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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445495

RESUMO

As the most common gene mutation found in cancers, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Meanwhile, mutant p53 overexpression is known to drive oncogenic phenotypes in cancer patients and to sustain the activation of EGFR signaling. Previously, we have demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways, by gefitinib and JNJ-26854165, exerts a strong synergistic lethal effect on HGSOC cells. In this study, we investigated whether the gain-of-function p53 mutation (p53R248Q) overexpression could affect EGFR-related signaling and the corresponding drug inhibition outcome in HGSOC. The targeted inhibition responses of gefitinib and JNJ-26854165, in p53R248Q-overexpressing cells, were extensively evaluated. We found that the phosphorylation of AKT increased when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several AKT-related regulatory proteins translocated in unique intracellular patterns. Subsequent analysis revealed that, under the combined inhibition of gefitinib and JNJ-26854165, the cytonuclear trafficking of EGFR and MDM2 was disrupted. Next, we analyzed the gefitinib and JNJ-26854165 responses and found differential sensitivity to the single- or combined-drug inhibitions in p53R248Q-overexpressing cells. Our findings suggested that the R248Q mutation of p53 in HGSOC caused significant changes in signaling protein function and trafficking, under EGFR/MDM2-targeted inhibition. Such knowledge could help to advance our understanding of the role of mutant p53 in ovarian carcinoma and to improve the prognosis of patients receiving EGFR/MDM2-targeted therapies.


Assuntos
Carcinoma Epitelial do Ovário/genética , Cistadenocarcinoma Seroso/genética , Mutação com Ganho de Função , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Carcinoma Epitelial do Ovário/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Triptaminas/farmacologia
2.
Molecules ; 26(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34443505

RESUMO

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18-11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.


Assuntos
Glutationa/metabolismo , Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Sulfóxidos/farmacologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Glucuronosiltransferase/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Neoplasias da Bexiga Urinária/enzimologia
3.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443374

RESUMO

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.


Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
4.
Molecules ; 26(14)2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34299620

RESUMO

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.


Assuntos
Glicemia/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Extratos Vegetais/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetulus , Diabetes Mellitus Tipo 2 , Transportador de Glucose Tipo 4/metabolismo , Células HeLa , Humanos , Resistência à Insulina/fisiologia , Camundongos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
5.
Commun Biol ; 4(1): 828, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211117

RESUMO

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Canais de Translocação SEC/metabolismo , Animais , Retículo Endoplasmático/efeitos dos fármacos , Glicoconjugados/farmacologia , Células HeLa , Humanos , Immunoblotting , Membranas Intracelulares/efeitos dos fármacos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Canais de Translocação SEC/genética , Partícula de Reconhecimento de Sinal/metabolismo
6.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203768

RESUMO

Mesembryanthemum crystallinum (common ice plant) is a halophyte species that has adapted to extreme conditions. In this study, we cloned a McHB7 transcription factor gene from the ice plant. The expression of McHB7 was significantly induced by 500 mM NaCl and it reached the peak under salt treatment for 7 days. The McHB7 protein was targeted to the nucleus. McHB7-overexpressing in ice plant leaves through Agrobacterium-mediated transformation led to 25 times more McHB7 transcripts than the non-transformed wild type (WT). After 500 mM NaCl treatment for 7 days, the activities of superoxide dismutase (SOD) and peroxidase (POD) and water content of the transgenic plants were higher than the WT, while malondialdehyde (MDA) was decreased in the transgenic plants. A total of 1082 and 1072 proteins were profiled by proteomics under control and salt treatment, respectively, with 22 and 11 proteins uniquely identified under control and salt stress, respectively. Among the 11 proteins, 7 were increased and 4 were decreased after salt treatment. Most of the proteins whose expression increased in the McHB7 overexpression (OE) ice plants under high salinity were involved in transport regulation, catalytic activities, biosynthesis of secondary metabolites, and response to stimulus. The results demonstrate that the McHB7 transcription factor plays a positive role in improving plant salt tolerance.


Assuntos
Mesembryanthemum/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Tolerância ao Sal/fisiologia , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Biologia Computacional , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Mesembryanthemum/efeitos dos fármacos , Mesembryanthemum/genética , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Salinidade , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismo
7.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206320

RESUMO

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 µg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography-mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.


Assuntos
Asteraceae/química , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes , Células Musculares/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Transportador de Glucose Tipo 4/genética , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos
8.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063066

RESUMO

The maintenance of intracellular NAD+/NADH homeostasis across multiple, subcellular compartments requires the presence of NADH-shuttling proteins, which circumvent the lack of permeability of organelle membranes to these cofactors. Very little is known regarding these proteins in the methylotrophic yeast, Pichia pastoris. During the study of the subcellular locations of these shuttling proteins, which often have dual subcellular locations, it became necessary to develop new ways to detect the weak peroxisomal locations of some of these proteins. We have developed a novel variation of the traditional Bimolecular Fluorescence Complementation (BiFC), called divergent BiFC, to detect intraorganellar colocalization of two noninteracting proteins based on their proximity-based protein crowding within a small subcellular compartment, rather than on the traditional protein-protein interactions expected for BiFC. This method is used to demonstrate the partially peroxisomal location of one such P. pastoris NADH-shuttling protein, malate dehydrogenase B, only when cells are grown in oleate, but not when grown in methanol or glucose. We discuss the mode of NADH shuttling in P. pastoris and the physiological basis of the medium-dependent compartmentalization of PpMdhB.


Assuntos
Proteínas Fúngicas/metabolismo , Malato Desidrogenase/metabolismo , Ácido Oleico/metabolismo , Peroxissomos/metabolismo , Saccharomycetales/enzimologia , Carbono/farmacologia , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , NAD/metabolismo , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes
9.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069257

RESUMO

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fluconazol/farmacologia , Ácido Láctico/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos
10.
Toxins (Basel) ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065185

RESUMO

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences.


Assuntos
Anti-Infecciosos/farmacologia , Histidina/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Anti-Infecciosos/química , Linhagem Celular Tumoral , Desenho de Fármacos , Fluoresceínas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Luciferases/metabolismo , Peptídeos/química , Saporinas/metabolismo
11.
J Bacteriol ; 203(16): e0020421, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34031040

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting ß-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. In contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted, and FtsZ relocalizes away from midcell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with ß-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the ß-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septum-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the ß-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the ß-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to ß-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas do Citoesqueleto/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Sinergismo Farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Transporte Proteico/efeitos dos fármacos , beta-Lactamas/farmacologia
12.
Commun Biol ; 4(1): 560, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980987

RESUMO

Accumulation and spread of tau in Alzheimer's disease and other tauopathies occur in a prion-like manner. However, the mechanisms and downstream consequences of tau trafficking remain largely unknown. We hypothesized that tau traffics from neurons to microglia via extracellular vesicles (EVs), leading to IL-6 generation and cognitive impairment. We assessed mice and neurons treated with anesthetics sevoflurane and desflurane, and applied nanobeam-sensor technology, an ultrasensitive method, to measure tau/p-tau amounts. Sevoflurane, but not desflurane, increased tau or p-tau amounts in blood, neuron culture medium, or EVs. Sevoflurane increased p-tau amounts in brain interstitial fluid. Microglia from tau knockout mice took up tau and p-tau when treated with sevoflurane-conditioned neuron culture medium, leading to IL-6 generation. Tau phosphorylation inhibitor lithium and EVs generation inhibitor GW4869 attenuated tau trafficking. GW4869 mitigated sevoflurane-induced cognitive impairment in mice. Thus, tau trafficking could occur from neurons to microglia to generate IL-6, leading to cognitive impairment.


Assuntos
Transporte Proteico/efeitos dos fármacos , Sevoflurano/farmacologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Anestésicos Inalatórios/farmacologia , Animais , Encéfalo/metabolismo , Disfunção Cognitiva/induzido quimicamente , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Feminino , Hipocampo/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Transporte Proteico/fisiologia , Sevoflurano/metabolismo , Tauopatias/metabolismo , Tauopatias/fisiopatologia , Proteínas tau/fisiologia
13.
Biochem Biophys Res Commun ; 557: 247-253, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33894410

RESUMO

Accumulation of amyloid-ß peptide (Aß) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aß accumulation offers a promising approach for therapeutic strategies against AD. Aß is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aß is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aß. mStx2a-treatment also inhibited the extracellular release of Aß. Therefore, mStx2a may provide a new strategy to inhibit the production of Aß by modulating the intracellular transport of APP.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/efeitos dos fármacos , Endossomos/metabolismo , Lisossomos/metabolismo , Transporte Proteico/efeitos dos fármacos , Toxina Shiga II/farmacologia , Animais , Células CHO , Domínio Catalítico/genética , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Globosídeos/química , Humanos , Mutação , Fosfatidilcolinas/química , Proteínas Recombinantes , Toxina Shiga II/química , Toxina Shiga II/genética , Triexosilceramidas/química
14.
Plant Cell ; 33(2): 420-438, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33866370

RESUMO

Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Boro/farmacologia , Proteólise/efeitos dos fármacos , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Antiporters/química , Proteínas de Arabidopsis/química , Sítios de Ligação , Testes Genéticos , Proteínas de Fluorescência Verde/metabolismo , Lisina/metabolismo , Modelos Biológicos , Poliubiquitina/metabolismo , Transporte Proteico/efeitos dos fármacos , Prótons , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Ubiquitinação/efeitos dos fármacos , Vacúolos/metabolismo
15.
Int J Mol Sci ; 22(8)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33924293

RESUMO

The study of cisplatin sensitivity is the key to the development of ovarian cancer treatment strategies. Mitochondria are one of the main targets of cisplatin, its self-clearing ability plays an important role in determining the fate of ovarian cancer cells. First, we proved that the sensitivity of ovarian cancer cells to cisplatin depends on mitophagy, and p62 acts as a broad autophagy receptor to regulate this process. However, p62's regulation of mitophagy does not depend on its location on the mitochondria. Our research shows that the mutation of the UBA domain of p62 increases the localisation of HK2 on the mitochondria, thereby increasing the phosphorylated ubiquitin form of parkin, then stabilising the process of mitophagy and ultimately cell survival. Collectively, our results showed that a mutation in the UBA domain of p62 regulates the level of apoptosis stimulated by cisplatin in ovarian cancer.


Assuntos
Cisplatino/farmacologia , Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Mutação/genética , Neoplasias Ovarianas/patologia , Proteína Sequestossoma-1/genética , Regulação para Cima/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Modelos Biológicos , Neoplasias Ovarianas/genética , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Proteína Sequestossoma-1/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
17.
Front Immunol ; 12: 663586, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859652

RESUMO

As of January 2021, SARS-CoV-2 has killed over 2 million individuals across the world. As such, there is an urgent need for vaccines and therapeutics to reduce the burden of COVID-19. Several vaccines, including mRNA, vector-based vaccines, and inactivated vaccines, have been approved for emergency use in various countries. However, the slow roll-out of vaccines and insufficient global supply remains a challenge to turn the tide of the pandemic. Moreover, vaccines are important tools for preventing the disease but therapeutic tools to treat patients are also needed. As such, since the beginning of the pandemic, repurposed FDA-approved drugs have been sought as potential therapeutic options for COVID-19 due to their known safety profiles and potential anti-viral effects. One of these drugs is ivermectin (IVM), an antiparasitic drug created in the 1970s. IVM later exerted antiviral activity against various viruses including SARS-CoV-2. In this review, we delineate the story of how this antiparasitic drug was eventually identified as a potential treatment option for COVID-19. We review SARS-CoV-2 lifecycle, the role of the nucleocapsid protein, the turning points in past research that provided initial 'hints' for IVM's antiviral activity and its molecular mechanism of action- and finally, we culminate with the current clinical findings.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Ivermectina/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/antagonistas & inibidores , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Reposicionamento de Medicamentos , Humanos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Células Vero , Replicação Viral/efeitos dos fármacos , alfa Carioferinas/antagonistas & inibidores , beta Carioferinas/antagonistas & inibidores
18.
J Biochem Mol Toxicol ; 35(6): 1-10, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33720461

RESUMO

Lung cancer is a noxious disease with substandard overall survival. Despite this, there are several treatment strategies for lung cancer include chemotherapy, radiotherapy, surgery; however, the overall survival remains poor. Punicalagin has been documented as a potential phytomedicine to selectively inhibit the progression and expansion of numerous cancers. In the present study, we evaluated the antiproliferative ability of punicalagin against lung cancer A549 cells by inducing apoptosis by inhibiting STAT-3 activation. Punicalagin induces toxic effects of A549 cells in a dose-associated manner after 24 h treatment. And we also observed that punicalagin (10, 20, and 30 µM) induced reactive oxygen species generation, alters the mitochondrion membrane potential and apoptotic morphological changes in A549 cells. The STAT-3 overexpression regulates apoptosis, proliferation, and angiogenesis. Here, the punicalagin inhibited STAT-3 translocation and thereby induces apoptosis by inhibiting expression Bcl-2 and enhanced expression of Bax, cytochrome-c, caspase-9, and caspase-3 in A549 cells. Hence, we stated that the punicalagin is a possible therapy for non-small cell lung, malignancies. Altogether, the punicalagin is a promising phytomedicine in malignancy treatment and further endeavors are needed to unveil the complete potential.


Assuntos
Apoptose/efeitos dos fármacos , Taninos Hidrolisáveis/farmacologia , Neoplasias Pulmonares , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Células A549 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transporte Proteico/efeitos dos fármacos
19.
Biochem Pharmacol ; 188: 114521, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33741329

RESUMO

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Angiotensina II/farmacologia , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptor Tipo 2 de Angiotensina/agonistas , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia
20.
Biochem Biophys Res Commun ; 553: 154-159, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33773137

RESUMO

The glucocorticoid receptor (GR) plays an important role in steroid-dependent regulation of metabolism, development, and the immune response in humans. Although GR is known to be activated by the binding of glucocorticoid, the mechanism of action is poorly understood. We investigated dimerization of GR in the cytoplasm and nuclear trans-localization in response to treatment with the ligand dexamethasone. GFP-tagged GR and FLAG-tagged GR were co-expressed in COS-1 cells, and cell lysates were subjected to co-immunoprecipitation assay with anti-GFP antibody to determine their dimerization. FLAG-GR was co-precipitated with GFP-GR in the cytoplasmic fraction of COS-1 cells. Treatment with the GR agonist dexamethasone significantly decreased the cytoplasmic interaction between FLAG- and GFP-GR, and significantly increased interaction of the GRs in the nuclear fraction. The two amino acids, Pro625 and Ile628 known to be located in GR-GR dimer interface, were mutated to alanine and the influence of the mutation on dimerization, ligand-dependent nuclear localization, and transcriptional activities were determined. Mutant GR showed a dramatic decrease in interaction in the cytoplasmic fraction and no detectable nuclear translocation in the presence or absence of dexamethasone. Furthermore, luciferase assays showed that mutant GR showed no detectable transcriptional activation via the GR-responsive DNA element (GRE) compared to the wild-type. Our results suggest that GR exists as a dimer in the cytoplasm and this dimerization may be essential for GRE-mediated transcriptional activation following ligand binding.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Multimerização Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Animais , Células COS , Núcleo Celular/efeitos dos fármacos , Chlorocebus aethiops , Citoplasma/efeitos dos fármacos , Dexametasona/metabolismo , Dexametasona/farmacologia , Humanos , Ligantes , Modelos Moleculares , Mutação , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores de Glucocorticoides/genética
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