ABSTRACT
The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
Subject(s)
Dipeptides , Lysosomes , Lysosomes/metabolism , Animals , Dipeptides/metabolism , Oocytes/metabolism , Cryoelectron Microscopy , Mice , Xenopus laevis , Humans , Mice, Knockout , Molecular Dynamics Simulation , Symporters/metabolism , Symporters/genetics , Symporters/chemistry , Female , Transient Receptor Potential ChannelsABSTRACT
Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.
Subject(s)
Membrane Glycoproteins , Neuronal Ceroid-Lipofuscinoses , Mice , Animals , Child , Humans , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism , Neuronal Ceroid-Lipofuscinoses/pathology , Lysosomes/metabolism , Phospholipases/metabolism , Glycerophospholipids/metabolism , Phospholipids/metabolismABSTRACT
Cancer cells need a steady supply of nutrients to evade cell death and proliferate. Depriving cancer cells of the amino acid cystine can trigger the non-apoptotic cell death process of ferroptosis. Here, we report that cancer cells can evade cystine deprivation-induced ferroptosis by uptake and catabolism of the cysteine-rich extracellular protein albumin. This protective mechanism is enhanced by mTORC1 inhibition and involves albumin degradation in the lysosome, predominantly by cathepsin B (CTSB). CTSB-dependent albumin breakdown followed by export of cystine from the lysosome via the transporter cystinosin fuels the synthesis of glutathione, which suppresses lethal lipid peroxidation. When cancer cells are grown under non-adherent conditions as spheroids, mTORC1 pathway activity is reduced, and albumin supplementation alone affords considerable protection against ferroptosis. These results identify the catabolism of extracellular protein within the lysosome as a mechanism that can inhibit ferroptosis in cancer cells.
Subject(s)
Ferroptosis , Cystine , Glutathione/metabolism , Mechanistic Target of Rapamycin Complex 1 , Lysosomes/metabolism , Albumins , Cell Line, TumorABSTRACT
Mucins are functionally implicated in a range of human pathologies, including cystic fibrosis, influenza, bacterial endocarditis, gut dysbiosis, and cancer. These observations have motivated the study of mucin biosynthesis as well as the development of strategies for inhibition of mucin glycosylation. Mammalian pathways for mucin catabolism, however, have remained underexplored. The canonical view, derived from analysis of N-glycoproteins in human lysosomal storage disorders, is that glycan degradation and proteolysis occur sequentially. Here, we challenge this view by providing genetic and biochemical evidence supporting mammalian proteolysis of heavily O-glycosylated mucin domains without prior deglycosylation. Using activity screening coupled with mass spectrometry, we ascribed mucin-degrading activity in murine liver to the lysosomal protease cathepsin D. Glycoproteomics of substrates digested with purified human liver lysosomal cathepsin D provided direct evidence for proteolysis within densely O-glycosylated domains. Finally, knockout of cathepsin D in a murine model of the human lysosomal storage disorder neuronal ceroid lipofuscinosis 10 resulted in accumulation of mucins in liver-resident macrophages. Our findings imply that mucin-degrading activity is a component of endogenous pathways for glycoprotein catabolism in mammalian tissues.
Subject(s)
Cathepsin D , Lysosomes , Mucins , Animals , Cathepsin D/genetics , Cathepsin D/metabolism , Glycoproteins/metabolism , Humans , Lysosomes/enzymology , Mammals/metabolism , Mice , Mucins/metabolism , Polysaccharides/metabolismABSTRACT
Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.
Subject(s)
Esters , Glycerophospholipids , Inositol Phosphates , Lysosomes , Membrane Glycoproteins , Molecular Chaperones , Animals , Biomarkers/cerebrospinal fluid , Biomarkers/metabolism , Child , Esters/metabolism , Glycerophospholipids/cerebrospinal fluid , Glycerophospholipids/metabolism , Humans , Inositol Phosphates/cerebrospinal fluid , Inositol Phosphates/metabolism , Lysosomal Storage Diseases/cerebrospinal fluid , Lysosomal Storage Diseases/genetics , Lysosomal Storage Diseases/metabolism , Lysosomes/metabolism , Lysosomes/pathology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/cerebrospinal fluid , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolismABSTRACT
SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Lysosomes/drug effects , Neurodegenerative Diseases/metabolism , Animals , Anti-Inflammatory Agents/chemistry , Biomarkers , Brain/drug effects , Brain/metabolism , Disease Models, Animal , Disease Susceptibility , Drug Development , Gene Expression Profiling , Humans , Mice , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/pathology , Neurons/drug effects , Neurons/metabolism , Smad Proteins/agonistsABSTRACT
The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/Bâ¢GTP-RagC/Dâ¢GDP) permits mTORC1 association. We used cryo-electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome.
Subject(s)
Lysosomes/chemistry , Mechanistic Target of Rapamycin Complex 1/chemistry , Monomeric GTP-Binding Proteins/chemistry , Regulatory-Associated Protein of mTOR/chemistry , Cryoelectron Microscopy , Humans , Molecular Docking Simulation , Protein Structure, Quaternary , Signal TransductionABSTRACT
The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.
Subject(s)
Amino Acids/deficiency , Autophagosomes/metabolism , Autophagy , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Ribosomes/metabolism , Animals , Cell Nucleus/metabolism , HEK293 Cells , Humans , Lysosomes/metabolism , Mice , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/genetics , Proteome/metabolism , Proteomics , RNA-Binding Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Transcription FactorsABSTRACT
The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H+-adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux.
Subject(s)
Amino Acids/metabolism , Lysosomes/metabolism , Metabolomics , Vacuolar Proton-Translocating ATPases/metabolism , Chemical Fractionation/methods , HEK293 Cells , Humans , Lysosomes/chemistry , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/metabolismABSTRACT
Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.
Subject(s)
Interleukin-4 Receptor alpha Subunit/antagonists & inhibitors , Macrophages/metabolism , MicroRNAs/biosynthesis , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Up-Regulation , Animals , Brugia malayi/immunology , Cell Line, Transformed , Cell Proliferation , Cells, Cultured , Gene Expression Profiling , Interleukin-4/metabolism , Interleukin-4 Receptor alpha Subunit/genetics , Interleukin-4 Receptor alpha Subunit/metabolism , Macrophage Activation , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-akt/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolismABSTRACT
Although the functional parameters of microRNAs (miRNAs) have been explored in some depth, the roles of these molecules in viral infections remain elusive. Here we report a general method for global analysis of miRNA function that compares the significance of both overexpressing and inhibiting each mouse miRNA on the growth properties of different viruses. Our comparative analysis of representatives of all three herpesvirus subfamilies identified host miRNAs with broad anti- and proviral properties which extend to a single-stranded RNA virus. Specifically, we demonstrate the broad antiviral capacity of miR-199a-3p and illustrate that this individual host-encoded miRNA regulates multiple pathways required and/or activated by viruses, including PI3K/AKT and ERK/MAPK signaling, oxidative stress signaling, and prostaglandin synthesis. Global miRNA expression analysis further demonstrated that the miR-199a/miR-214 cluster is down-regulated in both murine and human cytomegalovirus infection and manifests similar antiviral properties in mouse and human cells. Overall, we report a general strategy for examining the contributions of individual host miRNAs in viral infection and provide evidence that these molecules confer broad inhibitory potential against multiple viruses.