RESUMO
Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismoRESUMO
The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Multimerização Proteica , Piroptose , Transdução de Sinais , Aminoácidos/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas de Ligação a Fosfato/química , Domínios Proteicos , RNA Guia de Cinetoplastídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.
Assuntos
Ácido Araquidônico/análise , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Eicosanoides/metabolismo , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Citosol/metabolismo , Eicosanoides/fisiologia , Ativação Enzimática , Feminino , Humanos , Metabolismo dos Lipídeos/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases A2/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.
Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Membranas Mitocondriais/fisiologia , Animais , Autofagia/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Restrição Calórica , Células HEK293 , Humanos , Longevidade/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Permeabilidade , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Traumatismo por Reperfusão/metabolismo , Transdução de SinaisRESUMO
In mammals, endogenous circadian clocks sense and respond to daily feeding and lighting cues, adjusting internal â¼24 h rhythms to resonate with, and anticipate, external cycles of day and night. The mechanism underlying circadian entrainment to feeding time is critical for understanding why mistimed feeding, as occurs during shift work, disrupts circadian physiology, a state that is associated with increased incidence of chronic diseases such as type 2 (T2) diabetes. We show that feeding-regulated hormones insulin and insulin-like growth factor 1 (IGF-1) reset circadian clocks in vivo and in vitro by induction of PERIOD proteins, and mistimed insulin signaling disrupts circadian organization of mouse behavior and clock gene expression. Insulin and IGF-1 receptor signaling is sufficient to determine essential circadian parameters, principally via increased PERIOD protein synthesis. This requires coincident mechanistic target of rapamycin (mTOR) activation, increased phosphoinositide signaling, and microRNA downregulation. Besides its well-known homeostatic functions, we propose insulin and IGF-1 are primary signals of feeding time to cellular clocks throughout the body.
Assuntos
Relógios Circadianos/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Circadianas Period/metabolismo , Animais , Ritmo Circadiano/fisiologia , Feminino , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo , Transdução de SinaisRESUMO
Interactions between the gut microbiota, diet, and the host potentially contribute to the development of metabolic diseases. Here, we identify imidazole propionate as a microbially produced histidine-derived metabolite that is present at higher concentrations in subjects with versus without type 2 diabetes. We show that imidazole propionate is produced from histidine in a gut simulator at higher concentrations when using fecal microbiota from subjects with versus without type 2 diabetes and that it impairs glucose tolerance when administered to mice. We further show that imidazole propionate impairs insulin signaling at the level of insulin receptor substrate through the activation of p38γ MAPK, which promotes p62 phosphorylation and, subsequently, activation of mechanistic target of rapamycin complex 1 (mTORC1). We also demonstrate increased activation of p62 and mTORC1 in liver from subjects with type 2 diabetes. Our findings indicate that the microbial metabolite imidazole propionate may contribute to the pathogenesis of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Microbioma Gastrointestinal , Imidazóis/metabolismo , Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Diabetes Mellitus Tipo 2/microbiologia , Células HEK293 , Histidina/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Sequestossoma-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.
Assuntos
Citoplasma/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Difusão , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Nanopartículas/química , Nanopartículas/metabolismo , Tamanho da Partícula , Plasmídeos/genética , Plasmídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reologia , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/antagonistas & inibidores , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.
Assuntos
Células Progenitoras Endoteliais , Proteínas Supressoras de Tumor , Animais , Humanos , Serina-Treonina Quinases TOR/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Macrófagos Associados a Tumor/metabolismo , Células Progenitoras Endoteliais/metabolismo , Receptor de Proteína C Endotelial , Alvo Mecanístico do Complexo 1 de Rapamicina , Neovascularização Patológica , MamíferosRESUMO
The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.
Assuntos
Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento/metabolismo , Animais , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Humanos , Músculos/metabolismo , Neoplasias/metabolismoRESUMO
Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.
Assuntos
Proliferação de Células , Hidroximetilglutaril-CoA Sintase , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitinação , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células HEK293 , Hidroximetilglutaril-CoA Sintase/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Ácido Mevalônico/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/genética , Transdução de Sinais , Degrons , Proteínas Adaptadoras de Transdução de SinalRESUMO
Autophagy, an important quality control and recycling process vital for cellular homeostasis, is tightly regulated. The mTORC1 signaling pathway regulates autophagy under conditions of nutrient availability and scarcity. However, how mTORC1 activity is fine-tuned during nutrient availability to allow basal autophagy is unclear. Here, we report that the WD-domain repeat protein MORG1 facilitates basal constitutive autophagy by inhibiting mTORC1 signaling through Rag GTPases. Mechanistically, MORG1 interacts with active Rag GTPase complex inhibiting the Rag GTPase-mediated recruitment of mTORC1 to the lysosome. MORG1 depletion in HeLa cells increases mTORC1 activity and decreases autophagy. The autophagy receptor p62/SQSTM1 binds to MORG1, but MORG1 is not an autophagy substrate. However, p62/SQSTM1 binding to MORG1 upon re-addition of amino acids following amino acid's depletion precludes MORG1 from inhibiting the Rag GTPases, allowing mTORC1 activation. MORG1 depletion increases cell proliferation and migration. Low expression of MORG1 correlates with poor survival in several important cancers.
Assuntos
GTP Fosfo-Hidrolases , Proteínas Monoméricas de Ligação ao GTP , Humanos , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Lisossomos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismoRESUMO
Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.
Assuntos
Inflamação/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Purinas/biossíntese , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular , Citocinas/metabolismo , Metilação de DNA , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Transdução de SinaisRESUMO
Metformin has utility in cancer prevention and treatment, though the mechanisms for these effects remain elusive. Through genetic screening in C. elegans, we uncover two metformin response elements: the nuclear pore complex (NPC) and acyl-CoA dehydrogenase family member-10 (ACAD10). We demonstrate that biguanides inhibit growth by inhibiting mitochondrial respiratory capacity, which restrains transit of the RagA-RagC GTPase heterodimer through the NPC. Nuclear exclusion renders RagC incapable of gaining the GDP-bound state necessary to stimulate mTORC1. Biguanide-induced inactivation of mTORC1 subsequently inhibits growth through transcriptional induction of ACAD10. This ancient metformin response pathway is conserved from worms to humans. Both restricted nuclear pore transit and upregulation of ACAD10 are required for biguanides to reduce viability in melanoma and pancreatic cancer cells, and to extend C. elegans lifespan. This pathway provides a unified mechanism by which metformin kills cancer cells and extends lifespan, and illuminates potential cancer targets. PAPERCLIP.
Assuntos
Metformina/farmacologia , Acil-CoA Desidrogenase/genética , Envelhecimento , Animais , Tamanho Corporal , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Longevidade , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitocôndrias/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Neoplasias/tratamento farmacológico , Poro Nuclear/metabolismo , Fosforilação Oxidativa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Cyst(e)ine is a key precursor for the synthesis of glutathione (GSH), which protects cancer cells from oxidative stress. Cyst(e)ine is stored in lysosomes, but its role in redox regulation is unclear. Here, we show that breast cancer cells upregulate major facilitator superfamily domain containing 12 (MFSD12) to increase lysosomal cyst(e)ine storage, which is released by cystinosin (CTNS) to maintain GSH levels and buffer oxidative stress. We find that mTORC1 regulates MFSD12 by directly phosphorylating residue T254, while mTORC1 inhibition enhances lysosome acidification that activates CTNS. This switch modulates lysosomal cyst(e)ine levels in response to oxidative stress, fine-tuning redox homeostasis to enhance cell fitness. MFSD12-T254A mutant inhibits MFSD12 function and suppresses tumor progression. Moreover, MFSD12 overexpression correlates with poor neoadjuvant chemotherapy response and prognosis in breast cancer patients. Our findings reveal the critical role of lysosomal cyst(e)ine storage in adaptive redox homeostasis and suggest that MFSD12 is a potential therapeutic target.
RESUMO
The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14-3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron and leading to ubiquitination by CUL1ß-TrCP and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.
Assuntos
Aminoácidos , Fator de Transcrição Associado à Microftalmia , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Aminoácidos/metabolismo , Nutrientes , Estabilidade Proteica , Lisossomos/genética , Lisossomos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Mamíferos/metabolismoRESUMO
Human plasmacytoid dendritic cells (pDCs) are interleukin-3 (IL-3)-dependent cells implicated in autoimmunity, but the role of IL-3 in pDC biology is poorly understood. We found that IL-3-induced Janus kinase 2-dependent expression of SLC7A5 and SLC3A2, which comprise the large neutral amino acid transporter, was required for mammalian target of rapamycin complex 1 (mTORC1) nutrient sensor activation in response to toll-like receptor agonists. mTORC1 facilitated increased anabolic activity resulting in type I interferon, tumor necrosis factor, and chemokine production and the expression of the cystine transporter SLC7A11. Loss of function of these amino acid transporters synergistically blocked cytokine production by pDCs. Comparison of in vitro-activated pDCs with those from lupus nephritis lesions identified not only SLC7A5, SLC3A2, and SLC7A11 but also ectonucleotide pyrophosphatase-phosphodiesterase 2 (ENPP2) as components of a shared transcriptional signature, and ENPP2 inhibition also blocked cytokine production. Our data identify additional therapeutic targets for autoimmune diseases in which pDCs are implicated.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Sistemas de Transporte de Aminoácidos/metabolismo , Autoimunidade , Biomarcadores , Citocinas/genética , Citocinas/metabolismo , Suscetibilidade a Doenças , Metabolismo Energético , Humanos , Imunidade , Transdução de SinaisRESUMO
The lysosome has long been viewed as the recycling center of the cell. However, recent discoveries have challenged this simple view and have established a central role of the lysosome in nutrient-dependent signal transduction. The degradative role of the lysosome and its newly discovered signaling functions are not in conflict but rather cooperate extensively to mediate fundamental cellular activities such as nutrient sensing, metabolic adaptation, and quality control of proteins and organelles. Moreover, lysosome-based signaling and degradation are subject to reciprocal regulation. Transcriptional programs of increasing complexity control the biogenesis, composition, and abundance of lysosomes and fine-tune their activity to match the evolving needs of the cell. Alterations in these essential activities are, not surprisingly, central to the pathophysiology of an ever-expanding spectrum of conditions, including storage disorders, neurodegenerative diseases, and cancer. Thus, unraveling the functions of this fascinating organelle will contribute to our understanding of the fundamental logic of metabolic organization and will point to novel therapeutic avenues in several human diseases.
Assuntos
Lisossomos/metabolismo , Animais , Doença , Exocitose , Humanos , Transdução de SinaisRESUMO
mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.
Assuntos
Proteínas Ativadoras de GTPase , Proteínas Monoméricas de Ligação ao GTP , Aminoácidos/metabolismo , Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismoRESUMO
AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Glucose/metabolismo , Insulina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/química , Proteína rhoA de Ligação ao GTP/química , Células 3T3-L1 , Adipócitos/citologia , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dictyostelium , Transportador de Glucose Tipo 4/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Células HEK293 , Humanos , Camundongos , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
The TSC complex is a critical negative regulator of the small GTPase Rheb and mTORC1 in cellular stress signaling. The TSC2 subunit contains a catalytic GTPase activating protein domain and interacts with multiple regulators, while the precise function of TSC1 is unknown. Here we provide a structural characterization of TSC1 and define three domains: a C-terminal coiled-coil that interacts with TSC2, a central helical domain that mediates TSC1 oligomerization, and an N-terminal HEAT repeat domain that interacts with membrane phosphatidylinositol phosphates (PIPs). TSC1 architecture, oligomerization, and membrane binding are conserved in fungi and humans. We show that lysosomal recruitment of the TSC complex and subsequent inactivation of mTORC1 upon starvation depend on the marker lipid PI3,5P2, demonstrating a role for lysosomal PIPs in regulating TSC complex and mTORC1 activity via TSC1. Our study thus identifies a vital role of TSC1 in TSC complex function and mTORC1 signaling.