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Simple Nutrients Bypass the Requirement for HLH-30 in Coupling Lysosomal Nutrient Sensing to Survival John T

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Simple Nutrients Bypass the Requirement for HLH-30 in Coupling Lysosomal Nutrient Sensing to Survival John T Washington University School of Medicine Digital Commons@Becker Open Access Publications 2019 Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival John T. Murphy Haiyan Liu Xiucui Ma Alex Shaver Brian M. Egan See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Authors John T. Murphy, Haiyan Liu, Xiucui Ma, Alex Shaver, Brian M. Egan, Clara Oh, Alexander Boyko, Travis Mazer, Samuel Ang, Rohan Khopkar, Ali Javaheri, Sandeep Kumar, Xuntian Jiang, Daniel Ory, Kartik Mani, Scot J. Matkovich, Kerry Kornfeld, and Abhinav Diwan RESEARCH ARTICLE Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival 1☯ 1,2☯ 1,2 1 1,3 John T. Murphy , Haiyan Liu , Xiucui Ma , Alex ShaverID , Brian M. Egan , 1 1 1² 1 1 Clara OhID , Alexander Boyko , Travis Mazer , Samuel AngID , Rohan Khopkar , Ali Javaheri1, Sandeep Kumar3, Xuntian Jiang1, Daniel Ory1, Kartik Mani1,2, Scot 1 3☯ 1,2,4☯ J. Matkovich , Kerry Kornfeld , Abhinav DiwanID * a1111111111 1 Center for Cardiovascular Research and Division of Cardiology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 John Cochran a1111111111 VA Medical Center, St. Louis, Missouri, United States of America, 3 Department of Developmental Biology, a1111111111 Washington University School of Medicine, St. Louis, Missouri, United States of America, 4 Department of a1111111111 Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of a1111111111 America ☯ These authors contributed equally to this work. ² Deceased. * [email protected] OPEN ACCESS Citation: Murphy JT, Liu H, Ma X, Shaver A, Egan Abstract BM, Oh C, et al. (2019) Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular nutrient sensing to survival. PLoS Biol 17(5): e3000245. https://doi.org/10.1371/journal. material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to pbio.3000245 regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and sup- Academic Editor: Heidi A. Tissenbaum, University presses activity of the MiT/TFE family of transcription factors that control biogenesis of lyso- of Massachusetts Medical School, UNITED somes. In this study, we subjected worms lacking basic helix±loop±helix transcription factor STATES 30 (hlh-30), the Caenorhabditis elegans MiT/TFE ortholog, to starvation followed by refeed- Received: January 19, 2019 ing to understand how this pathway regulates survival with variable nutrient supply. Loss of Accepted: April 10, 2019 HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. ele- Published: May 14, 2019 gans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lyso- Copyright: This is an open access article, free of all somal acidification, TOR activation, and survival with refeeding despite the absence of HLH- copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or 30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcription- otherwise used by anyone for any lawful purpose. ally up-regulated in starvation in an HLH-30±dependent manner, also demonstrated The work is made available under the Creative increased mortality with starvation±refeeding that was partially rescued with glucose, sug- Commons CC0 public domain dedication. gesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced tran- Data Availability Statement: We have uploaded scription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of files named as S1 and S2 Data that contain all the vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 observations that are reported as summary data in main figures and supplementary data, respectively. (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C Each figure legend now includes callouts pointing homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation± to the raw data. We have uploaded all the RNA refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 sequencing data to the NCBI GEO server, accession number GSE126061. mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation± Funding: This study was supported by grants from the Children's Discovery Institute of Washington PLOS Biology | https://doi.org/10.1371/journal.pbio.3000245 May 14, 2019 1 / 42 HLH-30±independent lysosomal nutrient sensing in worms University School of Medicine and the St. Louis refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for Children's Hospital, the National Institutes of Health HLH-30 in coupling lysosome nutrient sensing to survival. (NIH) (R01HL107594 and R01NS094692), and the Department of Veterans Affairs (I01BX000448, 1l01BX004235) to AD; from the NIH to KK (RO1AG026561 and R01GM068598), to AJ (K08 HL138262); and a seed grant from the John Cochran Veterans Affairs Medical Center to KM. Some strains were provided by the CGC, which is Introduction funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The Washington Lysosomes are membrane-bound subcellular organelles that contain degradative enzymes and University metabolomics facility is supported by are ubiquitously present in all eukaryotic cell types. These specialized organelles compartmen- supported by NIDDK/NIH P30 DK020579. We also talize an acidified milieu for enzymatic degradation of complex macromolecules and organelles thank the Genome Technology Access Center in that are trafficked via autophagy for recycling intracellular substrates or taken up from the the Department of Genetics at Washington extracellular space via endocytosis, macropinocytosis, and phagocytosis. Transcription Factor University School of Medicine (supported by P30 EB (TFEB) and related members of the MiT/TFE (Microphthalmia-associated transcription CA91842 and UL1TR002345) for help with genomic analysis. The funders had no role in study factor) family of basic helix±loop±helix leucine zipper transcriptional activators (TFE3 and design, data collection and analysis, decision to MiTF) were discovered as master regulators of autophagy and lysosome biogenesis programs publish, or preparation of the manuscript. by analyzing lysosomal gene promoters [1, 2]. TFEB and its family members are activated Competing interests: None upon starvation and drive transcriptional up-regulation of autophagy±lysosome machinery genes to sustain survival of mammalian cells via ensuring continued nutrient availability by Abbreviations: AA, arachidonic acid; ama-1, autophagic breakdown of lipid droplets and intracellular material within lysosomes [1, 3, 4]. amanitin-binding subunit of RNA polymerase II; asp-1, aspartyl protease 1; atg, autophagy yeast Lysosomes play a critical role in sensing the cellular nutrient state and coupling the Atg homolog; au, arbitrary unit; Baf-A1, bafilomycin responses to activation of target of rapamycin (TOR), a nutrient-activated serine±threonine A1; bec-1, C. elegans ortholog of human BECN1; kinase that promotes anabolism and growth and suppresses catabolism [5]. Lysosomal nutri- bp, base pair; BSA, bovine serum albumin; CeMM, ent sensing (LYNUS) also regulates TFEB activation. In the fed state, TFEB associates with the Caenorhabditis elegans maintenance medium; LYNUS complex on the cytoplasmic face of the lysosome, which harbors mammalian target of CGC, Caenorhabditis Genetics Center; cpr-1, cysteine protease 1; Ct, cycle threshold; CTWF, rapamycin complex 1 (mTORC1) linked to vacuolar ATPase (V-ATPase) via Ragulator [6]. corrected total worm fluorescence; daf-15, The V-ATPase complex ªsensesº lysosomal levels of amino acids [6] and cholesterol [7] to ortholog of mammalian raptor; daf-16(lf), loss-of- transmit the ªfedº signal via its interaction with Ragulator, initiating guanine nucleotide function mutant for the sole member of the C. exchange factor (GEF) activity for Rag GTPases to activate mTOR. mTOR drives phosphoryla- elegans FOXO Forkhead transcription factor family; tion and inactivation of TFEB family members by inducing retention of these transcriptional DGLA, di-homo-gamma-linoleic acid; elo-2, fatty activators in the cytoplasm [6, 8]. Upon starvation, activation of GTPase activity of RagA/B [9] acid elongase; EPA, eicaso-pentaenoiec acid; fat, fatty acid desaturase; GAPDH, glyceraldehyde 3- facilitates Rag A degradation [10], whereby the mTOR-Ragulator complex dissociates and phosphate dehydrogenase; GEF, guanine relieves the tonic phosphorylation of TFEB family members. Simultaneous lysosomal calcium nucleotide exchange factor; GFP, green fluorescent channel opening activates calcineurin to dephosphorylate TFEB [11] (and presumably TFE3/ protein; GPC, glycerolphosphocholine; hlh-30(lf), MiTF) to unmask its nuclear localization signal, resulting in nuclear translocation where it loss-of-function tm1978 mutation hlh-30; hlh-30 activates transcription of lysosomal biogenesis and autophagy
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