RESUMO
Lysosomes catabolize and recycle lipids and other biological molecules to maintain cellular homeostasis in diverse nutrient environments. Lysosomal lipid catabolism relies on the stimulatory activity of bis(monoacylglycero)phosphate (BMP), an enigmatic lipid whose levels are altered across myriad lysosome-associated diseases. Here, we review the discovery of BMP over half a century ago and its structural properties that facilitate the activation of lipid hydrolases and recruitment of their coactivators. We further discuss the current, yet incomplete, understanding of BMP catabolism and anabolism. To conclude, we discuss its role in lysosome-associated diseases and the potential for modulating its levels by pharmacologically activating and inhibiting the BMP synthase to therapeutically target lysosomal storage disorders, drug-induced phospholipidosis, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, cancer, and viral infection.
Assuntos
Lisofosfolipídeos , Doenças por Armazenamento dos Lisossomos , Lisossomos , Monoglicerídeos , Humanos , Lisossomos/metabolismo , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Monoglicerídeos/química , Animais , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Metabolismo dos LipídeosRESUMO
The polyamines putrescine, spermidine, and spermine are abundant polycations of vital importance in mammalian cells. Their cellular levels are tightly regulated by degradation and synthesis, as well as by uptake and export. Here, we discuss the delicate balance between the neuroprotective and neurotoxic effects of polyamines in the context of Parkinson's disease (PD). Polyamine levels decline with aging and are altered in patients with PD, whereas recent mechanistic studies on ATP13A2 (PARK9) demonstrated a driving role of a disturbed polyamine homeostasis in PD. Polyamines affect pathways in PD pathogenesis, such as α-synuclein aggregation, and influence PD-related processes like autophagy, heavy metal toxicity, oxidative stress, neuroinflammation, and lysosomal/mitochondrial dysfunction. We formulate outstanding research questions regarding the role of polyamines in PD, their potential as PD biomarkers, and possible therapeutic strategies for PD targeting polyamine homeostasis.
Assuntos
Doença de Parkinson , Transtornos Parkinsonianos , Animais , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Poliaminas/metabolismo , Neuroproteção , Espermidina/metabolismo , Mamíferos/metabolismoRESUMO
Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.
Assuntos
Doença de Parkinson , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Canais de Potássio/metabolismo , PrótonsRESUMO
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Assuntos
Autofagossomos , Cálcio , Animais , Autofagossomos/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismoRESUMO
Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.
Assuntos
Demência Frontotemporal , Proteínas de Membrana , Proteínas do Tecido Nervoso , Doenças Neurodegenerativas , Amiloide , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/patologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Assuntos
Produtos Biológicos/uso terapêutico , Encéfalo/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Progranulinas/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Endossomos/metabolismo , Feminino , Demência Frontotemporal/sangue , Demência Frontotemporal/líquido cefalorraquidiano , Gliose/complicações , Gliose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Degeneração Neural/patologia , Fenótipo , Progranulinas/deficiência , Progranulinas/metabolismo , Receptores Imunológicos/metabolismo , Receptores da Transferrina/metabolismo , Distribuição TecidualRESUMO
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
Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Lisossomos/metabolismo , Mitofagia/fisiologia , Animais , Autofagia/fisiologia , Imunofluorescência/métodos , Corantes Fluorescentes/química , Humanos , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitofagia/genéticaRESUMO
Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.
Assuntos
Cisteína/toxicidade , Ferro/metabolismo , Mitocôndrias/metabolismo , Aminoácidos/metabolismo , Senescência Celular/fisiologia , Cisteína/metabolismo , Homeostase , Lisossomos/metabolismo , Mitocôndrias/fisiologia , Mitofagia/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismoRESUMO
ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.
Assuntos
COVID-19/metabolismo , SARS-CoV-2/metabolismo , Via Secretória , Liberação de Vírus , Fatores de Ribosilação do ADP/metabolismo , Animais , COVID-19/patologia , Feminino , Células HeLa , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Lisossomos , Camundongos , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Tratamento Farmacológico da COVID-19RESUMO
Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.
Assuntos
Quinase 3 da Glicogênio Sintase , Via de Sinalização Wnt , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Lisossomos/metabolismo , Fosforilação , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Assuntos
Anexinas/metabolismo , Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Lisossomos/metabolismo , RNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Animais Geneticamente Modificados , Anexinas/genética , Axônios/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Mutação , Ligação Proteica , Ratos/embriologia , Ratos Sprague-Dawley , Transfecção , Peixe-ZebraRESUMO
Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.
Assuntos
Endossomos/fisiologia , Biossíntese de Proteínas/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Axônios/metabolismo , Endossomos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/fisiologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/fisiologia , Ribossomos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/fisiologia , proteínas de unión al GTP Rab7RESUMO
Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.
Assuntos
Hipóxia Celular , Relógios Circadianos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos Dicarboxílicos/farmacologia , Animais , Proteínas CLOCK/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Relógios Circadianos/efeitos dos fármacos , Meios de Cultura/química , Fatores de Iniciação em Eucariotos , Concentração de Íons de Hidrogênio , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/metabolismo , Transcriptoma/efeitos dos fármacos , Proteína 2 do Complexo Esclerose Tuberosa/deficiência , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
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
Lysosomes are central to metabolic homeostasis. The microphthalmia bHLH-LZ transcription factors (MiT/TFEs) family members MITF, TFEB, and TFE3 promote the transcription of lysosomal and autophagic genes and are often deregulated in cancer. Here, we show that the GATOR2 complex, an activator of the metabolic regulator TORC1, maintains lysosomal function by protecting MiT/TFEs from proteasomal degradation independent of TORC1, GATOR1, and the RAG GTPase. We determine that in GATOR2 knockout HeLa cells, members of the MiT/TFEs family are ubiquitylated by a trio of E3 ligases and are degraded, resulting in lysosome dysfunction. Additionally, we demonstrate that GATOR2 protects MiT/TFE proteins in pancreatic ductal adenocarcinoma and Xp11 translocation renal cell carcinoma, two cancers that are driven by MiT/TFE hyperactivation. In summary, we find that the GATOR2 complex has independent roles in TORC1 regulation and MiT/TFE protein protection and thus is central to coordinating cellular metabolism with control of the lysosomal-autophagic system.
Assuntos
Neoplasias Renais , Fator de Transcrição Associado à Microftalmia , Humanos , Células HeLa , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteólise , Autofagia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas/metabolismo , Neoplasias Renais/metabolismo , 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/metabolismoRESUMO
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.
Assuntos
Glicoproteínas de Membrana , Lipofuscinoses Ceroides Neuronais , Camundongos , Animais , Criança , Humanos , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Lisossomos/metabolismo , Fosfolipases/metabolismo , Glicerofosfolipídeos/metabolismo , Fosfolipídeos/metabolismoRESUMO
Autophagy is a conserved intracellular pathway that delivers cytoplasmic contents to lysosomes for degradation via double-membrane autophagosomes. Autophagy substrates include organelles such as mitochondria, aggregate-prone proteins that cause neurodegeneration and various pathogens. Thus, this pathway appears to be relevant to the pathogenesis of diverse diseases, and its modulation may have therapeutic value. Here, we focus on the cell and molecular biology of mammalian autophagy and review the key proteins that regulate the process by discussing their roles and how these may be modulated by posttranslational modifications. We consider the membrane-trafficking events that impact autophagy and the questions relating to the sources of autophagosome membrane(s). Finally, we discuss data from structural studies and some of the insights these have provided.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Citoesqueleto/química , Citoesqueleto/metabolismo , Endocitose , Humanos , Lisossomos/metabolismo , Mamíferos , Modelos Moleculares , Fagossomos/metabolismo , Proteínas SNARE/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.
Assuntos
Cistos , Ferroptose , Humanos , Cisteína , Cistina , Ferroptose/genética , Aminoácidos , LisossomosRESUMO
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.