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• W2783201301 abstract "•Glutamine-deprived cells sustain proliferation via de novo glutamine biosynthesis•Proliferation and protein synthesis in the absence of glutamine requires asparagine•Mammalian cells do not catabolize asparagine•Asparaginase blocks the ability of cells to adapt to glutamine deprivation When mammalian cells are deprived of glutamine, exogenous asparagine rescues cell survival and growth. Here we report that this rescue results from use of asparagine in protein synthesis. All mammalian cell lines tested lacked cytosolic asparaginase activity and could not utilize asparagine to produce other amino acids or biosynthetic intermediates. Instead, most glutamine-deprived cell lines are capable of sufficient glutamine synthesis to maintain essential amino acid uptake and production of glutamine-dependent biosynthetic precursors, with the exception of asparagine. While experimental introduction of cytosolic asparaginase could enhance the synthesis of glutamine and increase tricarboxylic acid cycle anaplerosis and the synthesis of nucleotide precursors, cytosolic asparaginase suppressed the growth and survival of cells in glutamine-depleted medium in vitro and severely compromised the in vivo growth of tumor xenografts. These results suggest that the lack of asparaginase activity represents an evolutionary adaptation to allow mammalian cells to survive pathophysiologic variations in extracellular glutamine. When mammalian cells are deprived of glutamine, exogenous asparagine rescues cell survival and growth. Here we report that this rescue results from use of asparagine in protein synthesis. All mammalian cell lines tested lacked cytosolic asparaginase activity and could not utilize asparagine to produce other amino acids or biosynthetic intermediates. Instead, most glutamine-deprived cell lines are capable of sufficient glutamine synthesis to maintain essential amino acid uptake and production of glutamine-dependent biosynthetic precursors, with the exception of asparagine. While experimental introduction of cytosolic asparaginase could enhance the synthesis of glutamine and increase tricarboxylic acid cycle anaplerosis and the synthesis of nucleotide precursors, cytosolic asparaginase suppressed the growth and survival of cells in glutamine-depleted medium in vitro and severely compromised the in vivo growth of tumor xenografts. These results suggest that the lack of asparaginase activity represents an evolutionary adaptation to allow mammalian cells to survive pathophysiologic variations in extracellular glutamine. Most cultured mammalian cells fail to proliferate or even survive in the absence of exogenous glutamine, despite possessing the enzymatic machinery for its de novo synthesis from the tricarboxylic acid (TCA) cycle carbon and free ammonium. Studies from the past decade convincingly demonstrate that glutamine, a versatile donor of nitrogen and carbon atoms for diverse biosynthetic reactions, is consumed by proliferating cells in excess of other amino acids. Major biosynthetic products of glutamine include nucleotides, non-essential amino acids (NEAAs) and the anaplerotic substrates of the TCA cycle (Vander Heiden and DeBerardinis, 2017Vander Heiden M.G. DeBerardinis R.J. Understanding the intersections between metabolism and cancer biology.Cell. 2017; 168: 657-669Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). Indeed, glutamine-derived α-ketoglutarate (α-KG), a key intermediate of the TCA cycle, has been proposed to be the key determinant of cellular glutamine dependency. Thus, a cell permeable α-KG was shown to be sufficient to rescue glutamine depletion-induced apoptosis in MYC-transformed cells (Wise et al., 2008Wise D.R. DeBerardinis R.J. Mancuso A. Sayed N. Zhang X.Y. Pfeiffer H.K. Nissim I. Daikhin E. Yudkoff M. McMahon S.B. et al.Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction.Proc. Natl. Acad. Sci. USA. 2008; 105: 18782-18787Crossref PubMed Scopus (1413) Google Scholar, Yuneva et al., 2007Yuneva M. Zamboni N. Oefner P. Sachidanandam R. Lazebnik Y. Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells.J. Cell Biol. 2007; 178: 93-105Crossref PubMed Scopus (522) Google Scholar). More recently, asparagine, the amino acid most structurally similar to glutamine, was also found to be sufficient to reduce glutamine-depletion-induced apoptosis in MYC-transformed cells (Zhang et al., 2014Zhang J. Fan J. Venneti S. Cross J.R. Takagi T. Bhinder B. Djaballah H. Kanai M. Cheng E.H. Judkins A.R. et al.Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.Mol. Cell. 2014; 56: 205-218Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). In most species, either asparagine or glutamine can be used as substrates to maintain TCA cycle anaplerosis following intracellular deamidation initiated by cytosolic asparaginase or glutaminase, respectively. However, in mammals asparaginase expression is restricted to the liver, kidney, and smooth muscle, and is not observed in proliferative tissues or cancer cell lines. This raises the question of how exogenous asparagine is utilized to sustain cell survival and growth when extracellular glutamine becomes depleted. The cellular consumption of glutamine rises in concordance with the growth rate. Accumulating evidence suggests that glutamine becomes preferentially depleted in the milieu of a variety of tumor types in vivo, or in the tissues where the vascular supply is compromised (Kamphorst et al., 2015Kamphorst J.J. Nofal M. Commisso C. Hackett S.R. Lu W. Grabocka E. Vander Heiden M.G. Miller G. Drebin J.A. Bar-Sagi D. et al.Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein.Cancer Res. 2015; 75: 544-553Crossref PubMed Scopus (498) Google Scholar, Márquez et al., 1989Márquez J. Sánchez-Jiménez F. Medina M.A. Quesada A.R. Núñez de Castro I. Nitrogen metabolism in tumor bearing mice.Arch. Biochem. Biophys. 1989; 268: 667-675Crossref PubMed Scopus (72) Google Scholar, Rivera et al., 1988Rivera S. Azcón-Bieto J. López-Soriano F.J. Miralpeix M. Argilés J.M. Amino acid metabolism in tumour-bearing mice.Biochem. J. 1988; 249: 443-449Crossref PubMed Scopus (60) Google Scholar, Roberts et al., 1956Roberts E. Simonsen D.G. Tanaka K.K. Tanaka T. Free amino acids in growing and regressing ascites cell tumors: host resistance and chemical agents.Cancer Res. 1956; 16: 970-978PubMed Google Scholar). Even within the same tumor tissue, the levels of glutamine were found to be substantially lower in the tumor core compared with the peripheral regions (Pan et al., 2016Pan M. Reid M.A. Lowman X.H. Kulkarni R.P. Tran T.Q. Liu X. Yang Y. Hernandez-Davies J.E. Rosales K.K. Li H. et al.Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation.Nat. Cell Biol. 2016; 18: 1090-1101Crossref PubMed Scopus (212) Google Scholar). These results suggest that cell growth in vascularly compromised tissues may need to employ adaptive mechanisms to maintain glutamine levels necessary to sustain cell survival and proliferation. One potential mechanism to maintain glutamine levels under exogenous glutamine limitation is via its de novo biosynthesis (Bott et al., 2015Bott A.J. Peng I.C. Fan Y. Faubert B. Zhao L. Li J. Neidler S. Sun Y. Jaber N. Krokowski D. et al.Oncogenic Myc induces expression of glutamine synthetase through promoter demethylation.Cell Metab. 2015; 22: 1068-1077Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Carey et al., 2015Carey B.W. Finley L.W. Cross J.R. Allis C.D. Thompson C.B. Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (597) Google Scholar, Tardito et al., 2015Tardito S. Oudin A. Ahmed S.U. Fack F. Keunen O. Zheng L. Miletic H. Sakariassen P.O. Weinstock A. Wagner A. et al.Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.Nat. Cell Biol. 2015; 17: 1556-1568Crossref PubMed Scopus (334) Google Scholar). Here we show that asparagine rescued cell proliferation in the setting of exogenous glutamine deficit without being catabolized. The restoration of cell proliferation correlated with a restoration of global protein synthesis, and required glutamine synthetase (GLUL). Exogenous asparagine was used exclusively for protein synthesis and not for either TCA cycle anaplerosis or glutamine synthesis. In contrast to lower metazoan organisms, we found that mammalian cells lack the ability to catabolize asparagine to aspartate and free ammonia, which makes them unable to utilize asparagine’s carbon and nitrogen atoms toward the biosynthesis of glutamine when the latter is depleted. We demonstrate that the expression of the yeast (ASP1) or zebrafish (zASPG) asparaginase in mammalian cells restores the capacity of mammalian cells to use asparagine as a biosynthetic substrate. However, the cytosolic activity of these asparaginases becomes a liability in the setting of exogenous glutamine limitation in culture. Under physiological concentrations of asparagine (<0.1 mM), expression of a functional asparaginase rendered cancer cells incapable of engaging in protein synthesis and cell proliferation when exogenous glutamine became limiting. Expression of a cytosolic asparaginase also suppressed tumor cell growth in vivo, indicating altogether that the access to exogenous asparagine is critical for cell survival and growth as vascularly compromised tissues and tumors become depleted of glutamine. Glutamine depletion-induced apoptosis has been shown to be suppressed by exogenous asparagine (Zhang et al., 2014Zhang J. Fan J. Venneti S. Cross J.R. Takagi T. Bhinder B. Djaballah H. Kanai M. Cheng E.H. Judkins A.R. et al.Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.Mol. Cell. 2014; 56: 205-218Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). This pro-survival effect of asparagine was evident across a variety of cell lines, regardless of their tissue of origin or method of transformation (Figure S1A). For example, we found that, in a number of breast cancer cell lines, supplementation with asparagine allowed cell proliferation in the absence of exogenous glutamine (Figure 1A). In contrast, a cell-permeable form of α-KG, while restoring intracellular α-KG and citrate levels, did not rescue proliferation (Figures 1B and S1B, left panel), which suggests that asparagine-mediated rescue of proliferation in the absence of glutamine is independent of restoring the anaplerotic input into the TCA cycle. To determine whether the effect of asparagine on cell proliferation in the absence of glutamine is dose-dependent, we manipulated the concentration of glutamine or asparagine in the medium. We found that under glutamine depletion, asparagine restored the maximal proliferation at concentrations as low as 10–25 μM, while higher asparagine levels did not further enhance cell proliferation (Figure 1C, left). In addition, supplementation with additional ammonium and aspartate (alone or in combination) also failed to promote proliferation further (Figure S1B, middle and right panels). Equimolar concentrations (5–50 μM) of glutamine failed to maintain cell proliferation as well, indicating that asparagine is superior to glutamine in sustaining cell proliferation when present at low concentrations. Indeed, 250 μM glutamine was needed to surpass the level of cell proliferation supported by 25 μM asparagine (Figure 1C, right). Similar to glutamine, asparagine is utilized as a preferred nitrogen source to activate target of rapamycin (TOR)-dependent translation and growth in yeast (Cardenas et al., 1999Cardenas M.E. Cutler N.S. Lorenz M.C. Di Como C.J. Heitman J. The TOR signaling cascade regulates gene expression in response to nutrients.Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (479) Google Scholar, Godard et al., 2007Godard P. Urrestarazu A. Vissers S. Kontos K. Bontempi G. van Helden J. André B. Effect of 21 different nitrogen sources on global gene expression in the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 2007; 27: 3065-3086Crossref PubMed Scopus (184) Google Scholar, Hardwick et al., 1999Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.Proc. Natl. Acad. Sci. USA. 1999; 96: 14866-14870Crossref PubMed Scopus (466) Google Scholar). In addition, both glutamine and asparagine have been shown to facilitate the uptake of EAAs for protein synthesis (Krall et al., 2016Krall A.S. Xu S. Graeber T.G. Braas D. Christofk H.R. Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.Nat. Commun. 2016; 7: 11457Crossref PubMed Scopus (271) Google Scholar, Nicklin et al., 2009Nicklin P. Bergman P. Zhang B. Triantafellow E. Wang H. Nyfeler B. Yang H. Hild M. Kung C. Wilson C. et al.Bidirectional transport of amino acids regulates mTOR and autophagy.Cell. 2009; 136: 521-534Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar). To test the hypothesis that asparagine may rescue cell proliferation in the absence of glutamine via restoring the import of EAAs, we performed gas chromatography-mass spectrometry analysis to quantify the intracellular levels of EAAs in glutamine-deprived cells. Surprisingly, the levels of the leucine, isoleucine, methionine, phenylalanine, threonine, tyrosine, and valine were significantly increased in glutamine-deprived cells and returned to levels observed in cells grown in complete medium when glutamine-deficient medium was supplemented with asparagine (Figure 1D). A similar pattern (leucine as an example) is seen in every cell line we have tested (Figure S1C, left), and comparable effects were also observed with NEAAs that are present in the standard DMEM (such as serine) (Figure S1C, right). To determine whether the observed increase in amino acid levels was due to autophagy resulting from glutamine depletion (Cheong et al., 2011Cheong H. Lindsten T. Wu J. Lu C. Thompson C.B. Ammonia-induced autophagy is independent of ULK1/ULK2 kinases.Proc. Natl. Acad. Sci. USA. 2011; 108: 11121-11126Crossref PubMed Scopus (264) Google Scholar), which may stimulate protein catabolism and amino acid accumulation, we measured the autophagy flux when glutamine was withdrawn or replaced with asparagine. As measured by LC3 conjugation, autophagy flux was not altered under these conditions (Figure S1D). We also measured amino acid depletion in the culture medium in the conditions indicated. The depletion of leucine, serine, or methionine from the medium correlated in timing and extent with the accumulation of cellular protein as the cells proliferated (Figures S1E and S1F). We hypothesized that the influx of other amino acids could compromise the ability of cells to retain intracellular glutamine to support cell survival and proliferation due to the existing import/antiport exchanges between glutamine and other amino acids via system L (Nicklin et al., 2009Nicklin P. Bergman P. Zhang B. Triantafellow E. Wang H. Nyfeler B. Yang H. Hild M. Kung C. Wilson C. et al.Bidirectional transport of amino acids regulates mTOR and autophagy.Cell. 2009; 136: 521-534Abstract Full Text Full Text PDF PubMed Scopus (1274) Google Scholar). To test this, we supplemented low-glutamine-containing medium with varied concentrations of EAAs plus serine and glycine (MEM/S/G). Indeed, elevated extracellular levels of EAAs compromised cell proliferation and survival in T47D and SF188 cells under low-glutamine conditions, while no inhibitory effect was observed under glutamine-replete state (Figures S1G and S1H). However, this effect was relatively minor at 0.5× DMEM, which contains close to physiological levels of system L-imported amino acids (Table S1). The marked increase of intracellular amino acids in glutamine-depleted cells and its restoration back to glutamine-replete levels by asparagine supplementation prompted us to investigate whether this increase was due to the suppression of protein synthesis in glutamine-starved cells. We also hypothesized that asparagine supplementation may act via facilitating translation under glutamine-depleted conditions. Asparagine supplementation was able to restore translation in the absence of glutamine, as measured by the incorporation of puromycin, a tyrosyl-tRNA mimetic (Figure 1E). Even in cells in which asparagine primarily rescues survival, asparagine supplementation was able to suppress the translation inhibition induced by glutamine withdrawal (Figure S1I). In addition, we observed that accumulation of short-lived proteins, such as HIF1α and c-Myc (Figure 1F), triggered by blocking their targeted proteolytic degradation with a proteasome (in case of both HIF1α and c-Myc) or a lysosome inhibitor (in case of HIF1α) was profoundly compromised in glutamine-depleted cells. Asparagine supplementation resulted in a marked restoration of this accumulation. Protein synthesis cannot proceed in the absence of glutamine. Proliferating cells have to rely on de novo glutamine biosynthesis when the exogenous glutamine is limited. The rate-limiting enzyme for glutamine biosynthesis is GLUL, which has been shown to play a critical role for cell proliferation of murine embryonic stem cells and glioma cells in the absence of exogenous glutamine (Carey et al., 2015Carey B.W. Finley L.W. Cross J.R. Allis C.D. Thompson C.B. Intracellular alpha-ketoglutarate maintains the pluripotency of embryonic stem cells.Nature. 2015; 518: 413-416Crossref PubMed Scopus (597) Google Scholar, Tardito et al., 2015Tardito S. Oudin A. Ahmed S.U. Fack F. Keunen O. Zheng L. Miletic H. Sakariassen P.O. Weinstock A. Wagner A. et al.Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.Nat. Cell Biol. 2015; 17: 1556-1568Crossref PubMed Scopus (334) Google Scholar). Notably, GLUL protein was previously shown to be upregulated in glutamine-depleted cells in a post-transcriptional manner (Nguyen et al., 2016Nguyen T.V. Lee J.E. Sweredoski M.J. Yang S.J. Jeon S.J. Harrison J.S. Yim J.H. Lee S.G. Handa H. Kuhlman B. et al.Glutamine triggers acetylation-dependent degradation of glutamine synthetase via the thalidomide receptor cereblon.Mol. Cell. 2016; 61: 809-820Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Therefore, we asked whether the availability of asparagine, owing to its ability to facilitate translation in the absence of exogenous glutamine, may be critical for enabling adaptive upregulation of GLUL when extracellular glutamine is depleted. Strikingly, we found that GLUL protein levels showed little or no induction following glutamine depletion in the lines tested, unless exogenous asparagine was added. Asparagine supplementation significantly increased GLUL levels in all cell lines tested (Figures 2A and S2A). The accumulation of GLUL protein was not due to increased GLUL mRNA (Figure S2B), suggesting that increased transcription was not responsible for its accumulation. To further interrogate the critical role of GLUL in the adaptation of cells to glutamine limitation, we treated cells with methionine sulfoximine (MSO), a GLUL inhibitor. This resulted in a complete suppression of asparagine-induced translation (Figures 2B and S2C), as well as asparagine-dependent cell proliferation (Figure 2C) under glutamine-limited conditions, while having no effect on glutamine-replete cells. While asparagine facilitated GLUL protein expression in glutamine-depleted cells, replacing glutamine with other NEAAs (including alanine, aspartate, glutamate, or proline) individually had no effect on GLUL protein levels (Figure 2D), which is consistent with the inability of these amino acids to rescue proliferation of glutamine-depleted cells (Figure 2E). Furthermore, the observed effect on GLUL protein levels was specific to glutamine depletion, and not a feature of the general amino acid stress response induced by glutamine withdrawal. Indeed, depletion of valine triggered a comparable upregulation of ATF4, a principal amino acid stress-response effector, yet it did not induce GLUL protein, regardless of the presence or absence of asparagine (Figure 2F). In addition to suppressing the general amino acid response elicited by glutamine depletion, asparagine also markedly restored mTORC1 activity (Figure 2G), which has previously been shown to become compromised in the setting of glutamine withdrawal. Notably, the observed restoration was specific to the proteinogenic L-isoform of asparagine (Figure 2H). To determine whether GLUL is required for cell proliferation in glutamine-deficient medium supplemented with asparagine, we used CRISPR/Cas9 to genetically inactivate GLUL, leading to the elimination of GLUL protein (Figure S2D). GLUL depletion by three independent single-guide RNAs (sgRNAs) had no effect on cell proliferation when exogenous glutamine was provided (Figure 2I, left). In contrast, proliferation was profoundly compromised in GLUL-depleted cells when glutamine-deficient medium was supplemented with asparagine (Figure 2I, right). Introduction of an inducible mouse GLUL construct into GLUL-depleted cells resulted in a marked restoration of asparagine-driven proliferation (Figures 2J and 2K). Consistently with their effects on proliferation, MSO treatment, as well as sgRNAs directed against GLUL, also inhibited the short-lived protein accumulation under the same conditions (Figures S2E–S2G). Despite a marked restoration of protein synthesis and proliferation, asparagine supplementation did not restore intracellular glutamine levels in glutamine-deprived cells (Figure S2H, left), and did not cause any glutamine accumulation in conditioned medium (Figure S2H, right). In contrast, glutamate, which must be synthesized de novo when glutamine is absent, accumulated in the conditioned medium from glutamine-deprived cells. This accumulation was significantly repressed by asparagine supplementation (Figure S2I), suggesting that glutamate is both undergoing increased conversion to glutamine and being utilized to support on-going protein synthesis in asparagine-supplemented cells. Asparagine-driven repression of glutamate levels in the medium was dependent on GLUL activity, as no such effect was observed in GLUL-depleted cells (Figure S2J). To further verify that glutamine is being synthesized de novo in glutamine-deprived cells, we performed metabolic tracing experiments. We hypothesized that the majority of carbon for the de novo synthesis of glutamine must be derived from glucose, as glucose-derived pyruvate can serve as an anaplerotic substrate for the TCA cycle in cultured cells, when glutamine is unavailable. Indeed, exposing glutamine-deprived cells to [U-13C]glucose for 6 hr revealed that glucose carbons become readily incorporated into glutamine; moreover, asparagine supplementation facilitated this pattern (Figure S2K). Recently, it has been suggested that stromal cells might use asparagine as a carbon source to synthesize glutamine, and thus supply neighboring tumor cells with glutamine when tissue levels of glutamine are low (Yang et al., 2016Yang L. Achreja A. Yeung T.L. Mangala L.S. Jiang D. Han C. Baddour J. Marini J.C. Ni J. Nakahara R. et al.Targeting stromal glutamine synthetase in tumors disrupts tumor microenvironment-regulated cancer cell growth.Cell Metab. 2016; 24: 685-700Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). For asparagine-derived carbon to support glutamine biosynthesis, asparagine needs to be first catabolized to aspartate (Figure 3A). In bacteria and yeast, this reaction is catalyzed by cytosolic asparaginase (Dunlop and Roon, 1975Dunlop P.C. Roon R.J. L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.J. Bacteriol. 1975; 122: 1017-1024Crossref PubMed Google Scholar, Jones and Mortimer, 1970Jones G.E. Mortimer R.K. L-asparaginase-deficient mutants of yeast.Science. 1970; 167: 181-182Crossref PubMed Scopus (7) Google Scholar, Peterson and Ciegler, 1969Peterson R.E. Ciegler A. L-asparaginase production by various bacteria.Appl. Microbiol. 1969; 17: 929-930Crossref PubMed Google Scholar); however, cytosolic asparaginase activity has not been reliably reported in mammalian cells (Figure 3A). To determine whether asparagine can be catabolized to aspartate and free ammonium by cells in the setting of glutamine depletion, we measured the intracellular levels of aspartate and the free ammonium released into the medium. Glutamine depletion significantly reduced the intracellular aspartate and free ammonium release (Figures 3B and S3A). This was expected, as glutaminase-driven deamidation of glutamine is the main source of ammonium production, and the resulting glutamate is the driver of the transamination of oxaloacetate in proliferating cells. Neither the levels of intracellular aspartate nor extracellular free ammonium were restored by asparagine in T47D cells (Figures 3B and S3A). Indeed, none of the TCA cycle intermediates or the NEAA alanine and proline could be restored by asparagine in T47D cells under glutamine deprivation (Figures S3B and S3C). To determine whether the inability to catabolize asparagine is a general feature of mammalian cells, we tested a panel of mammalian cell lines from diverse tissues of origin, and found no meaningful restoration of aspartate by asparagine in any of the lines tested (Figure S3D). To rule out the possibility that the failure to detect asparagine catabolism is due to the artifact of mammalian cell culture, we infused mice with [U-13C]asparagine for 2 hr. Labeled asparagine readily entered various mouse tissues, with 30%–60% of tissue asparagine content becoming labeled after just 2 hr (Figure S3E). However, asparagine-derived carbons did not contribute meaningfully to the pools of aspartate or TCA cycle intermediates in any tissue (Figure S3F and data not shown). In contrast to mammalian tissues and cell lines, we found that cell lines derived from fruit fly or from zebrafish readily utilized asparagine to maintain intracellular aspartate, even beyond the levels normally achieved via the use of glutamine (Figure 3C). The human genome encodes two genes with homology to the cytosolic asparaginases of lower organisms. The first is ASPG, a human cytosolic asparaginase (Karamitros and Konrad, 2014Karamitros C.S. Konrad M. Human 60-kDa lysophospholipase contains an N-terminal L-asparaginase domain that is allosterically regulated by L-asparagine.J. Biol. Chem. 2014; 289: 12962-12975Crossref PubMed Scopus (30) Google Scholar). Expression of hASPG was found to be less than 1 per 106 transcripts in all mammalian cell lines tested (Uhlén et al., 2015Uhlén M. Fagerberg L. Hallström B.M. Lindskog C. Oksvold P. Mardinoglu A. Sivertsson Å. Kampf C. Sjöstedt E. Asplund A. et al.Proteomics. Tissue-based map of the human proteome.Science. 2015; 347: 1260419Crossref PubMed Scopus (7332) Google Scholar) (Figure S4F) and not detectable in the cell lines tested here. Furthermore, ectopically expressed hASPG did not display significant asparaginase activity in T47D cells under assay conditions, where comparably expressed zebrafish (zASPG) asparaginase exhibited robust and specific activity (Figure S4I). This is consistent with the high S0.5 of human asparaginase in comparison with other species; namely, 0.4 mM for bacterial and 11 mM for human isoforms (Karamitros and Konrad, 2014Karamitros C.S. Konrad M. Human 60-kDa lysophospholipase contains an N-terminal L-asparaginase domain that is allosterically regulated by L-asparagine.J. Biol. Chem. 2014; 289: 12962-12975Crossref PubMed Scopus (30) Google Scholar). Since the key catalytic residues are conserved between bacterial, yeast, and human asparaginases (Karamitros and Konrad, 2014Karamitros C.S. Konrad M. Human 60-kDa lysophospholipase contains an N-terminal L-asparaginase domain that is allosterically regulated by L-asparagine.J. Biol. Chem. 2014; 289: 12962-12975Crossref PubMed Scopus (30) Google Scholar), the reason behind the reduced enzymatic activity of human asparaginase remains to be elucidated. The other human gene with homology to asparaginases from lower organisms is asparaginase-like 1 (ASRGL1). Although its reported function is hydrolysis of β-aspartyl residues from the N termini of proteins (Cantor et al., 2009Cantor J.R. Stone E.M. Chantranupong L. Georgiou G. The human asparaginase-like protein 1 hASRGL1 is an Ntn hydrolase with beta-aspartyl peptidase activity.Biochemistry. 2009; 48: 11026-11031Crossref PubMed Scopus (63) Google Scholar), ASRGL1 is expressed in a wide variety of mammalian cell lines, including several of those studied here (Figures S4G and S4H, left). At the levels of endogenous expression of ASRGL1, we were unable to detect amidase activity in the cytosol of the studied cell lines, including T47D, MCF7, and MDA-MB-468 (Figure S4H, right). As a positive control, we transduced T47D cells with a retrovirus-encoded ASRGL1, achieving ∼100-fold increase in its expression (Figure S4J). At this level of ASRGL1 expression we were able to detect a modest amidase activity (Figure S4I). These results are consistent with a relatively high reported Km (∼3.4 mM) of ASRGL1 for asparagine (Cantor et al., 2009Cantor J.R. Stone E.M. Chantranupong L. Georgiou G. The human asparaginase-like protein 1 hASRGL1 is an Ntn hydrolase with beta-aspartyl peptidase activity.Biochemistry. 2009; 48: 11026-11031Crossref PubMed Scopus (63) Google Scholar). Taken together, at physiologic levels of expression and" @default.
• W2783201301 created "2018-01-26" @default.
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• W2783201301 date "2018-02-01" @default.
• W2783201301 modified "2023-10-11" @default.
• W2783201301 title "As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid" @default.
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