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Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100780

1083-351X

Kaito Mimura, Jun-Ichi Sakamaki, Hideaki Morishita, Masahito Kawazu, Hiroyuki Mano, Noboru Mizushima,

Mosquito-borne diseases and control

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy. Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy. Macroautophagy (hereafter, autophagy) is an intracellular degradation system in which cytoplasmic materials are delivered to and degraded by the lysosome (1Mizushima N. Komatsu M. Autophagy: Renovation of cells and tissues.Cell. 2011; 147: 728-741Abstract Full Text Full Text PDF PubMed Scopus (3226) Google Scholar). Autophagy is responsible for the turnover of proteins and organelles and provides cells with an alternate source of nutrients for cellular renovation and homeostasis. 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Russ C. et al.TMEM41B is a novel regulator of autophagy and lipid mobilization.EMBO Rep. 2018; 19e45889Crossref PubMed Scopus (56) Google Scholar). Our study aimed to identify additional negative regulators of autophagy by employing a genome-wide CRISPR screen. Our nonbiased screening identified the de novo purine synthesis enzyme phosphoribosylformylglycinamidine synthase (PFAS) as a negative regulator of autophagy. We demonstrate in this study that purine starvation, which is caused by the complete loss of purine supply, induces autophagy activation. Our data suggest that lack of nucleotide synthesis profoundly impacts autophagy, elucidating a novel aspect of autophagy regulation. We performed a genome-wide CRISPR screen for negative regulators of autophagy. In the screen, we used the GFP-LC3-RFP autophagic flux reporter (Fig. 1A) (35Kaizuka T. Morishita H. Hama Y. Tsukamoto S. Matsui T. Toyota Y. Kodama A. Ishihara T. Mizushima T. Mizushima N. An autophagic flux probe that releases an internal control.Mol. Cell. 2016; 64: 835-849Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Endogenous ATG4 proteins, which function as proteases, cleave the GFP-LC3-RFP reporter, yielding equimolar amounts of GFP-LC3 and RFP. Subsequently, GFP-LC3 is conjugated to phosphatidylethanolamine on autophagic membranes and delivered to lysosomes, where the GFP signal is quenched. RFP stays in the cytosol and serves as an internal control. Thus, autophagic flux can be visualized by measuring the GFP:RFP ratio using flow cytometry (Fig. 1B). To validate the screen, we performed a LentiCRISPR-mediated bulk knockout of a human embryonic kidney (HEK) 293T cell line stably expressing GFP-LC3-RFP and Cas9 (HEK293T GFP-LC3-RFP cells) with a LentiCRISPR vector comprising single-guide RNAs (sgRNAs) targeting previously known negative regulators of autophagy. After the introduction of sgRNAs targeting RRAGA (a gene coding RAGA, a positive regulator of mTORC1 (36Efeyan A. Schweitzer L.D. Bilate A.M. Chang S. Kirak O. Lamming D.W. Sabatini D.M. Raga, but not RagB, is essential for embryonic development and adult mice.Dev. Cell. 2014; 29: 321-329Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar)) and BRD4 (a gene coding BRD4, a transcriptional repressor of autophagy (17Sakamaki J.I. Wilkinson S. Hahn M. Tasdemir N. O'Prey J. Clark W. Hedley A. Nixon C. Long J.S. New M. Van Acker T. Tooze S.A. Lowe S.W. Dikic I. Ryan K.M. Bromodomain protein BRD4 is a transcriptional repressor of autophagy and lysosomal function.Mol. Cell. 2017; 66: 517-532.e519Abstract Full Text Full Text PDF PubMed Google Scholar)), the number of autophagy-activated cells with a low GFP:RFP ratio was increased (indicated by the region of interest) (Fig. 1C). For bulk knockouts, the phenotypes were observed according to the mutagenic efficiency of the transduced sgRNA, with limited effects on the GFP:RFP ratio of the entire population. For the screen, HEK293T GFP-LC3-RFP cells were transduced with the human genome-scale CRISPR knockout (GeCKO) library, a pooled LentiCRISPR library comprising 123,411 sgRNAs targeting 19,050 human genes (six sgRNAs per gene) (20Shalem O. Sanjana N.E. Hartenian E. Shi X. Scott D.A. Mikkelson T. Heckl D. Ebert B.L. Root D.E. Doench J.G. Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells.Science. 2014; 343: 84-87Crossref PubMed Scopus (2553) Google Scholar, 37Sanjana N.E. Shalem O. Zhang F. Improved vectors and genome-wide libraries for CRISPR screening.Nat. Methods. 2014; 11: 783-784Crossref PubMed Scopus (2034) Google Scholar). Infected cells were selected using puromycin. After 2 weeks of culture, the cells were sorted by flow cytometry. Cells gated in the autophagy-activated population, whose GFP signal decreased under nutrient-replete conditions, were collected. Sorting was repeated four times, at 2-week intervals (Fig. 1D). Genomic DNA was isolated from unsorted cells (control) and sorted cells (autophagy activated), and the sgRNA region was subjected to next-generation sequencing analysis. We performed the screen twice and plotted the abundance of each sgRNA (Fig. 1E and Table S1). GABARAP, a gene coding the mammalian Atg8 homolog γ-aminobutyric acid receptor-associated protein (GABARAP) (38Slobodkin M.R. Elazar Z. The Atg8 family: Multifunctional ubiquitin-like key regulators of autophagy.Essays Biochem. 2013; 55: 51-64Crossref PubMed Google Scholar), scored highly in both replicates. This is likely because GABARAP competes with the GFP-LC3-RFP reporter to associate with autophagosomal membranes (see Discussion). Genes coding previously reported negative regulators of autophagy, such as the components of mTORC1 and BRD4, were not detected (see Discussion). For the selection of candidate genes for the secondary screening, read counts were computationally analyzed by model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK) (39Li W. Xu H. Xiao T. Cong L. Love M.I. Zhang F. Irizarry R.A. Liu J.S. Brown M. Liu X.S. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.Genome Biol. 2014; 15: 554Crossref PubMed Google Scholar) (Table S2), and the top enriched genes were determined (Table S3). Additionally, based on the scatterplot, genes highly enriched in both replicates were defined as indicated in the rectangle (>−5 log2 fold-change in both replicates) (Fig. 1E). To avoid noise from low-count sgRNAs, sgRNAs with fewer than 30 read counts in the unsorted cells were filtered out (Fig. 1F). Genes with more than two corresponding sgRNAs remaining after processing were recorded (Table S4). From these lists, we selected a total of 21 candidate genes for the secondary screen (Fig. 1, E and F, Tables S2 and S3). Genes rarely expressed in HEK293T cells were excluded (using the published Human Protein Atlas database (40Thul P.J. Åkesson L. Wiking M. Mahdessian D. Geladaki A. Ait Blal H. Alm T. Asplund A. Björk L. Breckels L.M. Bäckström A. Danielsson F. Fagerberg L. Fall J. Gatto L. et al.A subcellular map of the human proteome.Science. 2017; 356eaal3321Crossref PubMed Scopus (882) Google Scholar)). For the secondary screen, HEK293T GFP-LC3-RFP cells were transduced with two independent sgRNAs against each candidate gene. In addition to the 21 candidate genes, RHEB (an activator of mTORC1 (41Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4369) Google Scholar)) was added as a positive control. The cells were analyzed by flow cytometry, and the ratio of the autophagy-activated population to the total population was measured (Fig. 2, A and B). A significant increase in the autophagy-activated population was observed in cells transduced with two sgRNAs for GABARAP and one sgRNA each for RHEB and PFAS. PFAS encodes PFAS, a de novo purine synthesis enzyme (Fig. 3A). Cells depleted of PFAS show defects in de novo purine synthesis and are dependent on the purine salvage pathway for cell growth (42Ali E.S. Sahu U. Villa E. O'Hara B.P. Gao P. Beaudet C. Wood A.W. Asara J.M. Ben-Sahra I. ERK2 phosphorylates PFAS to mediate posttranslational control of de novo purine synthesis.Mol. Cell. 2020; 78: 1178-1191.e6Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In this study, except for in the screens, cells lacking de novo purine synthesis were maintained in a medium supplemented with 100 μM hypoxanthine before analysis. To confirm the specific effect of PFAS knockout, we established a PFAS-KO HEK293T cell clone and then introduced the GFP-LC3-RFP autophagic flux reporter. When cultured in a normal culture medium (without hypoxanthine supplementation) for 2 days, the PFAS-KO clone showed a clear decline in the GFP:RFP signal ratio, which indicated upregulation of autophagic flux (Fig. 3B). This decline was restored by exogenous expression of PFAS-FLAG. These data confirm that the positive results of PFAS knockout in the second screen were not caused by off-target effects. In addition, LentiCRISPR-mediated bulk knockout of PFAS was performed in HeLa and U-2 OS cells expressing the GFP-LC3-RFP reporter. Autophagy activation was observed in both cell lines, suggesting that activation of autophagy by PFAS depletion is not specific to HEK293T (Fig. 3C) To specify the cause of autophagy activation in PFAS-depleted cells, we performed a LentiCRISPR-mediated bulk knockout of PPAT, a gene encoding phosphoribosyl pyrophosphate amidotransferase (PPAT), another essential enzyme in the de novo purine synthesis pathway (Fig. 3A). Additionally, pharmacological inhibition of de novo purine synthesis was performed using methotrexate (Fig. 3A). Bulk knockout of PPAT and methotrexate treatment both induced autophagy activation (Fig. 3D). Taken together, these findings suggest that the activation of autophagy observed in PFAS-KO cells involves the impairment of the de novo purine synthesis pathway. Hoxhaj et al. (43Hoxhaj G. Hughes-Hallett J. Timson R.C. Ilagan E. Yuan M. Asara J.M. Ben-Sahra I. Manning B.D. The mTORC1 signaling network senses changes in cellular purine nucleotide levels.Cell Rep. 2017; 21: 1331-1346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) previously reported that when exogenous purines are depleted, cells deficient in de novo purine synthesis show decreased intracellular purine levels. Therefore, we investigated whether autophagy is activated in response to the depletion of extracellular purine levels in PFAS-KO cells. To exclude the effects of exogenous purines contained in conventional culture media containing 10% serum (estimated to be approximately 7.5 μM (44Harkness R.A. Simmonds R.J. Gough P. Priscott P.K. Squire J.A. Purine base and nucleoside, cytidine and uridine concentrations in foetal calf and other sera [proceedings].Biochem. Soc. Trans. 1980; 8: 139Crossref PubMed Scopus (10) Google Scholar)), we used a purine-depleted medium supplemented with dialyzed serum. Activation of autophagy in PFAS-depleted cells was observed when they were cultured with less than 10 μM exogenous hypoxanthine but was canceled in the presence of sufficient hypoxanthine (more than 30 μM) (Fig. 4, A and B). Additionally, sufficient amounts of exogenous hypoxanthine suppressed the lysosomal turnover of endogenous LC3, observed by the accumulation of endogenous LC3-II upon treatment with lysosomal protease inhibitors, Pepstatin A and E64d (Fig. 4, C and D), and the formation of GFP-LC3 puncta (Fig. 4, E and F). Given that cells deficient in de novo purine synthesis are dependent on the salvage pathway, PFAS-KO cells cultured in a purine-insufficient medium should have been subjected to intracellular purine-starvation conditions. These data suggest that the decline of intracellular purine levels, caused by purine starvation, activates autophagy. Aside from hypoxanthine, sufficient amounts (more than 30 μM) of exogenous inosine, inosine monophosphate, and adenine could completely cancel the autophagy activation, whereas the effect of guanine was limited (Fig. 4G). Together with our findings on purine synthesis pathways (Fig. 3A), the unavailability of hypoxanthine or adenine and their derivatives could mediate purine starvation-induced autophagy. Next, we investigated the mechanism of autophagy activation in purine-starved cells. Recent studies have shown that depletion of intracellular purine bases via inhibition of purine synthesis pathways suppresses mTORC1 (43Hoxhaj G. Hughes-Hallett J. Timson R.C. Ilagan E. Yuan M. Asara J.M. Ben-Sahra I. Manning B.D. The mTORC1 signaling network senses changes in cellular purine nucleotide levels.Cell Rep. 2017; 21: 1331-1346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 45Emmanuel N. Ragunathan S. Shan Q. Wang F. Giannakou A. Huser N. Jin G. Myers J. Abraham R.T. Unsal-Kacmaz K. Purine nucleotide availability regulates mTORC1 activity through the Rheb GTPase.Cell Rep. 2017; 19: 2665-2680Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Therefore, we investigated whether the activation of autophagy is concomitant with the downregulation of mTORC1 in PFAS-depleted cells. Consistent with previous studies, mTORC1 activity was downregulated in PFAS-KO cells depleted of purines, as assessed from the phosphorylation status of its substrates p70 S6-kinase and eukaryotic translation initiation factor 4E-binding protein (Fig. 5A). mTORC1 activity was restored by hypoxanthine treatment or expression of PFAS–FLAG. Previous studies have suggested that purines regulate mTORC1 in a manner dependent on the tuberous sclerosis complex (TSC) protein complex and Ras homolog enriched in brain (RHEB) (43Hoxhaj G. Hughes-Hallett J. Timson R.C. Ilagan E. Yuan M. Asara J.M. Ben-Sahra I. Manning B.D. The mTORC1 signaling network senses changes in cellular purine nucleotide levels.Cell Rep. 2017; 21: 1331-1346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 45Emmanuel N. Ragunathan S. Shan Q. Wang F. Giannakou A. Huser N. Jin G. Myers J. Abraham R.T. Unsal-Kacmaz K. Purine nucleotide availability regulates mTORC1 activity through the Rheb GTPase.Cell Rep. 2017; 19: 2665-2680Abstract