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1.
Sci Adv ; 10(14): eadl5012, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38569033

RESUMO

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Proteínas do Envelope Viral/metabolismo , Fator de Transcrição AP-1/metabolismo , Pandemias , Replicação Viral , Lisossomos/metabolismo , Fatores de Ribosilação do ADP/metabolismo
2.
Int J Biol Sci ; 20(5): 1905-1926, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481802

RESUMO

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.


Assuntos
Nefropatias , Obstrução Ureteral , ATPases Vacuolares Próton-Translocadoras , Animais , Humanos , Camundongos , Apoptose , Autofagia/genética , 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 , Linhagem Celular Tumoral , Fibrose , Pontos de Checagem da Fase G2 do Ciclo Celular , Nefropatias/metabolismo , Lisossomos/metabolismo , Proteínas SNARE/metabolismo , Proteínas SNARE/farmacologia , Obstrução Ureteral/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/farmacologia
3.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474091

RESUMO

Ubiquitin-like modifier-activating enzyme 6 (UBA6) is a member of the E1 enzyme family, which initiates the ubiquitin-proteasome system (UPS). The UPS plays critical roles not only in protein degradation but also in various cellular functions, including neuronal signaling, myocardial remodeling, immune cell differentiation, and cancer development. However, the specific role of UBA6 in cellular functions is not fully elucidated in comparison with the roles of the UPS. It has been known that the E1 enzyme is associated with the motility of cancer cells. In this study, we verified the physiological roles of UBA6 in lung cancer cells through gene-silencing siRNA targeting UBA6 (siUBA6). The siUBA6 treatment attenuated the migration of H1975 cells, along with a decrease in lysosomal Ca2+ release. While autophagosomal proteins remained unchanged, lysosomal proteins, including TRPML1 and TPC2, were decreased in siUBA6-transfected cells. Moreover, siUBA6 induced the production of multivesicular bodies (MVBs), accompanied by an increase in MVB markers in siUBA6-transfected H1975 cells. Additionally, the expression of the exosomal marker CD63 and extracellular vesicles was increased by siUBA6 treatment. Our findings suggest that knock-down of UBA6 induces lysosomal TRPML1 depletion and inhibits endosomal trafficking to lysosome, and subsequently, leads to the accumulation of MVBs and enhanced exosomal secretion in lung cancer cells.


Assuntos
Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Lisossomos/metabolismo , Corpos Multivesiculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-38488191

RESUMO

Lysosomes play a central role in biochemical signal transduction and oxidative stress in cells. Inducing lysosome membrane penetration (LMP) to cause lysosomal-dependent cell death (LCD) in tumor cells is an effective strategy for cancer therapy. Chemical drugs can destroy the stability of lysosomes by neutralizing protons within the lysosomes or enhancing the fragility of the lysosomal membranes. However, there remain several unsolved problems of traditional drugs in LMP induction due to insufficient lysosomal targeting, fast metabolism, and toxicity in normal cells. With the development of nanotechnology, magnetic nanoparticles have been demonstrated to target lysosomes naturally, providing a versatile tool for lysosomal modulation. Combined with excellent tissue penetration and spatiotemporal manipulability of magnetic fields, magnetic modulation of lysosomes progresses rapidly in inducing LMP and LCD for cancer therapy. This review comprehensively discussed the strategies of magnetic modulation of lysosomes for cancer therapy. The intrinsic mechanisms of LMP-induced LCD were first introduced. Then, the modulation of lysosomes by diverse physical outputs of magnetic fields was emphatically discussed. Looking forward, this review will shed the light on the prospect of magnetic modulation of lysosomes, inspiring future research of magnetic modulation strategy in cancer therapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.


Assuntos
Membranas Intracelulares , Neoplasias , Humanos , Morte Celular/fisiologia , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fenômenos Magnéticos
5.
Cancer Lett ; 587: 216728, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38431036

RESUMO

Lysosomes are crucial organelles responsible for the degradation of cytosolic materials and bulky organelles, thereby facilitating nutrient recycling and cell survival. However, lysosome also acts as an executioner of cell death, including ferroptosis, a distinctive form of regulated cell death that hinges on iron-dependent phospholipid peroxidation. The initiation of ferroptosis necessitates three key components: substrates (membrane phospholipids enriched with polyunsaturated fatty acids), triggers (redox-active irons), and compromised defence mechanisms (GPX4-dependent and -independent antioxidant systems). Notably, iron assumes a pivotal role in ferroptotic cell death, particularly in the context of cancer, where iron and oncogenic signaling pathways reciprocally reinforce each other. Given the lysosomes' central role in iron metabolism, various strategies have been devised to harness lysosome-mediated iron metabolism to induce ferroptosis. These include the re-mobilization of iron from intracellular storage sites such as ferritin complex and mitochondria through ferritinophagy and mitophagy, respectively. Additionally, transcriptional regulation of lysosomal and autophagy genes by TFEB enhances lysosomal function. Moreover, the induction of lysosomal iron overload can lead to lysosomal membrane permeabilization and subsequent cell death. Extensive screening and individually studies have explored pharmacological interventions using clinically available drugs and phytochemical agents. Furthermore, a drug delivery system involving ferritin-coated nanoparticles has been specifically tailored to target cancer cells overexpressing TFRC. With the rapid advancements in understandings the mechanistic underpinnings of ferroptosis and iron metabolism, it is increasingly evident that lysosomes represent a promising target for inducing ferroptosis and combating cancer.


Assuntos
Ferro , Neoplasias , Humanos , Morte Celular , Ferro/metabolismo , Ferritinas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Lisossomos/metabolismo
6.
Biochem Pharmacol ; 222: 116111, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458329

RESUMO

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.


Assuntos
Autofagia , Neoplasias da Bexiga Urinária , Humanos , Autofagossomos/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Apoptose , Lisossomos/metabolismo
7.
Mol Cell ; 84(7): 1354-1364.e9, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38447580

RESUMO

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/metabolismo
8.
Talanta ; 273: 125894, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38461644

RESUMO

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that can be used as a marker for the occurrence of oxidative stress in the organism. Lysosomes serve as intracellular digestive sites, and when the concentration of H2O2 in them is abnormal, lysosomal function is often impaired, leading to the development of diseases. Hydrogen sulfide (H2S) acts as a gaseous signaling molecule that scavenges H2O2 from cells and tissues, thereby maintaining the redox environment of the body. However, most of the reported hydrogen peroxide fluorescent probes so far can only detect H2O2, but cannot maintain the intracellular redox environment. In this paper, an H2O2 fluorescent probe LN-HOD with lysosomal targeting properties was designed and synthesized by combining the H2O2 recognition site with a naphthylamine fluorophore via a thiocarbamate moiety. The probe has the advantages of large Stokes shift (110 nm), high sensitivity and good H2S release capability. The probe LN-HOD can be used to detect H2O2 in cells, zebrafish and plant roots. In addition, LN-HOD detects changes in the concentration of H2O2 in plant roots when Arabidopsis is stressed by cadmium ion (Cd2+). And through its ability to release H2S, it can help to remove excess H2O2 and maintain the redox environment in cells, zebrafish and plant roots. The present work provides new ideas for the detection and assisted removal of H2O2, which contributes to the in-depth study of the cellular microenvironment in organisms.


Assuntos
Corantes Fluorescentes , Sulfeto de Hidrogênio , Animais , Humanos , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Peixe-Zebra , Sulfeto de Hidrogênio/metabolismo , Oxirredução , Lisossomos/metabolismo , Células HeLa
9.
Eur J Med Chem ; 269: 116329, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38508117

RESUMO

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.


Assuntos
Catepsina B , Catepsina B/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Lisossomos/química , Lisossomos/metabolismo
10.
Int J Mol Sci ; 25(6)2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38542221

RESUMO

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.


Assuntos
Canabinoides , Infecções por HIV , HIV-1 , Humanos , Feminino , NF-kappa B/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Macrófagos/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Estresse Oxidativo , Exocitose , Lisossomos/metabolismo
11.
J Ethnopharmacol ; 328: 118076, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38521431

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: QiXian Granule (QXG) is an integrated traditional Chinese medicine formula used to treat postmenopausal atherosclerotic (AS) cardiovascular diseases. The previous studies have found that QXG inhibited isoproterenol (ISO)-induced myocardial remodeling. And its active ingredient, Icraiin, can inhibit ferroptosis by promoting oxidized low-density lipoprotein (xo-LDL)-induced vascular endothelial cell injury and autophagy in atherosclerotic mice. Another active ingredient, Salvianolic Acid B, can suppress ferroptosis and apoptosis during myocardial ischemia/reperfusion injury by reducing ubiquitin-proteasome degradation of Glutathione Peroxidase 4 (GPX4) and down-regulating the reactive oxygen species (ROS)- c-Jun N-terminal kinases (JNK)/mitogen-activated protein kinase (MAPK) pathway. AIM OF THE STUDY: The objective of this research was to assess the possible impact of QXG on atherosclerosis in postmenopausal individuals and investigate its underlying mechanisms. MATERIALS AND METHODS: Female ApoE-/- mice underwent ovariectomy and were subjected to a high-fat diet (HFD) to establish a postmenopausal atherosclerosis model. The therapeutic effects of QXG were observed in vivo and in vitro through intraperitoneal injection of erastin, G-protein Coupled Estrogen Receptor (GPER) inhibitor (G15), and silent Mucolipin Transient Receptor Potential Channel 1 (TRPML1) adenovirus injection via tail vein. UPLC-MS and molecular docking techniques identified and evaluated major QXG components, contributing to the investigation of QXG's anti-postmenopausal atherosclerotic effects. RESULTS: QXG increased serum Estradiol levels, decreased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, which indicated QXG had estrogen-like effects in Ovx/ApoE-/- mice. Furthermore, QXG demonstrated the potential to impede the progression of AS in Ovx/ApoE-/- mice, as evidenced by reductions in serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) levels. Additionally, QXG inhibited ferroptosis in Ovx/ApoE-/- mice. Notably, UPLC-MS analysis identified a total of 106 active components in QXG. The results of molecular docking analysis demonstrated that Epmedin B, Astragaloside II, and Orientin exhibit strong binding affinity towards TRPML1. QXG alleviates the progression of atherosclerosis by activating TRPML1 through the GPER pathway or directly activating TRPML1, thereby inhibiting GPX4 and ferritin heavy chain (FTH1)-mediated iron pendant disease. In vitro, QXG-treated serum suppressed proliferation, migration, and ox-LDL-induced MMP and ROS elevation in HAECs. CONCLUSION: QXG inhibited GPX4 and FTH1-mediated ferroptosis in vascular endothelial cells through up-regulating GPER/TRPML1 signaling, providing a potential therapeutic option for postmenopausal females seeking a safe and effective medication to prevent atherosclerosis. The study highlights QXG's estrogenic properties and its promising role in combating postmenopausal atherosclerosis.


Assuntos
Aterosclerose , Medicamentos de Ervas Chinesas , Ferroptose , Feminino , Animais , Camundongos , Células Endoteliais , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Pós-Menopausa , Cromatografia Líquida , Simulação de Acoplamento Molecular , Espectrometria de Massas em Tandem , Aterosclerose/tratamento farmacológico , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , LDL-Colesterol/metabolismo , Estrogênios/metabolismo , Apolipoproteínas E , Lisossomos/metabolismo
12.
Nat Commun ; 15(1): 2779, 2024 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-38555350

RESUMO

Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.


Assuntos
Resistência à Insulina , Obesidade , Masculino , Camundongos , Animais , Obesidade/complicações , Obesidade/genética , Tecido Adiposo/metabolismo , Inflamação/metabolismo , Dieta Hiperlipídica/efeitos adversos , Lisossomos/metabolismo , Lipídeos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL
13.
Cell Calcium ; 119: 102868, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38457907

RESUMO

The recent elegant study by Y. Yuan and colleagues examined functional relationships between the lysosomal two-pore channels 2 (TPC2) and IP3 receptors (IP3Rs) located in the endoplasmic reticulum [1]. The findings of this study suggest functional coupling of these channels and receptors. The study also describes interesting novel phenomena, which may indicate an additional coupling mechanism.


Assuntos
Sinalização do Cálcio , 60694 , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Cálcio/metabolismo , NADP/metabolismo
14.
Proc Natl Acad Sci U S A ; 121(13): e2319686121, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38507452

RESUMO

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.


Assuntos
Lisina , Proteínas de Membrana Transportadoras , Humanos , Arginina/metabolismo , Transporte Biológico , Dipeptídeos/metabolismo , Células HEK293 , Lisina/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfoproteínas/metabolismo
15.
Mol Biol Cell ; 35(5): ar63, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38446621

RESUMO

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Ratos , Animais , ATPases Vacuolares Próton-Translocadoras/metabolismo , beta-Frutofuranosidase/metabolismo , Endossomos/metabolismo , Transdução de Sinais , Lisossomos/metabolismo , Mamíferos/metabolismo
16.
J Cell Biol ; 223(4)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429999

RESUMO

Cholesterol from low-density lipoprotein (LDL) can be transported to many organelle membranes by non-vesicular mechanisms involving sterol transfer proteins (STPs). Fatty acid-binding protein (FABP) 7 was identified in our previous study searching for new regulators of intracellular cholesterol trafficking. Whether FABP7 is a bona fide STP remains unknown. Here, we found that FABP7 deficiency resulted in the accumulation of LDL-derived cholesterol in lysosomes and reduced cholesterol levels on the plasma membrane. A crystal structure of human FABP7 protein in complex with cholesterol was resolved at 2.7 Å resolution. In vitro, FABP7 efficiently transported the cholesterol analog dehydroergosterol between the liposomes. Further, the silencing of FABP3 and 8, which belong to the same family as FABP7, caused robust cholesterol accumulation in lysosomes. These two FABP proteins could transport dehydroergosterol in vitro as well. Collectively, our results suggest that FABP3, 7, and 8 are a new class of STPs mediating cholesterol egress from lysosomes.


Assuntos
Colesterol , Proteínas de Ligação a Ácido Graxo , Lisossomos , Humanos , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Lisossomos/metabolismo , Esteróis/metabolismo
17.
Mol Biol Cell ; 35(5): ar70, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38536415

RESUMO

Lysosome turnover and biogenesis are induced in response to treatment of cells with agents that cause membrane rupture, but whether other stress conditions engage similar homeostatic mechanisms is not well understood. Recently we described a form of selective turnover of lysosomes that is induced by metabolic stress or by treatment of cells with ionophores or lysosomotropic agents, involving the formation of intraluminal vesicles within intact organelles through microautophagy. Selective turnover involves noncanonical autophagy and the lipidation of LC3 onto lysosomal membranes, as well as the autophagy gene-dependent formation of intraluminal vesicles. Here, we find a form of microautophagy induction that requires activity of the lipid kinase PIKfyve and is associated with the nuclear translocation of TFEB, a known mediator of lysosome biogenesis. We show that LC3 undergoes turnover during this process, and that PIKfyve is required for the formation of intraluminal vesicles and LC3 turnover, but not for LC3 lipidation onto lysosomal membranes, demonstrating that microautophagy is regulated by PIKfyve downstream of noncanonical autophagy. We further show that TFEB activation requires noncanonical autophagy but not PIKfyve, distinguishing the regulation of biogenesis from microautophagy occurring in response to agents that induce lysosomal stress.


Assuntos
Lisossomos , Microautofagia , Lisossomos/metabolismo , Autofagia , Membranas Intracelulares/metabolismo , Ionóforos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos
18.
Sci Signal ; 17(829): eadk8249, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38530880

RESUMO

Mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GCase) are responsible for Gaucher disease (GD) and are considered the strongest genetic risk factor for Parkinson's disease (PD) and Lewy body dementia (LBD). GCase deficiency leads to extensive accumulation of glucosylceramides (GCs) in cells and contributes to the neuropathology of GD, PD, and LBD by triggering chronic neuroinflammation. Here, we investigated the mechanisms by which GC accumulation induces neuroinflammation. We found that GC accumulation within microglia induced by pharmacological inhibition of GCase triggered STING-dependent inflammation, which contributed to neuronal loss both in vitro and in vivo. GC accumulation in microglia induced mitochondrial DNA (mtDNA) leakage to the cytosol to trigger STING-dependent inflammation. Rapamycin, a compound that promotes lysosomal activity, improved mitochondrial function, thereby decreasing STING signaling. Furthermore, lysosomal damage caused by GC accumulation led to defects in the degradation of activated STING, further exacerbating inflammation mediated by microglia. Thus, limiting STING activity may be a strategy to suppress neuroinflammation caused by GCase deficiency.


Assuntos
Doença de Gaucher , Doença de Parkinson , Animais , Camundongos , alfa-Sinucleína/metabolismo , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidas/metabolismo , Inflamação/metabolismo , Lisossomos/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias , Doença de Parkinson/metabolismo
20.
Nat Commun ; 15(1): 2553, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519472

RESUMO

Lysosomal Storage Disorders (LSDs), which share common phenotypes, including enlarged lysosomes and defective lysosomal storage, are caused by mutations in lysosome-related genes. Although gene therapies and enzyme replacement therapies have been explored, there are currently no effective routine therapies against LSDs. During lysosome reformation, which occurs when the functional lysosome pool is reduced, lysosomal lipids and proteins are recycled to restore lysosome functions. Here we report that the sorting nexin protein SNX8 promotes lysosome tubulation, a process that is required for lysosome reformation, and that loss of SNX8 leads to phenotypes characteristic of LSDs in human cells. SNX8 overexpression rescued features of LSDs in cells, and AAV-based delivery of SNX8 to the brain rescued LSD phenotypes in mice. Importantly, by screening a natural compound library, we identified three small molecules that enhanced SNX8-lysosome binding and reversed LSD phenotypes in human cells and in mice. Altogether, our results provide a potential solution for the treatment of LSDs.


Assuntos
Doenças por Armazenamento dos Lisossomos , Camundongos , Animais , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/terapia , Doenças por Armazenamento dos Lisossomos/metabolismo , Proteínas/metabolismo , Encéfalo/metabolismo , Mutação , Lisossomos/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
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