RESUMO
The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos Essenciais/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Arginina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Alinhamento de SequênciaRESUMO
Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 µM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway.
Assuntos
Arginina/metabolismo , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: HIF (hypoxia inducible factor) regulates many aspects of cardiac function. We and others previously showed that chronic HIF activation in the heart in mouse models phenocopies multiple features of ischemic cardiomyopathy in humans, including mitochondrial loss, lipid accumulation, and systolic cardiac dysfunction. In some settings, HIF also causes the loss of peroxisomes. How, mechanistically, HIF promotes cardiac dysfunction is an open question. METHODS: We used mice lacking cardiac pVHL (von Hippel-Lindau protein) to investigate how chronic HIF activation causes multiple features of ischemic cardiomyopathy, such as autophagy induction and lipid accumulation. We performed immunoblot assays, RNA sequencing, mitochondrial and peroxisomal autophagy flux measurements, and live cell imaging on isolated cardiomyocytes. We used CRISPR-Cas9 gene editing in mice to validate a novel mediator of cardiac dysfunction in the setting of chronic HIF activation. RESULTS: We identify a previously unknown pathway by which cardiac HIF activation promotes the loss of mitochondria and peroxisomes. We found that DEPP1 (decidual protein induced by progesterone 1) is induced under hypoxia in a HIF-dependent manner and localizes inside mitochondria. DEPP1 is both necessary and sufficient for hypoxia-induced autophagy and triglyceride accumulation in cardiomyocytes ex vivo. DEPP1 loss increases cardiomyocyte survival in the setting of chronic HIF activation ex vivo, and whole-body Depp1 loss decreases cardiac dysfunction in hearts with chronic HIF activation caused by VHL loss in vivo. CONCLUSIONS: Our findings identify DEPP1 as a key component in the cardiac remodeling that occurs with chronic ischemia.
Assuntos
Autofagia , Cardiomiopatias , Animais , Camundongos , Cardiomiopatias/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/etiologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos Knockout , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Peroxissomos/metabolismo , Modelos Animais de Doenças , MasculinoRESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy. Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling. Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease.
Assuntos
Proteínas de Transporte/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Aminoácidos/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Feminino , Proteínas Ativadoras de GTPase , Glucose/deficiência , Glucose/metabolismo , Humanos , Cadeias alfa de Integrinas , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Especificidade por Substrato , Serina-Treonina Quinases TOR/antagonistas & inibidoresAssuntos
Metabolismo , Triptofano , Aminoácidos/metabolismo , Humanos , Cinurenina/metabolismo , Triptofano/metabolismoRESUMO
Chemoresistance to platinum therapy is a major obstacle that needs to be overcome in the treatment of ovarian cancer patients. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). This study demonstrates that the Notch signaling pathway and Notch3 in particular are critical for the regulation of CSCs and tumor resistance to platinum. We show that Notch3 overexpression in tumor cells results in expansion of CSCs and increased platinum chemoresistance. In contrast, γ-secretase inhibitor (GSI), a Notch pathway inhibitor, depletes CSCs and increases tumor sensitivity to platinum. Similarly, a Notch3 siRNA knockdown increases the response to platinum therapy, further demonstrating that modulation of tumor chemosensitivity by GSI is Notch specific. Most importantly, the cisplatin/GSI combination is the only treatment that effectively eliminates both CSCs and the bulk of tumor cells, indicating that a dual combination targeting both populations is needed for tumor eradication. In addition, we found that the cisplatin/GSI combination therapy has a synergistic cytotoxic effect in Notch-dependent tumor cells by enhancing the DNA-damage response, G(2)/M cell-cycle arrest, and apoptosis. Based on these results, we conclude that targeting the Notch pathway could significantly increase tumor sensitivity to platinum therapy. Our study suggests important clinical applications for targeting Notch as part of novel treatment strategies upon diagnosis of ovarian cancer and at recurrence. Both platinum-resistant and platinum-sensitive relapses may benefit from such an approach as clinical data suggest that all relapses after platinum therapy are increasingly platinum resistant.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Receptores Notch/metabolismo , Animais , Ciclo Celular , Morte Celular , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Receptor Notch3 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Kynurenic acid (KynA) is tissue protective in cardiac, cerebral, renal, and retinal ischemia models, but the mechanism is unknown. KynA can bind to multiple receptors, including the aryl hydrocarbon receptor, the a7 nicotinic acetylcholine receptor (a7nAChR), multiple ionotropic glutamate receptors, and the orphan G protein-coupled receptor GPR35. Here, we show that GPR35 activation was necessary and sufficient for ischemic protection by KynA. When bound by KynA, GPR35 activated Gi- and G12/13-coupled signaling and trafficked to the outer mitochondria membrane, where it bound, apparantly indirectly, to ATP synthase inhibitory factor subunit 1 (ATPIF1). Activated GPR35, in an ATPIF1-dependent and pertussis toxin-sensitive manner, induced ATP synthase dimerization, which prevented ATP loss upon ischemia. These findings provide a rationale for the development of specific GPR35 agonists for the treatment of ischemic diseases.
Assuntos
Ácido Cinurênico , Mitocôndrias Cardíacas , Isquemia Miocárdica , Receptores Acoplados a Proteínas G , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Ácido Cinurênico/uso terapêutico , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/prevenção & controle , Proteínas/metabolismo , Coelhos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Proteína Inibidora de ATPaseRESUMO
Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.
RESUMO
Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.
Assuntos
Antiporters/metabolismo , Conservadores da Densidade Óssea/metabolismo , Difosfonatos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Nitrogênio/metabolismo , Animais , Antiporters/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Testes Genéticos , Estudo de Associação Genômica Ampla , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas de Transporte de Monossacarídeos/genéticaRESUMO
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.
Assuntos
Aminoácidos/deficiência , Autofagossomos/metabolismo , Autofagia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteômica , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de TranscriçãoRESUMO
One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.
Assuntos
Mitocôndrias/metabolismo , Serina/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas , Carbono/metabolismo , Testes Genéticos , Humanos , Células Jurkat , Células K562 , Transportador 1 de Glucose-Sódio/genéticaRESUMO
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.
Assuntos
Aminoácidos/metabolismo , Lisossomos/metabolismo , Metabolômica , ATPases Vacuolares Próton-Translocadoras/metabolismo , Fracionamento Químico/métodos , Células HEK293 , Humanos , Lisossomos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Arginina/metabolismo , Lisossomos/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Arginina/deficiência , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The centrality of phosphoinositide-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intratumoral concentration, and an insulin resistance "class effect." This study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polythylene glycol)]. The supramolecular nanoparticles (SNP) that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-Ras(LSL/+)/Pten(fl/fl) ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the SNPs highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the SNPs exerted a temporally sustained inhibition of phosphorylation of Akt, mTOR, S6K, and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of SNPs abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer treatment.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Resistência à Insulina , Nanopartículas/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Terapia de Alvo Molecular , Peso Molecular , Nanopartículas/química , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
High-grade serous ovarian carcinoma presents significant clinical and therapeutic challenges. Although the traditional model of carcinogenesis has focused on the ovary as a tumor initiation site, recent studies suggest that there may be additional sites of origin outside the ovary, namely the secretory cells of the fallopian tube. Our study demonstrates that high-grade serous tumors can originate in fallopian tubal secretory epithelial cells and also establishes serous tubal intraepithelial carcinoma as the precursor lesion to high-grade serous ovarian and peritoneal carcinomas in animal models targeting the Brca, Tp53, and Pten genes. These findings offer an avenue to address clinically important questions that are critical for cancer prevention and early detection in women carrying BRCA1 and BRCA2 mutations.