impairs mIL-1β secretion and causes its accumulation in intracellular aggregates. PERK inhibition, on the other hand, controls caspase-1-mediated conversion of proIL-1β to mIL-1β. These findings reveal translation-independent functions of PERK and IRE1α: PERK controls the production of mIL-1β, which is then followed by GRASP55 and IRE1α activity to keep mIL-1β in a secretion-competent form.

Keywords:  GRASP; interleukin-1β (IL-1β); unconventional protein secretion (UPS); unfolded protein response (UPR)

DOI:  https://doi.org/10.1016/j.devcel.2019.02.011
Cell Death Dis. 2019 Mar 19. 10(4): 269

Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis.

Sarmistha Mahanty, Shruthi Shirur Dakappa, Rezwan Shariff, Saloni Patel, Mruthyunjaya Mathapathi Swamy, Amitabha Majumdar, Subba Rao Gangi Setty.

Keratinocytes maintain epidermal integrity through cellular differentiation. This process enhances intraorganelle digestion in keratinocytes to sustain nutritional and calcium-ionic stresses observed in upper skin layers. However, the molecular mechanisms governing keratinocyte differentiation and concomitant increase in lysosomal function is poorly understood. Here, by using primary neonatal human epidermal keratinocytes, we identified the molecular link between signaling pathways and cellular differentiation/lysosome biogenesis. Incubation of keratinocytes with CaCl2 induces differentiation with increased cell size and early differentiation markers. Further, differentiated keratinocytes display enhanced lysosome biogenesis generated through ATF6-dependent ER stress signaling, but independent of mTOR-MiT/TFE pathway. In contrast, chemical inhibition of mTORC1 accelerates calcium-induced keratinocyte differentiation, suggesting that activation of autophagy promotes the differentiation process. Moreover, differentiation of keratinocytes results in lysosome dispersion and Golgi fragmentation, and the peripheral lysosomes showed colocalization with Golgi-tethering proteins, suggesting that these organelles possibly derived from Golgi. In line, inhibition of Golgi function, but not the depletion of Golgi-tethers or altered lysosomal acidity, abolishes keratinocyte differentiation and lysosome biogenesis. Thus, ER stress regulates lysosome biogenesis and keratinocyte differentiation to maintain epidermal homeostasis.

DOI: https://doi.org/10.1038/s41419-019-1478-4
J Extracell Vesicles. 2019 ;8(1): 1588538

Extracellular vesicles derived from natural killer cells use multiple cytotoxic proteins and killing mechanisms to target cancer cells.

Chun-Hua Wu, Jingbo Li, Li Li, Jianping Sun, Muller Fabbri, Alan S Wayne, Robert C Seeger, Ambrose Y Jong.

  Extracellular vesicles (EVs) are secreted membrane vesicles, which play complex physiological and pathological functions in intercellular communication. Recently, we isolated natural killer (NK) cell-derived EVs (NK-EVs) from ex vivo expansion of NK cell cultures. The isolated NK-EVs contained cytotoxic proteins and several activated caspases, and they induced apoptosis in target cells. In this report, the protein levels of cytotoxic proteins from NK-EV isolates were analysed by ELISA. The mean values of perforin (PFN, 550 ng/mL), granzyme A (GzmA, 185 ng/mL), granzyme B (GzmB, 23.4 ng/mL), granulysin (GNLY, 56 ng/mL), and FasL (2.5 ng/mL) were obtained from >60 isolations using dot plots. The correlation between cytotoxicity and cytotoxic protein levels was examined by linear regression. PFN, GzmA, GzmB, GNLY all had a positive, moderate correlation with cytotoxicity, suggesting that there is not a single cytotoxic protein dominantly involved in killing and that all of these proteins may contribute to cytotoxicity. To further explore the possible killing mechanisms, cells were treated with NK-EVs, proteins extracted and lysates assessed by Western blotting. The levels of Gzm A substrates, SET and HMG2, were diminished in targeted cells, indicating that GzmA may induce a caspase-independent death pathway. Also, cytochrome C was released from mitochondria, a central hallmark of caspase-dependent death pathways. In addition, several ER-associated proteins were altered, suggesting that NK-EVs may induce ER stress resulting in cell death. Our results indicate that multiple killing mechanisms are activated by NK-derived EVs, including caspase-independent and -dependent cell death pathways, which can mediate cytotoxicity against cancer cells. Abbreviations: NK: natural killer cells; aNK: activated NK cells; EV: extracellular vesicles; ER: endoplasmic reticulum; ALL: acute lymphoblastic leukaemia; FBS: foetal bovine serum. GzmA: granzyme A; GzmB: granzyme B; GNLY: granulysin; PFN: perforin.

Keywords:  Scale-up isolation; cancer treatment; caspases; cytotoxicity; extracellular vesicles; natural killer cells

DOI:  https://doi.org/10.1080/20013078.2019.1588538
Cell Rep. 2019 Mar 19. pii: S2211-1247(19)30263-3. [Epub ahead of print]26(12): 3416-3428.e5

Oxidative Stress Triggers Selective tRNA Retrograde Transport in Human Cells during the Integrated Stress Response.

Hagen Schwenzer, Frank Jühling, Alexander Chu, Laura J Pallett, Thomas F Baumert, Mala Maini, Ariberto Fassati.

  In eukaryotes, tRNAs are transcribed in the nucleus and exported to the cytosol, where they deliver amino acids to ribosomes for protein translation. This nuclear-cytoplasmic movement was believed to be unidirectional. However, active shuttling of tRNAs, named tRNA retrograde transport, between the cytosol and nucleus has been discovered. This pathway is conserved in eukaryotes, suggesting a fundamental function; however, little is known about its role in human cells. Here we report that, in human cells, oxidative stress triggers tRNA retrograde transport, which is rapid, reversible, and selective for certain tRNA species, mostly with shorter 3' ends. Retrograde transport of tRNASeC, which promotes translation of selenoproteins required to maintain homeostatic redox levels in cells, is highly efficient. tRNA retrograde transport is regulated by the integrated stress response pathway via the PERK-REDD1-mTOR axis. Thus, we propose that tRNA retrograde transport is part of the cellular response to oxidative stress.

Keywords:  PKR; REDD1; fluorescence in situ hybridization; mTOR; nucleus; oxidative stress; retrograde transport; tRNA; unfolded protein response

DOI:  https://doi.org/10.1016/j.celrep.2019.02.077
Biochem Biophys Res Commun. 2019 Mar 15. pii: S0006-291X(19)30388-2. [Epub ahead of print]

Carbon monoxide induces the assembly of stress granule through the integrated stress response.

Yingqing Chen, Yeonsoo Joe, Jeongmin Park, Hyun-Chul Song, Uh-Hyun Kim, Hun Taeg Chung.

  Stress granules (SGs) are membraneless and phase-dense organelles that form transiently in response to a variety of harmful stimuli, including oxidative, heat, osmotic, ultraviolet light and chemotoxic stresses, and thus providing protective effects, allowing survivals. Carbon monoxide (CO), a gaseous second messenger, is synthesized by heme-oxygenases, and exerts anti-inflammatory, anti-proliferative and anti-apoptotic effects in a variety of cellular- and tissue-injury models. Several reports indicate that low levels of mitochondrial reactive oxygen species (mtROS) generated by CO can selectively activate PERK-eIF2α integrated stress response (ISR) to preserve the cellular homeostasis. Hence, CO can confer protection against cellular stresses. However, the mechanisms underlying the cyto-protective effects of CO against various harmful stimuli remain to be elucidated. Here, we sought to examine whether CO induces the SG assembly, and uncover its molecular mechanisms. We treated WI-38 cells and primary mouse embryonic fibroblasts (MEFs) with CO-releasing molecule 2 (CORM2) or CO gas, and found the SG assemblies were gradually increased in time and dose dependent manners. Next, we used Mito-TEMPO, an mtROS scavenger, to explore if mtROS might be involved in the CO-induced SG assembly. Furthermore, we confirmed the involvement of ISR consisted of PERK-eIF2α signaling pathway induced by CO for the SGs assembly. Finally, the inhibition of SG assembly by ISR inhibitor further verified CO-induced ISR might be responsible for SG. Taken together, in this study, we first demonstrated that CO is a novel SG inducer by activating ISR. Moreover, mtROS might be an initiator for the CO-induced ISR responsible for SG assembly.

Keywords:  Carbon monoxide; Integrated stress response; Mitochondrial reactive oxygen species; Stress granule

DOI:  https://doi.org/10.1016/j.bbrc.2019.03.017