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Choline kinase inhibition promotes ER-phagy

Choline kinase inhibition promotes ER-phagy Journal Pre-proof Mahtab Tavasoli, Sandhya Chipurupalli, Christopher R. McMaster PII: S0022-2275(22)00046-3 DOI: https://doi.org/10.1016/j.jlr.2022.100213 Reference: JLR 100213 To appear in: Journal of Lipid Research Received Date: 31 March 2022 Revised Date: 10 April 2022 Accepted Date: 11 April 2022 Please cite this article as: Tavasoli M, Chipurupalli S, McMaster CR, Choline kinase inhibition promotes ER-phagy, Journal of Lipid Research (2022), doi: https://doi.org/10.1016/j.jlr.2022.100213. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. Mahtab Tavasoli, Sandhya Chipurupalli, and Christopher R. McMaster Departments of Pharmacology and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada B3H 4H7 Corresponding author: [email protected] Journal Pre-proof The major membrane phospholipid phosphatidylcholine (PC) is predominantly synthesized through the CDP-choline (Kennedy) pathway at the endoplasmic reticulum (ER). The first step in this pathway is the phosphorylation of choline by choline kinase. Humans contain two choline kinase isoforms, CHKA and CHKB. Variants in the CHKA gene that decrease enzymatic activity have been shown to cause an inherited neurodevelopmental disorder with epilepsy, while recessive inheritance of non-functional -/- CHKB alleles causes an inherited rostro-caudal muscular dystrophy (1,2). In affected muscle of Chkb mice, we previously determined that CHKB protein is absent and CHKA expression was severely downregulated, resulting in an almost complete absence of choline kinase activity (2). It is known that stress to the ER activates several ER quality-control processes, namely the unfolded protein response, ER- associated degradation, and the relatively newly described ER-phagy. ER-phagy results in engulfment of sections of ER into autophagosomes and transport to lysosomes to aid in replenishment/recycling of -/- damaged ER (3). (Panel A) Transmission electron microscopy of Chkb muscle demonstrates extensive ER injury, including ER vacuolization and expansion (asterisk) and evidence of damaged ER (grey arrow) that is engulfed by autophagosomes and fused with lysosomes (red arrow). These findings are highly suggestive of ER-phagy; scale bar, 500 nm. (Panel B) Human bone osteosarcoma epithelial cells (U2OS) were treated with the choline kinase inhibitor EB3D (15 µM for 48 h) and ER-phagy monitored using the ER autophagy tandem reporter (EATR) (3). The EATR reporter is a tandem eGFP–mCherry ER resident protein where both reporters normally face the cytoplasm and fluoresce. In the setting of ER- phagy, mCherry preferentially fluoresces due to the acidic pH of the lysosome (3). As expected, and as previously reported, EATR localizes only to the ER when cells are fed, while under starvation (to induce ER-phagy) mCherry-only puncta were observed (3). The mCherry puncta were also observed when fed cells were treated with the choline kinase inhibitor EB3D. These data suggest that ER-phagy occurs upon inhibition of choline kinase and suggests that ER-phagy could play a role in the etiology of diseases associated with choline kinase inhibition. EQUIPMENT: Transmission Electron Microscope (JEOL JEM 1230) with Hamamatsu ORCA-HR digital camera. Zeiss Axio Observer Z.1 Spinning Disk Confocal Microscope. Journal Pre-proof REAGENTS: TetOn-mCherry-eGFP-RAMP4 (EATR reporter) was a gift from Jacob Corn (Addgene plasmid#109014), Lipofectamine 3000 (ThermoFisher Scientific), EB3D (MCA® MedChemExpress, Cat#HY-115463), doxycycline (Sigma Aldrich, Cat#D9891), EBSS (ThermoFisher, Cat#24010043). REFERENCES 1. Tavasoli, M., T. Feridooni, H. Feridooni, S. Sokolenko, A. Mishra, A. Lefsay, S. Srinivassane, S. A. Reid, J. Rowsell, M. Praest, A. MacKinnon, M. Mammoliti, A. A. Maloney, M. Moraca, K. Uaesoontrachoon, K. Nagaraju, E. P. Hoffman, K. B. S. Pasumarthi, and C. R. McMaster. 2022. A mouse model of inherited choline kinase beta-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia. J Biol Chem: 101716. 2. Tavasoli, M., S. Lahire, S. Sokolenko, R. Novorolsky, S. A. Reid, A. Lefsay, M. O. C. Otley, K. Uaesoontrachoon, J. Rowsell, S. Srinivassane, M. Praest, A. MacKinnon, M. S. Mammoliti, A. A. Maloney, M. Moraca, J. Pedro Fernandez-Murray, M. McKenna, C. J. Sinal, K. Nagaraju, G. S. Robertson, E. P. Hoffman, and C. R. McMaster. 2022. Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism. Nat Commun 13: 1559. 3. Liang, J. R., E. Lingeman, S. Ahmed, and J. E. Corn. 2018. Atlastins remodel the endoplasmic reticulum for selective autophagy. J Cell Biol 217: 3354-3367. Journal Pre-proof AA BB Journal Pre-proof CRediT author statement Manuscript JLR-D-22-00138   Mahtab Tavasoli: Conceptualization, Methodology, Validation, Investigation, Writing – Original Draft and Review and Editing Sandhya Chipurupalli: Conceptualization, Methodology, Writing – Original Draft Christopher R. McMaster: Conceptualization, Writing – Review and Editing, Supervision, Funding acquisition Journal Pre-proof http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Lipid Research American Society for Biochemistry and Molecular Biology

Choline kinase inhibition promotes ER-phagy

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Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2022 Elsevier Inc.
ISSN
0022-2275
eISSN
1539-7262
DOI
10.1016/j.jlr.2022.100213
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Abstract

Journal Pre-proof Mahtab Tavasoli, Sandhya Chipurupalli, Christopher R. McMaster PII: S0022-2275(22)00046-3 DOI: https://doi.org/10.1016/j.jlr.2022.100213 Reference: JLR 100213 To appear in: Journal of Lipid Research Received Date: 31 March 2022 Revised Date: 10 April 2022 Accepted Date: 11 April 2022 Please cite this article as: Tavasoli M, Chipurupalli S, McMaster CR, Choline kinase inhibition promotes ER-phagy, Journal of Lipid Research (2022), doi: https://doi.org/10.1016/j.jlr.2022.100213. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. Mahtab Tavasoli, Sandhya Chipurupalli, and Christopher R. McMaster Departments of Pharmacology and Biochemistry & Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada B3H 4H7 Corresponding author: [email protected] Journal Pre-proof The major membrane phospholipid phosphatidylcholine (PC) is predominantly synthesized through the CDP-choline (Kennedy) pathway at the endoplasmic reticulum (ER). The first step in this pathway is the phosphorylation of choline by choline kinase. Humans contain two choline kinase isoforms, CHKA and CHKB. Variants in the CHKA gene that decrease enzymatic activity have been shown to cause an inherited neurodevelopmental disorder with epilepsy, while recessive inheritance of non-functional -/- CHKB alleles causes an inherited rostro-caudal muscular dystrophy (1,2). In affected muscle of Chkb mice, we previously determined that CHKB protein is absent and CHKA expression was severely downregulated, resulting in an almost complete absence of choline kinase activity (2). It is known that stress to the ER activates several ER quality-control processes, namely the unfolded protein response, ER- associated degradation, and the relatively newly described ER-phagy. ER-phagy results in engulfment of sections of ER into autophagosomes and transport to lysosomes to aid in replenishment/recycling of -/- damaged ER (3). (Panel A) Transmission electron microscopy of Chkb muscle demonstrates extensive ER injury, including ER vacuolization and expansion (asterisk) and evidence of damaged ER (grey arrow) that is engulfed by autophagosomes and fused with lysosomes (red arrow). These findings are highly suggestive of ER-phagy; scale bar, 500 nm. (Panel B) Human bone osteosarcoma epithelial cells (U2OS) were treated with the choline kinase inhibitor EB3D (15 µM for 48 h) and ER-phagy monitored using the ER autophagy tandem reporter (EATR) (3). The EATR reporter is a tandem eGFP–mCherry ER resident protein where both reporters normally face the cytoplasm and fluoresce. In the setting of ER- phagy, mCherry preferentially fluoresces due to the acidic pH of the lysosome (3). As expected, and as previously reported, EATR localizes only to the ER when cells are fed, while under starvation (to induce ER-phagy) mCherry-only puncta were observed (3). The mCherry puncta were also observed when fed cells were treated with the choline kinase inhibitor EB3D. These data suggest that ER-phagy occurs upon inhibition of choline kinase and suggests that ER-phagy could play a role in the etiology of diseases associated with choline kinase inhibition. EQUIPMENT: Transmission Electron Microscope (JEOL JEM 1230) with Hamamatsu ORCA-HR digital camera. Zeiss Axio Observer Z.1 Spinning Disk Confocal Microscope. Journal Pre-proof REAGENTS: TetOn-mCherry-eGFP-RAMP4 (EATR reporter) was a gift from Jacob Corn (Addgene plasmid#109014), Lipofectamine 3000 (ThermoFisher Scientific), EB3D (MCA® MedChemExpress, Cat#HY-115463), doxycycline (Sigma Aldrich, Cat#D9891), EBSS (ThermoFisher, Cat#24010043). REFERENCES 1. Tavasoli, M., T. Feridooni, H. Feridooni, S. Sokolenko, A. Mishra, A. Lefsay, S. Srinivassane, S. A. Reid, J. Rowsell, M. Praest, A. MacKinnon, M. Mammoliti, A. A. Maloney, M. Moraca, K. Uaesoontrachoon, K. Nagaraju, E. P. Hoffman, K. B. S. Pasumarthi, and C. R. McMaster. 2022. A mouse model of inherited choline kinase beta-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia. J Biol Chem: 101716. 2. Tavasoli, M., S. Lahire, S. Sokolenko, R. Novorolsky, S. A. Reid, A. Lefsay, M. O. C. Otley, K. Uaesoontrachoon, J. Rowsell, S. Srinivassane, M. Praest, A. MacKinnon, M. S. Mammoliti, A. A. Maloney, M. Moraca, J. Pedro Fernandez-Murray, M. McKenna, C. J. Sinal, K. Nagaraju, G. S. Robertson, E. P. Hoffman, and C. R. McMaster. 2022. Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism. Nat Commun 13: 1559. 3. Liang, J. R., E. Lingeman, S. Ahmed, and J. E. Corn. 2018. Atlastins remodel the endoplasmic reticulum for selective autophagy. J Cell Biol 217: 3354-3367. Journal Pre-proof AA BB Journal Pre-proof CRediT author statement Manuscript JLR-D-22-00138   Mahtab Tavasoli: Conceptualization, Methodology, Validation, Investigation, Writing – Original Draft and Review and Editing Sandhya Chipurupalli: Conceptualization, Methodology, Writing – Original Draft Christopher R. McMaster: Conceptualization, Writing – Review and Editing, Supervision, Funding acquisition Journal Pre-proof

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Journal of Lipid Research – American Society for Biochemistry and Molecular Biology

Published: Apr 18, 2022

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