Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.786
Filtrar
1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360998

RESUMO

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Oligopeptídeos/farmacologia , Antifúngicos/química , Candida albicans/fisiologia , Cardiolipinas/metabolismo , Membrana Celular/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Oligopeptídeos/química , Fosfatidilgliceróis/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Ácidos Teicoicos/metabolismo , Triptofano/química
2.
FASEB J ; 35(9): e21808, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390515

RESUMO

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA-induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration- and time-dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA-treated EC, which is crucial for UA-induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA-treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F-mediated PS exposure and MPs release.


Assuntos
Anoctaminas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/metabolismo , Hiperuricemia/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperuricemia/sangue , Peroxidação de Lipídeos/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Ácido Úrico/sangue
3.
FASEB J ; 35(9): e21835, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34449927

RESUMO

Circulating neutrophil extracellular traps (NETs) resistant to t-PA have not been studied completely although NETs in thrombi may contribute to tissue plasminogen activator (t-PA) resistance. This research intended to elucidate whether circulating NETs are associated with t-PA resistance and the underlying mechanism. The levels of NETs were detected in the circulating neutrophils, ischemic brain tissue of acute ischemic stroke (AIS) patients, and transient middle cerebral artery occlusion (tMCAO) models. NET formation in blood, thrombi, and ischemic brain tissue of mice were analyzed by immunofluorescence. Exposed phosphatidylserine (PS) was assessed using flow cytometry and confocal microscopy. Procoagulant activity (PCA) was evaluated using fibrin formation assays, thrombin, and purified coagulation complex. The plasma levels of NETs in AIS patients were significantly higher than those in healthy individuals. After thrombolysis, a significant increase was noted in NET markers in no-improvement patients, while the changes in improvement patients were not significant. Importantly, NETs were decorated with von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1) in the blood and thrombi, which could reverse the fibrinolytic effects. In addition, NETs activated platelets (PLTs) and endothelial cells (ECs), stimulating a procoagulant phenotype and facilitating vWF and PAI-1 release. DNase I, activated protein C (APC), and sivelestat markedly inhibited these effects. Furthermore, targeting NETs protected mice from tMCAO-induced cerebral ischemia, possibly by regulating vWF and PAI-1. In summary, NETs may contribute to t-PA resistance in AIS through activation of PLTs and ECs. Strategies against NETs may present a promising therapeutic approach to improve the thrombolysis efficiency of t-PA in AIS patients.


Assuntos
Isquemia Encefálica/metabolismo , Armadilhas Extracelulares/metabolismo , AVC Isquêmico/metabolismo , Neutrófilos/metabolismo , Acidente Vascular Cerebral/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Idoso , Animais , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo , Trombina/metabolismo , Trombose/metabolismo
4.
FEBS Lett ; 595(17): 2197-2207, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339521

RESUMO

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.


Assuntos
Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/citologia , Vacúolos/metabolismo , Autofagossomos/química , Autofagossomos/metabolismo , Autofagia , Congelamento , Membranas Intracelulares/química , Lipídeos de Membrana/química , Microscopia Eletrônica , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/química , Receptores de Esteroides/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Vacúolos/química
5.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372582

RESUMO

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.


Assuntos
Ebolavirus/genética , Proteínas da Matriz Viral/genética , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cisteína/genética , Ebolavirus/metabolismo , Humanos , Lipídeos/fisiologia , Simulação de Dinâmica Molecular , Fosfatidilserinas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Multimerização Proteica , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/ultraestrutura , Vírion/metabolismo , Montagem de Vírus/genética , Liberação de Vírus/genética
6.
Nat Commun ; 12(1): 4339, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267198

RESUMO

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.


Assuntos
Fosfatidilinositóis/metabolismo , Domínios de Homologia à Plecstrina , Proteínas/química , Proteínas/metabolismo , Algoritmos , Animais , Sítios de Ligação , Biologia Computacional/métodos , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Fosfatidilinositóis/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteínas/genética , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sensibilidade e Especificidade , Proteína rhoA de Ligação ao GTP/metabolismo
7.
PLoS Biol ; 19(5): e3001259, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34038417

RESUMO

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed "find-me" signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other "eat-me" signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein-coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.


Assuntos
Apoptose/imunologia , Apoptose/fisiologia , Quimiocinas/metabolismo , Animais , Vesícula/metabolismo , Células CHO , Membrana Celular/metabolismo , Quimiocinas/fisiologia , Quimiotaxia , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagócitos/metabolismo , Fagocitose/fisiologia , Fosfatidilserinas/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/fisiologia
8.
Methods Mol Biol ; 2255: 27-42, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033092

RESUMO

Cellular signals to resist apoptosis have been attributed as one of the mechanisms of tumorigenesis. Hence, apoptosis is a cardinal target for drug development in cancers, and several antitumor drugs have been designed to induce apoptosis in tumor cells. Recently, venetoclax, a Bcl2 inhibitor that induces apoptosis, has been approved by the FDA for the treatment of CLL and SLL patients. Proapoptotic antitumor drugs have been traditionally developed and tested, targeting apoptosis in tumor cells. The mechanism of such drug actions has been functionally connected to the mechanism of apoptosis. The identification of apoptosis in a tumor cell takes into account different characteristics in several steps of apoptosis. Thus, it is understandable that modes of identification of apoptosis observed in tumor cells in a laboratory have also been tuned to different characteristics in several parameters of apoptosis. Here, we present a detailed methodology for a triple-parameter-based co-fluorescence imaging to identify apoptosis in live tumor cells. The procedure involves co-fluorescence staining specific for three cardinal features of apoptosis in live cells. The procedure is simple, time-sensitive, and can be performed successfully in a laboratory-friendly manner.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Fluorescência , Laboratórios/estatística & dados numéricos , Mitocôndrias/metabolismo , Imagem Óptica/métodos , Neoplasias Ovarianas/patologia , Neoplasias da Mama/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Neoplasias Ovarianas/metabolismo , Fosfatidilserinas/metabolismo , Células Tumorais Cultivadas
9.
Theranostics ; 11(13): 6507-6521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995671

RESUMO

Ischemic stroke remains a major cause of death, and anti-inflammatory strategies hold great promise for preventing major brain injury during reperfusion. In the past decade, stem cell-derived extracellular vesicles (EVs) have emerged as novel therapeutic effectors in immune modulation. However, the intravenous delivery of EVs into the ischemic brain remains a challenge due to poor targeting of unmodified EVs, and the costs of large-scale production of stem cell-derived EVs hinder their clinical application. Methods: EVs were isolated from a human neural progenitor cell line, and their anti-inflammatory effects were verified in vitro. To attach targeting ligands onto EVs, we generated a recombinant fusion protein containing the arginine-glycine-aspartic acid (RGD)-4C peptide (ACDCRGDCFC) fused to the phosphatidylserine (PS)-binding domains of lactadherin (C1C2), which readily self-associates onto the EV membrane. Subsequently, in a middle cerebral artery occlusion (MCAO) mouse model, the RGD-C1C2-bound EVs (RGD-EV) were intravenously injected through the tail vein, followed by fluorescence imaging and assessment of proinflammatory cytokines expression and microglia activation. Results: The neural progenitor cell-derived EVs showed intrinsic anti-inflammatory activity. The RGD-EV targeted the lesion region of the ischemic brain after intravenous administration, and resulted in a strong suppression of the inflammatory response. Furthermore, RNA sequencing revealed a set of 7 miRNAs packaged in the EVs inhibited MAPK, an inflammation related pathway. Conclusion: These results point to a rapid and easy strategy to produce targeting EVs and suggest a potential therapeutic agent for ischemic stroke.


Assuntos
Isquemia Encefálica/terapia , Vesículas Extracelulares/fisiologia , Infarto da Artéria Cerebral Média/terapia , Inflamação/prevenção & controle , Células-Tronco Neurais/citologia , Animais , Antígenos de Superfície/química , Antígenos de Superfície/farmacologia , Isquemia Encefálica/complicações , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Genes Reporter , Células HEK293 , Humanos , Infarto da Artéria Cerebral Média/complicações , Inflamação/etiologia , Injeções Intravenosas , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas do Leite/química , Proteínas do Leite/farmacologia , Nanopartículas , Células-Tronco Neurais/química , Oligopeptídeos/farmacologia , Fosfatidilserinas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
10.
Front Immunol ; 12: 634497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868255

RESUMO

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.


Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Crassostrea/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Animais , Toxinas Bacterianas/isolamento & purificação , Caspases/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Crassostrea/imunologia , Crassostrea/metabolismo , Quebras de DNA de Cadeia Dupla , Hemócitos/imunologia , Hemócitos/metabolismo , Hemócitos/patologia , Fosfatidilserinas/metabolismo , Vibrio/metabolismo , Vibrio parahaemolyticus/metabolismo
11.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33929485

RESUMO

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.


Assuntos
Autofagossomos , Autofagia , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Transporte Proteico
12.
Mol Cell ; 81(9): 2031-2040.e8, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33909989

RESUMO

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de Sinais
13.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805426

RESUMO

Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by clonal expansion of abnormal hematopoietic stem cells leading to hyperproliferation of one or more myeloid lineages. The main complications in MPNs are high risk of thrombosis and progression to myelofibrosis and leukemia. MPN patients with high risk scores are treated by hydroxyurea (HU), interferon-α, or ruxolitinib, a tyrosine kinase inhibitor. Polycythemia vera (PV) is an MPN characterized by overproduction of red blood cells (RBCs). ABCG2 is a member of the ATP-binding cassette superfamily transporters known to play a crucial role in multidrug resistance development. Proteome analysis showed higher ABCG2 levels in PV RBCs compared to RBCs from healthy controls and an additional increase of these levels in PV patients treated with HU, suggesting that ABCG2 might play a role in multidrug resistance in MPNs. In this work, we explored the role of ABCG2 in the transport of ruxolitinib and HU using human cell lines, RBCs, and in vitro differentiated erythroid progenitors. Using stopped-flow analysis, we showed that HU is not a substrate for ABCG2. Using transfected K562 cells expressing three different levels of recombinant ABCG2, MPN RBCs, and cultured erythroblasts, we showed that ABCG2 potentiates ruxolitinib-induced cytotoxicity that was blocked by the ABCG2-specific inhibitor KO143 suggesting ruxolitinib intracellular import by ABCG2. In silico modeling analysis identified possible ruxolitinib-binding site locations within the cavities of ABCG2. Our study opens new perspectives in ruxolitinib efficacy research targeting cell types depending on ABCG2 expression and polymorphisms among patients.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Eritrócitos/metabolismo , Proteínas de Neoplasias/metabolismo , Policitemia Vera/tratamento farmacológico , Pirazóis/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Simulação por Computador , Dicetopiperazinas/farmacologia , Eritrócitos/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hidroxiureia/metabolismo , Hidroxiureia/farmacologia , Interferon-alfa/farmacologia , Células K562 , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Fosfatidilserinas/metabolismo , Policitemia Vera/sangue , Policitemia Vera/patologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacocinética
14.
Front Immunol ; 12: 630170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717160

RESUMO

Rheumatoid arthritis (RA) is a chronic immune-mediated disease managed by conventional synthetic drugs, such as methotrexate (MTX), and targeted drugs including biological agents. Cell-based therapeutic approaches are currently developed in RA, mainly mesenchymal stroma cell-based approaches. Early-stage apoptotic cells possess direct and indirect anti-inflammatory properties. During the elimination of dying cells (a process called efferocytosis), specific mechanisms operate to control immune responses. There are compelling evidences in experimental models of arthritis indicating that apoptotic cell administration may benefit joint inflammation, and may even have therapeutic effects on arthritis. Additionally, it has been demonstrated that apoptotic cells could be administered with standard treatments of RA, such as MTX or TNF inhibitors (TNFi), given even a synergistic response with TNFi. Interestingly, apoptotic cell infusion has been successfully experienced to prevent acute graft-vs.-host disease after hematopoietic cell transplantation in patients with hematologic malignancies, with a good safety profile. In this mini-review, the apoptotic cell-based therapy development in arthritis is discussed, as well as its transfer in the short-term to an innovative treatment for patients with RA. The use of apoptotic cell-derived factors, including secretome or phosphatidylserine-containing liposomes, in RA are also discussed.


Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Inflamação/terapia , Lipossomos/uso terapêutico , Fosfatidilserinas/uso terapêutico , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Animais , Antirreumáticos/uso terapêutico , Apoptose , Humanos , Imunomodulação , Lipossomos/metabolismo , Fosfatidilserinas/metabolismo
15.
Ren Fail ; 43(1): 465-476, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33678127

RESUMO

OBJECTIVES: To explore the mechanism of calcium-sensing receptors (CaSRs) during the development of nephrolithiasis. MATERIALS AND METHODS: Wistar rats were treated with ethylene glycol to induce calcium oxalate crystallization, and gadolinium chloride (GdCl3, an agonist of CaSR) and NPS 2390 (an antagonist of CaSR) were added. Oxidative stress (OS) and calcium oxalate crystals in the kidney were observed. CaSR expression and the expression of extracellular signal-regulated protein kinase (ERK), OPN, and KIM-1 were determined by western blotting. In addition, renal tubular epithelial cells were isolated from the kidney to observe phosphatidylserine (PS) ectropion using flow cytometric analysis. Various biochemical parameters were assessed in serum and urine at the end of the experiment. RESULTS: Calcium oxalate increased OS, crystal adhesion, PS ectropion, and the expression of CaSR and ERK, OPN, and KIM-1 in vivo. In addition, lower levels of urine citrate as well as increased serum creatinine and urea levels were observed after treatment with calcium oxalate (p < .05). Compared with calcium oxalate treatment alone, the above deleterious changes were further significantly confirmed by GdCl3 but were reversed by NPS-2390. However, urine calcium excretion was decreased after ethylene glycol treatment but was significantly reduced by NPS 2390 and increased by GdCl3 (p < .05). CONCLUSIONS: The results suggest that CaSR might play significant roles in the induction of nephrolithiasis in rats by regulating reactive oxygen species (ROS) and PS ectropion and the composition of urine, OPN, KIM-1, and ERK expression.


Assuntos
Oxalato de Cálcio/análise , Nefrolitíase/etiologia , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Proteínas do Citoesqueleto/metabolismo , Ectrópio/patologia , Etilenoglicol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Receptor Celular 1 do Vírus da Hepatite A , Túbulos Renais/patologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Detecção de Cálcio/genética
16.
Phytomedicine ; 85: 153536, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33765552

RESUMO

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PURPOSE: This study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. METHODS: SOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. RESULTS: In silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10-160 µM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 µM) and 2 × the IC50 (69 µM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 µM), THP-1 human monocytes (CC50 of 1321 µM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 µM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. CONCLUSION: SOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.


Assuntos
Apoptose/efeitos dos fármacos , Furanos/farmacologia , Leishmania/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Naftalenos/farmacologia , Animais , Antiprotozoários/farmacologia , Linhagem Celular , Humanos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Óxido Nítrico/metabolismo , Fosfatidilserinas/metabolismo , Raízes de Plantas/química , Espécies Reativas de Oxigênio/metabolismo , Ovinos , Solidago/química , Células THP-1
17.
J Vis Exp ; (169)2021 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-33779604

RESUMO

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.


Assuntos
Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Transporte Biológico , Humanos
18.
Oxid Med Cell Longev ; 2021: 6463281, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33763169

RESUMO

The protective effects of Porphyra yezoensis polysaccharides (PYPs) with molecular weights of 576.2 (PYP1), 105.4 (PYP2), 22.47 (PYP3), and 3.89 kDa (PYP4) on the oxidative damage of human kidney proximal tubular epithelial (HK-2) cells and the differences in adherence and endocytosis of HK-2 cells to calcium oxalate monohydrate crystals before and after protection were investigated. Results showed that PYPs can effectively reduce the oxidative damage of oxalic acid to HK-2 cells. Under the preprotection of PYPs, cell viability increased, cell morphology improved, reactive oxygen species levels decreased, mitochondrial membrane potential increased, S phase cell arrest was inhibited, the cell apoptosis rate decreased, phosphatidylserine exposure reduced, the number of crystals adhered to the cell surface reduced, but the ability of cells to endocytose crystals enhanced. The lower the molecular weight, the better the protective effect of PYP. The results in this article indicated that PYPs can reduce the risk of kidney stone formation by protecting renal epithelial cells from oxidative damage and reducing calcium oxalate crystal adhesion, and PYP4 with the lowest molecular weight may be a potential drug for preventing kidney stone formation.


Assuntos
Oxalato de Cálcio/toxicidade , Endocitose/efeitos dos fármacos , Células Epiteliais/patologia , Rim/patologia , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/farmacologia , Porphyra/química , Substâncias Protetoras/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Células Epiteliais/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fase S/efeitos dos fármacos
19.
J Biol Chem ; 296: 100411, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33581114

RESUMO

Formations of myofibers, osteoclasts, syncytiotrophoblasts, and fertilized zygotes share a common step, cell-cell fusion. Recent years have brought about considerable progress in identifying some of the proteins involved in these and other cell-fusion processes. However, even for the best-characterized cell fusions, we still do not know the mechanisms that regulate the timing of cell-fusion events. Are they fully controlled by the expression of fusogenic proteins or do they also depend on some triggering signal that activates these proteins? The latter scenario would be analogous to the mechanisms that control the timing of exocytosis initiated by Ca2+ influx and virus-cell fusion initiated by low pH- or receptor interaction. Diverse cell fusions are accompanied by the nonapoptotic exposure of phosphatidylserine at the surface of fusing cells. Here we review data on the dependence of membrane remodeling in cell fusion on phosphatidylserine and phosphatidylserine-recognizing proteins and discuss the hypothesis that cell surface phosphatidylserine serves as a conserved "fuse me" signal regulating the time and place of cell-fusion processes.


Assuntos
Fusão Celular , Fosfatidilserinas/metabolismo , Transdução de Sinais , Exocitose , Humanos , Internalização do Vírus
20.
PLoS Genet ; 17(2): e1009066, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33571185

RESUMO

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.


Assuntos
Caenorhabditis elegans/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Fosfatidilserinas/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Dantroleno/farmacologia , Canais de Sódio Degenerina/genética , Canais de Sódio Degenerina/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Relaxantes Musculares Centrais/farmacologia , Necrose/genética , Necrose/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismo , Tapsigargina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...