ABSTRACT
Cancer cells frequently alter their lipids to grow and adapt to their environment1-3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.
ABSTRACT
Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as "PS"), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery.
ABSTRACT
Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.
Subject(s)
Carcinoma, Hepatocellular , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits , HSP40 Heat-Shock Proteins , Liver Neoplasms , Humans , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/metabolism , Cyclic AMP-Dependent Protein Kinase Catalytic Subunits/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Gene Expression Regulation, Neoplastic , Sodium-Potassium-Exchanging ATPaseABSTRACT
Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.
Subject(s)
Bacteria , Bacteriophage T4 , DNA Glycosylases , Bacteria/classification , Bacteria/enzymology , Bacteria/genetics , Bacteria/immunology , Bacteria/virology , Bacteriophage T4/growth & development , Bacteriophage T4/immunology , Bacteriophage T4/metabolism , DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Escherichia coli/genetics , Escherichia coli/virology , Gene Library , Metagenomics/methods , Soil Microbiology , Virus ReplicationABSTRACT
Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.
Subject(s)
Disulfides , Disulfides/chemistry , Disulfides/metabolism , Humans , Mass Spectrometry/methods , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Peptides/chemistry , Peptides/metabolism , Proteomics/methodsABSTRACT
Cancers commonly reprogram translation and metabolism, but little is known about how these two features coordinate in cancer stem cells. Here we show that glioblastoma stem cells (GSCs) display elevated protein translation. To dissect underlying mechanisms, we performed a CRISPR screen and identified YRDC as the top essential transfer RNA (tRNA) modification enzyme in GSCs. YRDC catalyzes the formation of N6-threonylcarbamoyladenosine (t6A) on ANN-decoding tRNA species (A denotes adenosine, and N denotes any nucleotide). Targeting YRDC reduced t6A formation, suppressed global translation and inhibited tumor growth both in vitro and in vivo. Threonine is an essential substrate of YRDC. Threonine accumulated in GSCs, which facilitated t6A formation through YRDC and shifted the proteome to support mitosis-related genes with ANN codon bias. Dietary threonine restriction (TR) reduced tumor t6A formation, slowed xenograft growth and augmented anti-tumor efficacy of chemotherapy and anti-mitotic therapy, providing a molecular basis for a dietary intervention in cancer treatment.
Subject(s)
Glioblastoma , Threonine , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/metabolism , Humans , Animals , Mice , Threonine/metabolism , Threonine/genetics , Protein Biosynthesis , Neoplastic Stem Cells/metabolism , Cell Line, Tumor , Codon/genetics , RNA, Transfer/genetics , Xenograft Model Antitumor Assays , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Brain Neoplasms/metabolismABSTRACT
Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. We recently reported a family with a paternally inherited intragenic ASTN2 duplication with a range of neurodevelopmental disorders, including autism spectrum disorder (ASD), learning difficulties, and speech and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity and repetitive behaviors, altered social behaviors, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors were also prominent in Astn2 cKO animals. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrates a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents (IPSCs) in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.
ABSTRACT
Arginine and lysine, frequently appearing as a pair on histones, have been proven to carry diverse modifications and execute various epigenetic regulatory functions. However, the most context-specific and transient effectors of these marks, while significant, have evaded study as detection methods have thus far not reached a standard to capture these ephemeral events. Herein, a pair of complementary photo-arginine/δ-photo-lysine (R-dz/K-dz) probes is developed and involve these into histone peptide, nucleosome, and chromatin substrates to capture and explore the interactomes of Arg and Lys hPTMs. By means of these developed tools, this study identifies that H3R2me2a can recruit MutS protein homolog 6 (MSH6), otherwise repelDouble PHD fingers 2 (DPF2), Retinoblastoma binding protein 4/7 (RBBP4/7). And it is disclosed that H3R2me2a inhibits the chromatin remodeling activity of the cBAF complex by blocking the interaction between DPF2 (one component of cBAF) and the nucleosome. In addition, the novel pairs of H4K5 PTMs and respective readers are highlighted, namely H4K5me-Lethal(3)malignant brain tumor-like protein 2 (L3MBTL2), H4K5me2-L3MBTL2, and H4K5acK8ac-YEATS domain-containing protein 4 (YEATS4). These powerful tools pave the way for future investigation of related epigenetic mechanisms including but not limited to hPTMs.
Subject(s)
Lysine , Nucleosomes , Lysine/metabolism , Protein Processing, Post-Translational , Histones/metabolism , Chromatin , Arginine/metabolismABSTRACT
The T-cell receptor (TCR) is central to the ligand-dependent activation of T lymphocytes and as such orchestrates both adaptive and pathologic immune processes 1 . However, major questions remain regarding the structure and function of the human TCR 2-4 . Here, we present cryogenic electron microscopy structures for the unliganded human TCR-CD3 complex in a native-like lipid bilayer, revealing two related conformations that are distinct from its structure in detergent. These new "closed and compacted" conformations afford insights into the interactions between the TCR-CD3 and the membrane, including conserved surface patches that make extensive outer leaflet contact, and suggest novel conformational regulation by glycans. We show that the closed/compacted conformations, not the extended one previously reported in detergent 5-8 , represent the unliganded resting state for the TCR-CD3 in vivo , underscoring the importance of structural interrogation of membrane proteins in native-like environments. We use conformation-locking disulfide mutants to show that ectodomain opening is necessary for maximal ligand-dependent TCR-CD3 activation, demonstrating that TCR-intrinsic conformational change is necessary for full TCR-CD3 activation and opening numerous avenues for immunoreceptor engineering.
ABSTRACT
Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms. For complete details on the use and execution of this protocol, please refer to Hoshino et al.1.
Subject(s)
Extracellular Vesicles , Proteomics , Humans , Animals , Mice , Blotting, Western , Computational Biology , Mass SpectrometryABSTRACT
Mitochondria must maintain adequate amounts of metabolites for protective and biosynthetic functions. However, how mitochondria sense the abundance of metabolites and regulate metabolic homeostasis is not well understood. In this work, we focused on glutathione (GSH), a critical redox metabolite in mitochondria, and identified a feedback mechanism that controls its abundance through the mitochondrial GSH transporter, SLC25A39. Under physiological conditions, SLC25A39 is rapidly degraded by mitochondrial protease AFG3L2. Depletion of GSH dissociates AFG3L2 from SLC25A39, causing a compensatory increase in mitochondrial GSH uptake. Genetic and proteomic analyses identified a putative iron-sulfur cluster in the matrix-facing loop of SLC25A39 as essential for this regulation, coupling mitochondrial iron homeostasis to GSH import. Altogether, our work revealed a paradigm for the autoregulatory control of metabolic homeostasis in organelles.
Subject(s)
ATP-Dependent Proteases , ATPases Associated with Diverse Cellular Activities , Glutathione , Mitochondria , Mitochondrial Proteins , Phosphate Transport Proteins , Glutathione/metabolism , Homeostasis , Iron/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Proteomics , Feedback, Physiological , Mitochondrial Proteins/metabolism , Phosphate Transport Proteins/metabolism , Humans , Iron-Sulfur Proteins/metabolism , Proteolysis , HEK293 Cells , ATP-Dependent Proteases/genetics , ATP-Dependent Proteases/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolismABSTRACT
Fibrolamellar hepatocellular carcinoma (FLC) is a usually lethal primary liver cancer driven by a somatic dysregulation of protein kinase A. We show that the proteome of FLC tumors is distinct from that of adjacent nontransformed tissue. These changes can account for some of the cell biological and pathological alterations in FLC cells, including their drug sensitivity and glycolysis. Hyperammonemic encephalopathy is a recurrent problem in these patients, and established treatments based on the assumption of liver failure are unsuccessful. We show that many of the enzymes that produce ammonia are increased and those that consume ammonia are decreased. We also demonstrate that the metabolites of these enzymes change as expected. Thus, hyperammonemic encephalopathy in FLC may require alternative therapeutics.
Subject(s)
Brain Diseases , Carcinoma, Hepatocellular , Liver Neoplasms , Neurotoxicity Syndromes , Humans , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Proteome , AmmoniaABSTRACT
Genome-wide association studies (GWASs) of serum metabolites have the potential to uncover genes that influence human metabolism. Here, we combined an integrative genetic analysis that associates serum metabolites to membrane transporters with a coessentiality map of metabolic genes. This analysis revealed a connection between feline leukemia virus subgroup C cellular receptor 1 (FLVCR1) and phosphocholine, a downstream metabolite of choline metabolism. Loss of FLVCR1 in human cells strongly impairs choline metabolism due to the inhibition of choline import. Consistently, CRISPR-based genetic screens identified phospholipid synthesis and salvage machinery as synthetic lethal with FLVCR1 loss. Cells and mice lacking FLVCR1 exhibit structural defects in mitochondria and upregulate integrated stress response (ISR) through heme-regulated inhibitor (HRI) kinase. Finally, Flvcr1 knockout mice are embryonic lethal, which is partially rescued by choline supplementation. Altogether, our findings propose FLVCR1 as a major choline transporter in mammals and provide a platform to discover substrates for unknown metabolite transporters.
Subject(s)
Genome-Wide Association Study , Receptors, Virus , Humans , Animals , Mice , Receptors, Virus/metabolism , Mutation , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mammals/metabolism , CholineABSTRACT
Plasma extracellular vesicles and particles (EVPs) are enriched in biomolecules that reflect individuals' physiological and pathological states. Several studies have demonstrated the potential of human plasma EVPs as a novel liquid biopsy. Here we describe a protocol for human plasma EVPs isolation and proteomic characterization. We isolated human plasma EVPs by the classical ultracentrifugation method and performed mass spectrometry-based proteomic profiling. Using this protocol, researchers can reveal the plasma EVPs proteome and explore the clinical application of plasma EVPs proteins for developing disease biomarkers.
Subject(s)
Extracellular Vesicles , Proteomics , Humans , Proteomics/methods , Mass Spectrometry , Ultracentrifugation , Blood Proteins/metabolism , Extracellular Vesicles/metabolism , Proteome/metabolismABSTRACT
Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/ß-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/ß immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/ß. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/ß-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/ß-dependent antiviral immunity.
Subject(s)
COVID-19 , Mycobacterium , Child , Humans , Interferon-gamma , SARS-CoV-2 , Interferon-alpha , Interferon Regulatory Factor-1ABSTRACT
Utilization of specific codons varies between organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine limitation-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced an adaptive proteomic shift toward low-arginine codon-containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.
Subject(s)
Arginine , Colorectal Neoplasms , Humans , Base Sequence , Arginine/genetics , Arginine/metabolism , Protein Biosynthesis , Proteomics , Escherichia coli/metabolism , Codon/metabolism , Colorectal Neoplasms/genetics , Tumor MicroenvironmentABSTRACT
Utilization of specific codons varies significantly across organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine restriction-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced a proteomic shift towards low arginine codon containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.
ABSTRACT
Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals1-4. However, these interactions have not been studied systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality.
Subject(s)
Ants , Body Fluids , Molting , Pupa , Social Behavior , Animals , Ants/growth & development , Ants/physiology , Larva/physiology , Molting/physiology , Pupa/physiology , Body Fluids/physiologyABSTRACT
The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.
Subject(s)
Bacterial Toxins , Cri-du-Chat Syndrome , Endopeptidases , Haploinsufficiency , Hemolysin Proteins , Staphylococcal Infections , Staphylococcus aureus , Bacterial Toxins/immunology , Cri-du-Chat Syndrome/genetics , Cri-du-Chat Syndrome/immunology , Endopeptidases/genetics , Haploinsufficiency/genetics , Haploinsufficiency/immunology , Hemolysin Proteins/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Cellular/genetics , Necrosis , Staphylococcal Infections/genetics , Staphylococcal Infections/immunology , Staphylococcal Infections/pathologyABSTRACT
Tumourigenesis and cancer progression require enhanced global protein translation1-3. Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.