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The Journal of Clinical Investigation Apr 2023The nonessential amino acid asparagine can only be synthesized de novo by the enzymatic activity of asparagine synthetase (ASNS). While ASNS and asparagine have been...
The nonessential amino acid asparagine can only be synthesized de novo by the enzymatic activity of asparagine synthetase (ASNS). While ASNS and asparagine have been implicated in the response to numerous metabolic stressors in cultured cells, the in vivo relevance of this enzyme in stress-related pathways remains unexplored. Here, we found ASNS to be expressed in pericentral hepatocytes, a population of hepatic cells specialized in xenobiotic detoxification. ASNS expression was strongly enhanced in 2 models of acute liver injury: carbon tetrachloride (CCl4) and acetaminophen. We found that mice with hepatocyte-specific Asns deletion were more prone to pericentral liver damage than their control littermates after toxin exposure. This phenotype could be reverted by i.v. administration of asparagine. Unexpectedly, the stress-induced upregulation of ASNS involved an ATF4-independent, noncanonical pathway mediated by the nuclear receptor, liver receptor homolog 1 (LRH-1; NR5A2). Altogether, our data indicate that the induction of the asparagine-producing enzyme ASNS acts as an adaptive mechanism to constrain the necrotic wave that follows toxin administration and provide proof of concept that i.v. delivery of asparagine can dampen hepatotoxin-induced pericentral hepatocellular death.
Topics: Animals; Mice; Asparagine; Hepatocytes; Amino Acids; Liver
PubMed: 36719750
DOI: 10.1172/JCI163508 -
Cell Metabolism May 2021Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an...
Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.
Topics: Activating Transcription Factor 4; Animals; Asparagine; Aspartic Acid; Cell Line, Tumor; Cell Proliferation; Diet; Electron Transport Chain Complex Proteins; Humans; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Inbred NOD; Mitochondria; Neoplasms; Nucleotides; Survival Rate
PubMed: 33609439
DOI: 10.1016/j.cmet.2021.02.001 -
Nature Oct 2022The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide, therapeutic agents used in the treatment of haematopoietic...
The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide, therapeutic agents used in the treatment of haematopoietic malignancies and as ligands for targeted protein degradation. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.
Topics: Humans; Asparagine; Dipeptides; Glutamine; Imides; Lenalidomide; Ligands; Peptide Hydrolases; Proteolysis; Proteome; Thalidomide; Ubiquitination; Ubiquitin-Protein Ligase Complexes; Amino Acid Motifs; Cyclization
PubMed: 36261529
DOI: 10.1038/s41586-022-05333-5 -
Cancer Research Jul 2023Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has a poor prognosis. Pituitary tumor transforming gene 1 (PTTG1) is highly expressed...
UNLABELLED
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and has a poor prognosis. Pituitary tumor transforming gene 1 (PTTG1) is highly expressed in HCC, suggesting it could play an important role in hepatocellular carcinogenesis. Here, we evaluated the impact of PTTG1 deficiency on HCC development using a diethylnitrosamine (DEN)-induced HCC mouse model and a hepatitis B virus (HBV) regulatory X protein (HBx)-induced spontaneous HCC mouse model. PTTG1 deficiency significantly suppressed DEN- and HBx-induced hepatocellular carcinogenesis. Mechanistically, PTTG1 promoted asparagine synthetase (ASNS) transcription by binding to its promoter, and asparagine (Asn) levels were correspondingly increased. The elevated levels of Asn subsequently activated the mTOR pathway to facilitate HCC progression. In addition, asparaginase treatment reversed the proliferation induced by PTTG1 overexpression. Furthermore, HBx promoted ASNS and Asn metabolism by upregulating PTTG1 expression. Overall, PTTG1 is involved in the reprogramming of Asn metabolism to promote HCC progression and may serve as a therapeutic and diagnostic target for HCC.
SIGNIFICANCE
PTTG1 is upregulated in hepatocellular carcinoma and increases asparagine production to stimulate mTOR activity and promote tumor progression.
Topics: Animals; Mice; Asparagine; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hepatitis B virus; Liver Neoplasms; Prognosis; TOR Serine-Threonine Kinases
PubMed: 37159932
DOI: 10.1158/0008-5472.CAN-22-3561 -
Blood Sep 2019Tumor cells rewire metabolic pathways to adapt to their increased nutritional demands for energy, reducing equivalents, and cellular biosynthesis. Alternations in amino... (Review)
Review
Tumor cells rewire metabolic pathways to adapt to their increased nutritional demands for energy, reducing equivalents, and cellular biosynthesis. Alternations in amino acid metabolism are 1 modality for satisfying those demands. Amino acids are not only components of proteins but also intermediate metabolites fueling multiple biosynthetic pathways. Amino acid-depletion therapies target amino acid uptake and catabolism using heterologous enzymes or recombinant or engineered human enzymes. Notably, such therapies have minimal effect on normal cells due to their lower demand for amino acids compared with tumor cells and their ability to synthesize the targeted amino acids under conditions of nutrient stress. Here, we review novel aspects of amino acid metabolism in hematologic malignancies and deprivation strategies, focusing on 4 key amino acids: arginine, asparagine, glutamine, and cysteine. We also present the roles of amino acid metabolism in the immunosuppressive tumor microenvironment and in drug resistance. This summary also offers an argument for the reclassification of amino acid-depleting enzymes as targeted therapeutic agents.
Topics: Amino Acids; Animals; Antineoplastic Agents; Arginine; Asparagine; Cysteine; Glutamine; Hematologic Neoplasms; Humans; Metabolic Networks and Pathways; Molecular Targeted Therapy; Tumor Microenvironment
PubMed: 31416801
DOI: 10.1182/blood.2019001034 -
Nature Aug 2021Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and...
Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.
Topics: Antidepressive Agents; Asparagine; Binding Sites; Cryoelectron Microscopy; Glutamic Acid; Glycine; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Ketamine; Leucine; Molecular Dynamics Simulation; Nerve Tissue Proteins; Receptors, N-Methyl-D-Aspartate
PubMed: 34321660
DOI: 10.1038/s41586-021-03769-9 -
Nature Cancer Nov 2022The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and...
The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin.
Topics: Animals; Mice; Humans; Pancreatic Neoplasms; Asparagine; Adenocarcinoma; Symbiosis; Tumor Microenvironment
PubMed: 36411320
DOI: 10.1038/s43018-022-00463-1 -
Cell Metabolism Apr 2020Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress....
Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value.
Topics: Animals; Asparagine; Aspartate-Ammonia Ligase; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation; Humans; Leukemia; Mice, Inbred NOD; Mice, SCID; Repressor Proteins; Transcription, Genetic
PubMed: 32268116
DOI: 10.1016/j.cmet.2020.03.008 -
Nature Cell Biology Jan 2021Nutrient availability is central for T-cell functions and immune responses. Here we report that CD8 T-cell activation and anti-tumour responses are strongly potentiated...
Nutrient availability is central for T-cell functions and immune responses. Here we report that CD8 T-cell activation and anti-tumour responses are strongly potentiated by the non-essential amino acid Asn. Increased Asn levels enhance CD8 T-cell activation and effector functions against tumour cells in vitro and in vivo. Conversely, restriction of dietary Asn, ASNase administration or inhibition of the Asn transporter SLC1A5 impairs the activity and responses of CD8 T cells. Mechanistically, Asn does not directly alter cellular metabolic fluxes; it instead binds the SRC-family protein tyrosine kinase LCK and orchestrates LCK phosphorylation at Tyr 394 and 505, thereby leading to enhanced LCK activity and T-cell-receptor signalling. Thus, our findings reveal a critical and metabolism-independent role for Asn in the direct modulation of the adaptive immune response by controlling T-cell activation and efficacy, and further uncover that LCK is a natural Asn sensor signalling Asn sufficiency to T-cell functions.
Topics: Animals; Asparagine; CD8-Positive T-Lymphocytes; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Phosphorylation; Signal Transduction; Tumor Cells, Cultured; Tyrosine; src-Family Kinases
PubMed: 33420490
DOI: 10.1038/s41556-020-00615-4 -
Molecular Cell May 2022GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis....
GLS1 orchestrates glutaminolysis and promotes cell proliferation when glutamine is abundant by regenerating TCA cycle intermediates and supporting redox homeostasis. CB-839, an inhibitor of GLS1, is currently under clinical investigation for a variety of cancer types. Here, we show that GLS1 facilitates apoptosis when glutamine is deprived. Mechanistically, the absence of exogenous glutamine sufficiently reduces glutamate levels to convert dimeric GLS1 to a self-assembled, extremely low-K filamentous polymer. GLS1 filaments possess an enhanced catalytic activity, which further depletes intracellular glutamine. Functionally, filamentous GLS1-dependent glutamine scarcity leads to inadequate synthesis of asparagine and mitogenome-encoded proteins, resulting in ROS-induced apoptosis that can be rescued by asparagine supplementation. Physiologically, we observed GLS1 filaments in solid tumors and validated the tumor-suppressive role of constitutively active, filamentous GLS1 mutants K320A and S482C in xenograft models. Our results change our understanding of GLS1 in cancer metabolism and suggest the therapeutic potential of promoting GLS1 filament formation.
Topics: Apoptosis; Asparagine; Glutaminase; Glutamine; Humans; Reactive Oxygen Species
PubMed: 35381197
DOI: 10.1016/j.molcel.2022.03.016