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1.
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 , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Metiltransferases/metabolismo , Proteínas do Tecido Nervoso/genética , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Internalização do Vírus
2.
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
3.
Plant Sci ; 289: 110215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31623776

RESUMO

14-3-3 proteins are a family of conserved proteins present in eukaryotes as several isoforms, playing a regulatory role in many cellular and physiological processes. In plants, 14-3-3 proteins have been reported to be involved in the response to stress conditions, such as drought, salt and cold. In the present study, 14-3-3ε and 14-3-3ω isoforms, which were representative of ε and non-ε phylogenetic groups, were overexpressed in Arabidopsis thaliana plants; the effect of their overexpression was investigated on H+-ATPase activation and plant response to cold stress. Results demonstrated that H+-ATPase activity was increased in 14-3-3ω-overexpressing plants, whereas overexpression of both 14-3-3 isoforms brought about cold stress tolerance, which was evaluated through ion leakage, lipid peroxidation, osmolyte synthesis, and ROS production assays. A dedicated tandem mass tag (TMT)-based proteomic analysis demonstrated that different proteins involved in the plant response to cold or oxidative stress were over-represented in 14-3-3ε-overexpressing plants.


Assuntos
Proteínas 14-3-3/genética , Arabidopsis/genética , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Proteínas 14-3-3/metabolismo , Aclimatação/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
4.
Int J Mol Sci ; 20(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623139

RESUMO

The vacuolar H+-ATPase (V-ATPase) plays many important roles in cell growth and in response to stresses in plants. The V-ATPase subunit H (VHA-H) is required to form a stable and active V-ATPase. Genome-wide analyses of VHA-H genes in crops contribute significantly to a systematic understanding of their functions. A total of 22 VHA-H genes were identified from 11 plants representing major crops including cotton, rice, millet, sorghum, rapeseed, maize, wheat, soybean, barley, potato, and beet. All of these VHA-H genes shared exon-intron structures similar to those of Arabidopsis thaliana. The C-terminal domain of VHA-H was shorter and more conserved than the N-terminal domain. The VHA-H gene was effectively used as a genetic marker to infer the phylogenetic relationships among plants, which were congruent with currently accepted taxonomic groupings. The VHA-H genes from six species of crops (Gossypium raimondii, Brassica napus, Glycine max, Solanum tuberosum, Triticum aestivum, and Zea mays) showed high gene structural diversity. This resulted from the gains and losses of introns. Seven VHA-H genes in six species of crops (Gossypium raimondii, Hordeum vulgare, Solanum tuberosum, Setaria italica, Triticum aestivum, and Zea mays) contained multiple transcript isoforms arising from alternative splicing. The study of cis-acting elements of gene promoters and RNA-seq gene expression patterns confirms the role of VHA-H genes as eco-enzymes. The gene structural diversity and proteomic diversity of VHA-H genes in our crop sampling facilitate understanding of their functional diversity, including stress responses and traits important for crop improvement.


Assuntos
Produtos Agrícolas/genética , Genoma de Planta , Estudo de Associação Genômica Ampla , Família Multigênica , Subunidades Proteicas/genética , ATPases Vacuolares Próton-Translocadoras/genética , Processamento Alternativo , Sequência de Aminoácidos , Produtos Agrícolas/classificação , Genômica/métodos , Motivos de Nucleotídeos , Filogenia , Regiões Promotoras Genéticas , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo
5.
Mol Med Rep ; 20(3): 2796-2804, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31524252

RESUMO

The renin­angiotensin system (RAS) serves an essential role in hypertension. MicroRNAs (miRs) have been reported to be important regulators in angiotensin (Ang) II­dependent hypertension. We aimed to explore the roles of Ang II and miR­133a in the mechanism underlying hypertension. Human umbilical vein endothelial cells (HUVECs) were identified by immunofluorescence staining. Cell viability and miR­133a expression under the inhibition of Ang II of various concentrations were determined by an MTT assay and reverse transcription­quantitative polymerase chain reaction (RT­qPCR), respectively. The effects of HUVECs transfected with miR­133a mimic or inhibitor on Ang II­induced apoptosis were measured using flow cytometry. The potential targeting of miR­133a to the 3' untranslated region of (pro) renin receptor (PRR) was assessed using TargetScan and a dual­luciferase assay. The effects of PRR interference using small interfering (si)RNA on PRR expression and the rate of apoptosis were determined by RT­qPCR, western blotting and flow cytometry, respectively. Ang II at a concentration of 10­5 M significantly inhibited the cell viability (P<0.05) and miR­133a expression (P<0.01); Downregulation of miR­133a suppressed cell viability. HUVECs transfected with miR­133a mimic reduced the rate of Ang II­induced apoptosis from 21.99 to 12.38%, but miR­133a inhibitor promoted Ang II­induced apoptosis (apoptosis rate, 28.9%). PRR was predicted to be a target gene of miR­133a. Transfection with siPRR decreased the apoptotic rate in Ang II + negative control and Ang II + miR­133a inhibitor group to 11.39 and 12.94%, respectively. Our findings also suggested that Ang II promoted PRR expression to enhance the apoptotic rate of HUVECs via the suppression of miR­133a. Furthermore, siPRR efficiently decreased the Ang II­induced apoptosis.


Assuntos
Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , MicroRNAs/genética , Interferência de RNA , Regiões 3' não Traduzidas , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/etiologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina , ATPases Vacuolares Próton-Translocadoras/metabolismo
6.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470498

RESUMO

Cancer cells overexpress proton exchangers at the plasma membrane in order acidify the extracellular matrix and maintain the optimal pH for sustaining cancer growth. Among the families of proton exchangers implicated in carcinogenesis, carbonic anhydrases (CAs), monocarboxylate transporters (MCTs), Na+/H+ exchangers (NHEs), sodium bicarbonate cotransporters (NBCs), and vacuolar ATPases (V-ATPases) are highlighted. Considerable research has been carried out into the utility of the understanding of these machineries in the diagnosis and prognosis of several solid tumors. In addition, as therapeutic targets, the interference of their functions has contributed to the discovery or optimization of cancer therapies. According to recent reports, the study of these mechanisms seems promising in the particular case of oral squamous cell carcinoma (OSCC). In the present review, the latest advances in these fields are summarized, in particular, the usefulness of proton exchangers as potential prognostic biomarkers and therapeutic targets in OSCC.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Neoplasias Bucais/metabolismo , Prótons , Carcinoma de Células Escamosas/patologia , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Bucais/patologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Nanomedicina Teranóstica/métodos , ATPases Vacuolares Próton-Translocadoras/metabolismo
7.
Nat Chem Biol ; 15(8): 776-785, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285595

RESUMO

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.


Assuntos
Autofagia/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-akt
8.
PLoS One ; 14(7): e0219602, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291376

RESUMO

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+-ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed V-ATPase sub-complex.


Assuntos
Vesículas Extracelulares/metabolismo , Osteoclastos/metabolismo , Proteoma/metabolismo , Animais , Células da Medula Óssea , Remodelação Óssea , Células Cultivadas , Camundongos , Osteogênese , Cultura Primária de Células , Proteômica , ATPases Vacuolares Próton-Translocadoras/metabolismo
9.
PLoS One ; 14(7): e0219979, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31335886

RESUMO

The halophyte Suaeda salsa displayed strong resistance to salinity. Up to date, molecular mechanisms underlying tolerance of S. salsa to salinity have not been well understood. In the present study, S. salsa seedlings were treated with 30‰ salinity and then leaves and roots were subjected to Illumina sequencing. Compared with the control, 68,599 and 77,250 unigenes were significantly differentially expressed in leaves and roots in saline treatment, respectively. KEGG enrichment analyses indicated that photosynthesis process, carbohydrate, lipid and amino acid metabolisms were all downregulated in saline treatment, which should inhibit growth of S. salsa. Expression levels of Na+/H+ exchanger, V-H+ ATPase, choline monooxygenase, potassium and chloride channels were upregulated in saline treatment, which could relieve reduce over-accumulation of Na+ and Cl-. Fe-SOD, glutathione, L-ascorbate and flavonoids function as antioxidants in plants. Genes in relation to them were all upregulated, suggesting that S. salsa initiated various antioxidant mechanisms to tolerate high salinity. Besides, plant hormones, especially auxin, ethylene and jasmonic acid signaling transduction pathways were all upregulated in response to saline treatment, which were important to gene regulations of ion transportation and antioxidation. These changes might comprehensively contribute to tolerance of S. salsa to salinity. Overall, the present study provided new insights to understand the mechanisms underlying tolerance to salinity in halophytes.


Assuntos
Chenopodiaceae/genética , Estresse Salino , Transcriptoma , Chenopodiaceae/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Flavonoides/genética , Flavonoides/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Reguladores de Crescimento de Planta/genética , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
10.
Mediators Inflamm ; 2019: 4143604, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31275057

RESUMO

A small group of only seven transcription factors known as STATs (signal transducer and activator of transcription) are considered to be canonical determinants of specific gene activation for a plethora of ligand/receptor systems. The activation of STATs involves a family of four tyrosine kinases called JAK kinases. JAK1 and JAK2 activate STAT1 in the cytoplasm at the heterodimeric gamma interferon (IFNγ) receptor, while JAK1 and TYK2 activate STAT1 and STAT2 at the type I IFN heterodimeric receptor. The same STATs and JAKs are also involved in signaling by functionally different cytokines, growth factors, and hormones. Related to this, IFNγ-activated STAT1 binds to the IFNγ-activated sequence (GAS) element, but so do other STATs that are not involved in IFNγ signaling. Activated JAKs such as JAK2 and TYK2 are also involved in the epigenetics of nucleosome unwrapping for exposure of DNA to transcription. Furthermore, activated JAKs and STATs appear to function coordinately for specific gene activation. These complex events have not been addressed in canonical STAT signaling. Additionally, the function of noncoding enhancer RNAs, including their role in enhancer/promoter interaction is not addressed in the canonical STAT signaling model. In this perspective, we show that JAK/STAT signaling, involving membrane receptors, is essentially a variation of cytoplasmic nuclear receptor signaling. Focusing on IFN signaling, we showed that ligand, IFN receptor, the JAKs, and the STATs all undergo endocytosis and ATP-dependent nuclear translocation to promoters of genes specifically activated by IFNs. We argue here that the vacuolar ATPase (V-ATPase) proton pump probably plays a key role in endosomal membrane crossing by IFNs for receptor cytoplasmic binding. Signaling of nuclear receptors such as those of estrogen and dihydrotestosterone provides templates for making sense of the specificity of gene activation by closely related cytokines, which has implications for lymphocyte phenotypes.


Assuntos
Esteroides/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Humanos , Interferons/metabolismo , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , TYK2 Quinase/metabolismo
11.
BMC Dev Biol ; 19(1): 12, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226923

RESUMO

BACKGROUND: Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. RESULTS: Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl--channels (9-anthroic acid) and Na+/K+/2Cl--cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl--cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl--cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. CONCLUSIONS: Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.


Assuntos
Epitélio/fisiologia , Transporte de Íons/fisiologia , Folículo Ovariano/citologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster , Eletroquímica , Feminino , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Oogênese , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia
12.
J Agric Food Chem ; 67(32): 8950-8957, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31189310

RESUMO

To determine how nutritional restriction compromised milk synthesis, sows were fed 100% (control) or 76% (restricted) of the recommended feed allowance from postpartum day (PD)-1 to PD-28. In comparison to the control, more body reserves loss, increased plasma triglyceride and high-density lipoprotein cholesterol levels, and decreased plasma methionine concentrations were observed in the restricted group at PD-21. The increased plasma malondialdehyde level, decreased plasma histidine and taurine concentrations, and decreased glutathione peroxidase activity were observed at PD-28 when backfat loss further increased in the restricted group. In mammary glands, vacuolar H+-adenosine triphosphatase (v-ATPase), as the upstream of the mechanistic target of rapamycin (mTOR) signaling, showed decreased activity, while phosphorylation of mTOR, S6 kinase, and eukaryotic translation initiation factor 4E-binding protein 1 and ß-casein abundance all decreased following feed restriction. Altogether, long-term nutrition restriction could induce progressively aggravated oxidative stress and compromise mammary protein synthesis through repression of v-ATPase/mTORC1 signaling.


Assuntos
Glândulas Mamárias Animais/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Estresse Oxidativo , Biossíntese de Proteínas , Suínos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Leite/metabolismo , Fosforilação , Período Pós-Parto/metabolismo , Gravidez , Transdução de Sinais , Suínos/genética , ATPases Vacuolares Próton-Translocadoras/genética
13.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(6): 261-277, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31189779

RESUMO

Vacuolar-type ATPase (V-ATPase), initially identified in yeast and plant vacuoles, pumps protons into the lumen of organelles coupled with ATP hydrolysis. The mammalian counterpart is found ubiquitously in endomembrane organelles and the plasma membrane of specialized cells such as osteoclasts. V-ATPase is also present in unique organelles such as insulin secretory granules, neural synaptic vesicles, and acrosomes of spermatozoa. Consistent with its diverse physiological roles and unique localization, the seven subunits of V-ATPase have 2-4 isoforms that are organelle- or cell-specific. Subunits of the enzyme function in trafficking organelles and vesicles by interacting with small molecule GTPases. During osteoclast differentiation, one of the four isoforms of subunit a, a3, is indispensable for secretory lysosome trafficking to the plasma membrane. Diseases such as osteopetrosis, renal acidosis, and hearing loss are related to V-ATPase isoforms. In addition to its role as an enzyme, V-ATPase has versatile physiological roles in eukaryotic cells.


Assuntos
Lisossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Osteoclastos/citologia
14.
Toxicol Mech Methods ; 29(8): 561-568, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31161845

RESUMO

Cleistanthus collinus is a poisonous shrub used for deliberate self-harm in rural areas of South India and intake of boiled decoction of leaves is a common method of self-harm. Distal renal tubular acidosis (dRTA) is an important clinical symptom observed in C. collinus poisoning, and renal V-ATPases may be potential targets of damage. However, a lack of understanding of molecular mediators involved hampers medical management, which is mainly supportive. We hypothesized that C. collinus poisoning induces renal oxidative stress; probably by inducing mitochondrial uncoupling, which compromises V-ATPase activity to ultimately produce dRTA. This was tested by exposing renal BBMV, kidney cells in culture, and Wistar rats to C. collinus poisoning. Exposure to C. collinus aqueous extract resulted in significant elevations in the lipid peroxidation marker, conjugated dienes, in cell culture and in vivo. A significant decrease in mitochondrial respiratory control ratio was observed in kidneys from C. collinus-treated animals suggesting that mitochondrial oxidative phosphorylation is uncoupled. This was accompanied by significant increase in ADP levels and a decrease in proton pump activity. Thus, these results demonstrate that C. collinus poisoning induces oxidative stress which influences proton pump activity, probably due to feedback inhibition by elevated ADP levels because of mitochondrial dysfunction in the rat kidney.


Assuntos
Acidose Tubular Renal/induzido quimicamente , Euphorbiaceae/envenenamento , Rim/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Acidose Tubular Renal/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Mitocôndrias Musculares/metabolismo , Fosforilação Oxidativa , Extratos Vegetais/envenenamento , Ratos Wistar
15.
J Endod ; 45(7): 898-906, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104818

RESUMO

INTRODUCTION: Endodontic disease, 1 of the most prevalent chronic infectious diseases worldwide, occurs when the dental pulp becomes infected and inflamed, leading to bone destruction around the tooth root, severe pain, and tooth loss. Although many studies have tried to develop therapies to alleviate the bone erosion and inflammation associated with endodontic disease, there is an urgent need for an effective treatment. METHODS: In this study, we used a gene-based therapy approach by administering recombinant adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown to target both periapical bone resorption and inflammation in the mouse model of endodontic disease. RESULTS: The results showed that Atp6v1c1 knockdown is simultaneously capable of reducing bone resorption by 70% through impaired osteoclast activation, inhibiting inflammation by decreasing T-cell infiltration in the periapical lesion by 75%, and protecting the periodontal ligament from destruction caused by inflammation. Notably, AAV-mediated gene therapy of Atp6v1c1 knockdown significantly reduced proinflammatory cytokine expression, including tumor necrosis factor α, interleukin 1α, interleukin 17, interleukin 12, and interleukin 6 levels in periapical tissues caused by bacterial infection. Quantitative real-time polymerase chain reaction revealed that Atp6v1c1 knockdown reduced osteoclast-specific functional genes (ie, Ctsk) in periapical tissues. CONCLUSIONS: Our results showed that AAV-mediated Atp6v1c1 knockdown in periapical tissues slowed endodontic disease progression, prevented bone erosion, and alleviated inflammation in the periapical tissues and periodontal ligament potentially through regulation of toll-like receptor signaling, indicating that targeting Atp6v1c1 may facilitate the design of novel therapeutic approaches to reduce inflammation and bone erosion in endodontic disease.


Assuntos
Perda do Osso Alveolar , Reabsorção Óssea , ATPases Vacuolares Próton-Translocadoras , Perda do Osso Alveolar/genética , Animais , Reabsorção Óssea/genética , Dependovirus , Inativação Gênica , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
16.
Cells ; 8(5)2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108937

RESUMO

ß-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and ß-amyloid (Aß) production, a critical step in the pathogenesis of Alzheimer's disease (AD). It is thus of considerable interest to investigate how BACE1 activity is regulated. BACE1 has its maximal activity at acidic pH and GFP variant-pHluorin-displays pH dependence. In light of these observations, we generated three tandem fluorescence-tagged BACE1 fusion proteins, named pHluorin-BACE1-mCherry, BACE1-mCherry-pHluorin and BACE1-mCherry-EGFP. Comparing the fluorescence characteristics of these proteins in response to intracellular pH changes induced by chloroquine or bafilomycin A1, we found that pHluorin-BACE1-mCherry is a better pH sensor for BACE1 because its fluorescence intensity responds to pH changes more dramatically and more quickly. Additionally, we found that (pro)renin receptor (PRR), a subunit of the v-ATPase complex, which is critical for maintaining vesicular pH, regulates pHluorin's fluorescence and BACE1 activity in pHluorin-BACE1-mCherry expressing cells. Finally, we found that the expression of Swedish mutant APP (APPswe) suppresses pHluorin fluorescence in pHluorin-BACE1-mCherry expressing cells in culture and in vivo, implicating APPswe not only as a substrate but also as an activator of BACE1. Taken together, these results suggest that the pHluorin-BACE1-mCherry fusion protein may serve as a useful tool for visualizing active/inactive BACE1 in culture and in vivo.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Cloroquina/farmacologia , Feminino , Fluorescência , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Macrolídeos/farmacologia , Masculino , Camundongos , Receptores de Superfície Celular/metabolismo , Transfecção , ATPases Vacuolares Próton-Translocadoras/metabolismo
17.
Molecules ; 24(10)2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31137574

RESUMO

Malaria is an infectious disease caused by Plasmodium group. The mechanisms of antimalarial drugs DHA/CQ are still unclear today. The inhibitory effects (IC50) of single treatments with DHA/CQ or V-ATPase inhibitor Baf-A1 or combination treatments by DHA/CQ combined with Baf-A1 on the growth of Plasmodium falciparum strain 3D7 was investigated. Intracellular cytoplasmic pH and labile iron pool (LIP) were labeled by pH probe BCECF, AM and iron probe calcein, AM, the fluorescence of the probes was measured by FCM. The effects of low doses of DHA (0.2 nM, 0.4 nM, 0.8 nM) on gene expression of V-ATPases (vapE, vapA, vapG) located in the membrane of DV were tested by RT-qPCR. DHA combined with Baf-A1 showed a synergism effect (CI = 0.524) on the parasite growth in the concentration of IC50. Intracellular pH and irons were effected significantly by different doses of DHA/Baf-A1. Intracellular pH was decreased by CQ combined with Baf-A1 in the concentration of IC50. Intracellular LIP was increased by DHA combined with Baf-A1 in the concentration of 20 IC50. The expression of gene vapA was down-regulated by all low doses of DHA (0.2/0.4/0.8 nM) significantly (p < 0.001) and the expression of vapG/vapE were up-regulated by 0.8 nM DHA significantly (p < 0.001). Interacting with ferrous irons, affecting the DV membrane proton pumping and acidic pH or cytoplasmic irons homeostasis may be the antimalarial mechanism of DHA while CQ showed an effect on cytoplasmic pH of parasite in vitro. Lastly, this article provides us preliminary results and a new idea for antimalarial drugs combination and new potential antimalarial combination therapies.


Assuntos
Artemisininas/farmacologia , Cloroquina/farmacologia , Eritrócitos/parasitologia , Homeostase , Estágios do Ciclo de Vida/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antimaláricos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/parasitologia , Quimioterapia Combinada , Eritrócitos/efeitos dos fármacos , Fluorescência , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Ferro/metabolismo , Macrolídeos/farmacologia , Parasitos/efeitos dos fármacos , Parasitos/crescimento & desenvolvimento , Testes de Sensibilidade Parasitária , Plasmodium falciparum/efeitos dos fármacos , Trofozoítos/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
18.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091659

RESUMO

Dysregulation of cellular energy metabolism is closely linked to cancer development and progression. Calorie or glucose restriction (CR or GR) inhibits energy-dependent pathways, including IGF-1/PI3K/Akt/mTOR, in cancer cells. However, alterations in proton dynamics and reversal of the pH gradient across the cell membrane, which results in intracellular alkalinization and extracellular acidification in cancer tissues, have emerged as important etiopathogenic factors. We measured glucose, lactate, and ATP production after GR, plant-derived CR-mimetic curcumin treatment, and curcumin plus GR in human hepatoma cells. Intracellular pH regulatory effects, in particular, protein-protein interactions within mTOR complex-1 and its structural change, were investigated. Curcumin treatment or GR mildly inhibited Na+/H+ exchanger-1 (NHE1). vATPase, monocarboxylate transporter (MCT)-1, and MCT4 level. Combination treatment with curcumin and GR further enhanced the inhibitory effects on these transporters and proton-extruding enzymes, with intracellular pH reduction. ATP and lactate production decreased according to pH change. Modeling of mTOR protein revealed structural changes upon treatments, and curcumin plus GR decreased binding of Raptor and GßL to mTOR, as well as of Rag A and Rag B to Raptor. Consequently, 4EBP1 phosphorylation was decreased and cell migration and proliferation were inhibited in a pH-dependent manner. Autophagy was increased by curcumin plus GR. In conclusion, curcumin treatment combined with GR may be a useful supportive approach for preventing intracellular alkalinization and cancer progression.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Curcumina/farmacologia , Glucose/deficiência , Neoplasias Hepáticas/metabolismo , Álcalis/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Glucose/metabolismo , Células Hep G2 , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
19.
Dev Cell ; 49(3): 393-408.e7, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30982660

RESUMO

The NAD+-dependent deacetylase Sirtuin 1 (SIRT1) is down-regulated in triple-negative breast cancer. To determine the mechanistic basis by which reduced SIRT1 expression influences processes related to certain aggressive cancers, we examined the consequences of depleting breast cancer cells of SIRT1. We discovered that reducing SIRT1 levels decreased the expression of one particular subunit of the vacuolar-type H+ ATPase (V-ATPase), which is responsible for proper lysosomal acidification and protein degradation. This impairment in lysosomal function caused a reduction in the number of multi-vesicular bodies (MVBs) targeted for lysosomal degradation and resulted in larger MVBs prior to their fusing with the plasma membrane to release their contents. Collectively, these findings help explain how reduced SIRT1 expression, by disrupting lysosomal function and generating a secretome comprising exosomes with unique cargo and soluble hydrolases that degrade the extracellular matrix, can promote processes that increase breast-cancer-cell survival and invasion.


Assuntos
Neoplasias da Mama/metabolismo , Lisossomos/metabolismo , Sirtuína 1/deficiência , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Exossomos/metabolismo , Feminino , Homeostase , Humanos , Corpos Multivesiculares/metabolismo , Invasividade Neoplásica , Sirtuína 1/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
20.
J Biol Chem ; 294(23): 9161-9171, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31023825

RESUMO

The low-level endo-lysosomal signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), is required for full assembly and activity of vacuolar H+-ATPases (V-ATPases) containing the vacuolar a-subunit isoform Vph1 in yeast. The cytosolic N-terminal domain of Vph1 is also recruited to membranes in vivo in a PI(3,5)P2-dependent manner, but it is not known if its interaction with PI(3,5)P2 is direct. Here, using biochemical characterization of isolated yeast vacuolar vesicles, we demonstrate that addition of exogenous short-chain PI(3,5)P2 to Vph1-containing vacuolar vesicles activates V-ATPase activity and proton pumping. Modeling of the cytosolic N-terminal domain of Vph1 identified two membrane-oriented sequences that contain clustered basic amino acids. Substitutions in one of these sequences (231KTREYKHK) abolished the PI(3,5)P2-dependent activation of V-ATPase without affecting basal V-ATPase activity. We also observed that vph1 mutants lacking PI(3,5)P2 activation have enlarged vacuoles relative to those in WT cells. These mutants exhibit a significant synthetic growth defect when combined with deletion of Hog1, a kinase important for signaling the transcriptional response to osmotic stress. The results suggest that PI(3,5)P2 interacts directly with Vph1, and that this interaction both activates V-ATPase activity and protects cells from stress.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sequência de Aminoácidos , Mutagênese , Pressão Osmótica , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/química , Vacúolos/metabolismo
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