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1.
Nat Commun ; 12(1): 858, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558502

RESUMO

Synaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by 'crowding' in the vesicle membrane from stable interaction modules.


Assuntos
Reagentes para Ligações Cruzadas/química , Espectrometria de Massas , Membranas Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Fusão de Membrana , Ligação Proteica , Mapas de Interação de Proteínas , Proteolipídeos , Proteoma/metabolismo , Ratos , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
2.
Int J Mol Sci ; 22(1)2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-33374906

RESUMO

Plant vacuoles are unique compartments that play a critical role in plant growth and development. The vacuolar H+-ATPase (V-ATPase), together with the vacuolar H+-pyrophosphatase (V-PPase), generates the proton motive force that regulates multiple cell functions and impacts all aspects of plant life. We investigated the effect of V-ATPase activity in the vacuole on plant growth and development. We used an Arabidopsisthaliana (L.) Heynh. double mutant, vha-a2 vha-a3, which lacks two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a. The mutant is viable but exhibits impaired growth and leaf chlorosis. Nitrate assimilation led to excessive ammonium accumulation in the shoot and lower nitrogen uptake, which exacerbated growth retardation of vha-a2 vha-a3. Ion homeostasis was disturbed in plants with missing VHA-a2 and VHA-a3 genes, which might be related to limited growth. The reduced growth and excessive ammonium accumulation of the double mutant was alleviated by potassium supplementation. Our results demonstrate that plants lacking the two tonoplast-localized subunits of V-ATPase can be viable, although with defective growth caused by multiple factors, which can be alleviated by adding potassium. This study provided a new insight into the relationship between V-ATPase, growth, and ammonium accumulation, and revealed the role of potassium in mitigating ammonium toxicity.


Assuntos
Compostos de Amônio/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Homeostase/efeitos dos fármacos , Homeostase/genética , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Mutação , Nitrogênio/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Potássio/farmacologia , Força Próton-Motriz , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/genética
3.
Mol Cell ; 80(3): 379-380, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33157012

RESUMO

In this issue of Molecular Cell, Wang et al. (2020a) present near-atomic resolution cryoEM structures of a proton-pumping vacuolar ATPase from human cells, illuminating the glycosylation of integral subunits and identifying a novel and conserved glycolipid ligand.


Assuntos
Açúcares , ATPases Vacuolares Próton-Translocadoras , Microscopia Crioeletrônica , Humanos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
4.
Int J Nanomedicine ; 15: 6385-6399, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922007

RESUMO

Purpose: The mononuclear phagocyte system (MPS) presents a formidable obstacle that hampers the delivery of various nanopreparations to tumors. Therefore, there is an urgent need to improve the off-MPS targeting ability of nanomedicines. In the present study, we present a novel preconditioning strategy to substantially increase the circulation times and tumor targeting of nanoparticles by regulating nanocarrier-MPS interactions. Methods: In vitro, the effect of different vacuolar H+-ATPase inhibitors on macrophage uptake of targeted or nontargeted lipid vesicles was evaluated. Specifically, the clinically approved proton-pump inhibitor esomeprazole (ESO) was selected as a preconditioning agent. Then, we further investigated the blocking effect of ESO on the macrophage endocytosis of nanocarriers. In vivo, ESO was first intravenously administered into A549-tumor-bearing nude mice, and 24 h later, the c(RGDm7)-modified vesicles co-loaded with doxorubicin and gefitinib were intravenously injected. Results: In vitro, ESO was found to reduce the interactions between macrophages and c(RGDm7)-modified vesicles by interfering with the latter's lysosomal trafficking. Studies conducted in vivo confirmed that ESO pretreatment greatly decreased the liver and spleen distribution of the targeted vesicles, enhanced their tumor accumulation, and improved the therapeutic outcome of the drug-loaded nanomedicines. Conclusion: Our findings indicate that ESO can regulate the nanoparticle-MPS interaction, which provides a feasible option for enhancing the off-MPS targeting of nanomedicines.


Assuntos
Portadores de Fármacos/química , Esomeprazol/farmacologia , Sistema Fagocitário Mononuclear/citologia , Nanopartículas/química , Células A549 , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transporte Biológico , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Endocitose , Esomeprazol/farmacocinética , Esomeprazol/uso terapêutico , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/administração & dosagem , Neoplasias/tratamento farmacológico , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Distribuição Tecidual/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
PLoS One ; 15(9): e0237268, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886660

RESUMO

Endosomal sorting complexes required for transport proteins (ESCRT) catalyze the fission of cellular membranes during budding of membrane away from the cytosol. Here we have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the recruitment of ESCRTs specifically, ALIX, CHMP4b and VPS4 onto the budding HIV Gag virus-like particles (VLPs). We imaged the budding VLPs with 200 millisecond time resolution for 300 frames. Our data shows three phases for ESCRT dynamics: 1) recruitment in which subunits of ALIX, CHMP4b and VPS4 are recruited with constant proportions on the budding sites of HIV Gag virus like particles for nearly 10 seconds, followed by 2) disassembly of ALIX and CHMP4b while VPS4 signal remains constant for nearly 20 seconds followed by 3) disassembly of VPS4. We hypothesized that the disassembly observed in step 2 was catalyzed by VPS4 and powered by ATP hydrolysis. To test this hypothesis, we performed ATP depletion using (-) glucose medium, deoxyglucose and oligomycin. Imaging ATP depleted cells, we show that the disassembly of CHMP4b and ALIX observed in step 2 is ATP dependent. ATP depletion resulted in the recruitment of approximately 2-fold as many subunits of all ESCRTs. Resuming ATP production in cells, resulted in disassembly of the full ESCRT machinery which had been locked in place during ATP depletion. With some caveats, our experiments provide insight into the formation of the ESCRT machinery at the budding site of HIV during budding.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Liberação de Vírus , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Células HeLa , Humanos , Ligação Proteica , Vírion/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
6.
Nat Commun ; 11(1): 3921, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764564

RESUMO

The vacuolar-type H+-ATPases (V-ATPase) hydrolyze ATP to pump protons across the plasma or intracellular membrane, secreting acids to the lumen or acidifying intracellular compartments. It has been implicated in tumor metastasis, renal tubular acidosis, and osteoporosis. Here, we report two cryo-EM structures of the intact V-ATPase from bovine brain with all the subunits including the subunit H, which is essential for ATPase activity. Two type-I transmembrane proteins, Ac45 and (pro)renin receptor, along with subunit c", constitute the core of the c-ring. Three different conformations of A/B heterodimers suggest a mechanism for ATP hydrolysis that triggers a rotation of subunits DF, inducing spinning of subunit d with respect to the entire c-ring. Moreover, many lipid molecules have been observed in the Vo domain to mediate the interactions between subunit c, c", (pro)renin receptor, and Ac45. These two structures reveal unique features of mammalian V-ATPase and suggest a mechanism of V1-Vo torque transmission.


Assuntos
Encéfalo/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Hidrólise , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Prótons , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/ultraestrutura
7.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
8.
Nat Commun ; 11(1): 2663, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471988

RESUMO

Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membranas Artificiais , Estresse Mecânico , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Fenômenos Bioquímicos , Microscopia Crioeletrônica , Humanos , Nanotubos , Polimerização , Ligação Proteica/fisiologia , Multimerização Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismo
9.
PLoS One ; 15(5): e0225356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437440

RESUMO

High plasma LDL cholesterol (LDL-c) concentration is a major risk factor for atherosclerosis. Hepatic LDL receptor (LDLR) regulates LDL metabolism, and thereby plasma LDL-c concentration. Recently, we have identified the (pro)renin receptor [(P)RR] as a novel regulator of LDL metabolism, which regulates LDLR degradation and hence its protein abundance and activity. In silico analysis suggests that the (P)RR is a target of miR-148a. In this study we determined whether miR-148a could regulate LDL metabolism by regulating (P)RR expression in HepG2 and Huh7 cells. We found that miR-148a suppressed (P)RR expression by binding to the 3'-untranslated regions (3'-UTR) of the (P)RR mRNA. Mutating the binding sites for miR-148a in the 3'-UTR of (P)RR mRNA completely abolished the inhibitory effects of miR-148a on (P)RR expression. In line with our recent findings, reduced (P)RR expression resulted in decreased cellular LDL uptake, likely as a consequence of decreased LDLR protein abundance. Overexpressing the (P)RR prevented miR-148a-induced reduction in LDLR abundance and cellular LDL uptake. Our study supports a new concept that miR-148a is a regulator of (P)RR expression. By reducing (P)RR abundance, miR-148a decreases LDLR protein abundance and consequently cellular LDL uptake.


Assuntos
Lipoproteínas LDL/metabolismo , MicroRNAs/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células HEK293 , Células Hep G2 , Humanos
10.
Nat Struct Mol Biol ; 27(6): 589-597, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424347

RESUMO

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vibrio parahaemolyticus/metabolismo , Proteínas de Bactérias/genética , Membrana Celular , Microscopia Crioeletrônica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , ATPases Vacuolares Próton-Translocadoras/genética
11.
Parasitol Res ; 119(7): 2245-2255, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32447515

RESUMO

This is the first study showing an in vivo microautophagy upregulation by Leishmania infantum in dogs. Both Leishmania amastigotes and promastigotes were detected in the cytoplasm of many professional and nonprofessional phagocytic cells of popliteal lymph node of three dogs suffering from chronic cutaneous leishmaniasis. Ultrastructurally, parasites appeared to be wrapped by lysosomes and/or multivesicular bodies. Neither phagophores nor double-membraned vacuoles consistent with autophagosomes were observed. Transcription factor EB (TFEB), a key factor involved in lysosome biogenesis, showed a statistically significant increase in the total component when examined by western blot in samples from leishmaniotic dogs compared with samples from healthy dogs. Instead, phosphorylated TFEB showed unmodified expression levels both in leishmaniotic and healthy dogs. Furthermore, Hsc70 and endosomal sorting complex required for transport (ESCRT)-I, which are known to play a role in microautophagy, showed no variation in expression levels both in diseased and healthy animals. Vps4A/B, an evolutionary conserved ATPase responsible for ESCRT-I complex disassembly and MVB maturation, was statistically significantly overexpressed in lymph nodal samples from leishmaniotic dogs. Bag3 was downregulated in diseased dogs whereas CHIP, p62, and LC3-II did not show any variation in expression levels. The altered expression profile of Bag3 suggested an altered interaction of Bag3 with Hsc70 and CHIP, which usually form a molecular complex involved in autophagosome-lysosome pathways. Ultrastructural and molecular findings suggested that the microautophagy pathway is upregulated in lymph nodes of dogs suffering from a chronic natural infection by Leishmania infantum.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/veterinária , Linfonodos/parasitologia , Microautofagia/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Doenças do Cão/parasitologia , Cães , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Leishmaniose Visceral/parasitologia , Pele/parasitologia , Ativação Transcricional , Regulação para Cima/imunologia , ATPases Vacuolares Próton-Translocadoras/metabolismo
12.
Genes Cells ; 25(7): 483-497, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32314441

RESUMO

(Pro)renin receptor ((P)RR) regulates the renin-angiotensin system and functions as an essential accessory subunit of vacuolar H+ -ATPase. There is accumulating evidence that shows close relationship between (P)RR and autophagy. Soluble (P)RR consisting of the extracellular domain of (P)RR is generated from (P)RR by proteolytic enzymes. The aim of the present study was to clarify the influence of autophagy inhibition on soluble (P)RR expression in cancer cells. Autophagy was inhibited by treatment of bafilomycin A1 or chloroquine in MCF-7 and A549 cells for 72 hr. Western blot analysis showed that protein levels of soluble (P)RR were markedly elevated by autophagy inhibition, whereas no noticeable increases were observed in full-length (P)RR. Secretion of soluble (P)RR into the medium was increased dose-dependently by bafilomycin A1 or chloroquine. Autophagy inhibition was confirmed by enhanced accumulation of autophagy-related proteins, LC3, p62 and LAMP1 in intracellular vesicles. Increased amount of soluble (P)RR by autophagy inhibition was decreased by site-1 protease inhibitor, whereas no noticeable increase in site-1 protease immunoreactivity was observed in cells with autophagy inhibition by immunocytochemistry. These findings suggest that soluble (P)RR protein accumulates by autophagy inhibition, possibly because of the reduced degradation of soluble (P)RR in the intracellular vesicles during autophagy inhibition.


Assuntos
Autofagia/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Renina/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Autofagia/genética , Linhagem Celular Tumoral , Cloroquina/farmacologia , Vesículas Citoplasmáticas/metabolismo , Inibidores Enzimáticos/farmacologia , Furina/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Macrolídeos/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas de Ligação a RNA/metabolismo , Receptores de Superfície Celular/genética , ATPases Vacuolares Próton-Translocadoras/genética
13.
Am J Physiol Cell Physiol ; 318(6): C1107-C1122, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267718

RESUMO

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Túbulos de Malpighi/metabolismo , Tetraspaninas/metabolismo , Junções Íntimas/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Impedância Elétrica , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Túbulos de Malpighi/embriologia , Túbulos de Malpighi/ultraestrutura , Via Secretória , Transdução de Sinais , Tetraspaninas/genética , Junções Íntimas/genética , Junções Íntimas/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
14.
Int J Mol Sci ; 21(5)2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32121289

RESUMO

Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.


Assuntos
Adenosina/análogos & derivados , Diferenciação Celular , Núcleo Celular/metabolismo , Metiltransferases/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Estabilidade de RNA/genética , Transporte Ativo do Núcleo Celular , Adenosina/metabolismo , Animais , Reabsorção Óssea/patologia , Proliferação de Células , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , NF-kappa B/metabolismo , Osteogênese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/metabolismo , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
15.
Biochem Pharmacol ; 175: 113858, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32061774

RESUMO

Monocytes are professional immune cells that produce abundant levels of pro-inflammatory eicosanoids including prostaglandins and leukotrienes during inflammation. Vacuolar (H+)-ATPase (V-ATPase) is critically involved in a variety of inflammatory processes including cytokine trafficking and lipid mediator biosynthesis. However, its role in eicosanoid biosynthetic pathways in monocytes remains elusive. Here, we present a differential role of V-ATPase in the expression and in the activity of cyclooxygenase (COX)-2 in human monocytes. Pharmacological targeting of V-ATPase increased the expression of COX-2 protein in lipopolysaccharide-stimulated primary monocytes, which was paralleled by enhanced phosphorylation of p38 MAPK and ERK-1/2, without impacting the NF-κB and SAPK/JNK pathways. Targeting of both p38 MAPK and ERK-1/2 pathways showed that the kinase pathways are crucial for COX-2 expression in human monocytes. Despite increased COX-2 protein levels, however, suppression of V-ATPase activity impaired the biosynthesis of COX- and also of 5-lipoxygenase (LOX)-derived lipid mediators in monocytes without affecting 12-/15-LOX products, assessed by a metabololipidomics approach using UPLC-MS-MS analysis. Our results indicate that changes in the intracellular pH may contribute to suppression of COX-2 and 5-LOX activities. We suggest that V-ATPase on one hand limits COX-2 protein levels via restricting p38 MAPK and ERK-1/2 activation, while on the other hand it governs the cellular activity of COX-2 through appropriate adjustment of the intracellular pH.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Adolescente , Adulto , Idoso , Células Cultivadas , Ciclo-Oxigenase 2/genética , Voluntários Saudáveis , Humanos , Concentração de Íons de Hidrogênio , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucotrienos/biossíntese , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade , Prostaglandinas/biossíntese , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Adulto Jovem
16.
EBioMedicine ; 51: 102581, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31901859

RESUMO

BACKGROUND: V-ATPases are hetero-oligomeric enzymes consisting of 13 subunits and playing key roles in ion homeostasis and signaling. Differential expression of these proton pumps has been implicated in carcinogenesis and metastasis. To elucidate putative molecular signatures underlying these phenomena, we evaluated the expression of V-ATPase genes in esophageal squamous cell carcinoma (ESCC) and extended the analysis to other cancers. METHODS: Expression of all V-ATPase genes were analyzed in ESCC by a microarray data and in different types of tumors available from public databases. Expression of C isoforms was validated by qRT-PCR in paired ESCC samples. FINDINGS: A differential expression pattern of V-ATPase genes was found in different tumors, with combinations in up- and down-regulation leading to an imbalance in the expression ratios of their isoforms. Particularly, a high C1 and low C2 expression pattern accurately discriminated ESCC from normal tissues. Structural modeling of C2a isoform uncovered motifs for oncogenic kinases in an additional peptide stretch, and an actin-biding domain downstream to this sequence. INTERPRETATION: Altogether these data revealed that the expression ratios of subunits/isoforms could form a conformational code that controls the H+ pump regulation and interactions related to tumorigenesis. This study establishes a paradigm change by uncovering multi-cancer molecular signatures present in the V-ATPase structure, from which future studies must address the complexity of the onco-related V-ATPase assemblies as a whole, rather than targeting changes in specific subunit isoforms. FUNDING: This work was supported by grants from CNPq and FAPERJ-Brazil.


Assuntos
Neoplasias Esofágicas/enzimologia , Neoplasias Esofágicas/genética , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Idoso , Sequência de Aminoácidos , Biomarcadores Tumorais/metabolismo , Neoplasias Esofágicas/diagnóstico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC , Homologia Estrutural de Proteína , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo
17.
Cell ; 180(2): 296-310.e18, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31978346

RESUMO

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/metabolismo
18.
J Biol Chem ; 295(8): 2259-2269, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31941791

RESUMO

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1 Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.


Assuntos
Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Membranas Intracelulares/metabolismo , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Vacúolos/metabolismo
19.
Nat Commun ; 11(1): 164, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919360

RESUMO

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.


Assuntos
Predisposição Genética para Doença/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/patologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Antivirais/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dibenzotiepinas , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Metiltransferases/metabolismo , Morfolinas , Proteínas do Tecido Nervoso/genética , Oxazinas/farmacologia , Piridinas/farmacologia , Piridonas , Tiepinas/farmacologia , Triazinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Internalização do Vírus
20.
Cancer Sci ; 111(2): 477-488, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31840304

RESUMO

ATP6L, the C subunit of the V-ATPase V0 domain, is involved in regulating the acidic tumor micro-environment and may promote tumor progression. However, the expression and functional role of ATP6L in tumors have not yet been well explored. In this study, we found that ATP6L protein overexpression was related to colorectal cancer histological differentiation (P < 0.001), presence of metastasis (P < 0.001) and recurrence (P = 0.02). ATP6L expression in the liver metastatic foci was higher than in the primary foci (P = 0.04). ATP6L expression was notably concomitant with epithelial-mesenchymal transition (EMT) immunohistochemical features, such as reduced expression of the epithelial marker E-cadherin (P = 0.021) and increased expression of the mesenchymal marker vimentin (P = 0.004). Results of in vitro and in vivo experiments showed that ATP6L expression could alter cell morphology, regulate EMT-associated protein expression, and enhance migration and invasion. The effect of ATP6L on metastasis was further demonstrated in a tail vein injection mice model. In addition, the mouse xenograft model showed that ATP6L-overexpressing HCT116 cells grew into larger tumor masses, showed less necrosis and formed more micro-vessels than the control cells. Taken together, our results suggest that ATP6L promotes metastasis of colorectal cancer by inducing EMT and angiogenesis, and is a potential target for tumor therapy.


Assuntos
Neoplasias Colorretais/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Células HCT116 , Células HT29 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Transplante de Neoplasias , Prognóstico , Vimentina/metabolismo
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