Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.845
Filtrar
1.
Int J Nanomedicine ; 14: 4867-4880, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308663

RESUMO

Background: The demand for an effective vaccine delivery system that drives a suitable immune response is increasing. The oxidized carbon nanosphere (OCN), a negatively charged carbon nanoparticle, has the potential to fulfill this requirement because it can efficiently deliver macromolecules into cells and allows endosomal leakage. However, fundamental insights into how OCNs are taken up by antigen-presenting cells, and the intracellular behavior of delivered molecules is lacking. Furthermore, how immune responses are stimulated by OCN-mediated delivery has not been investigated. Purpose: In this study, the model protein antigen ovalbumin (OVA) was used to investigate the uptake mechanism and intracellular fate of OCN-mediated delivery of protein in macrophages. Moreover, the immune response triggered by OVA delivered by OCNs was characterized. Methods: Bone-marrow-derived macrophages (BMDMs) from mice were used to study antigen uptake and intracellular trafficking. Mice were immunized using OCN-OVA combined with known adjuvants, and the specific immune response was measured. Results: OCNs showed no cytotoxicity against BMDMs. OCN-mediated delivery of OVA into BMDMs was partially temperature independent process. Using specific inhibitors, it was revealed that intracellular delivery of OCN-OVA does not rely on phagocytosis or the clathrin- and lipid raft/caveolae-mediated pathways. Delivered OVA was found to colocalize with compartments containing MHC class I, but not with early endosomes, lysosomes, and autophagosomes. Immunization of OVA using OCNs in combination with the known adjuvant monophosphoryl lipid A specifically enhanced interferon gamma (IFNγ)- and granzyme B-producing cytotoxic T cells (CTLs). Conclusion: OCNs effectively delivered protein antigens into macrophages that localized with compartments containing MHC class I partially by the temperature independent, but not clathrin- and lipid raft/caveolae-mediated pathways. Increased CD8+ T-cell activity was induced by OCN-delivered antigens, suggesting antigen processing toward antigen presentation for CTLs. Taken together, OCNs are a potential protein antigen delivery system that stimulates the cell-mediated immune response.


Assuntos
Antígenos/administração & dosagem , Carbono/química , Sistemas de Liberação de Medicamentos , Imunidade Celular , Nanopartículas/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/imunologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Feminino , Imunidade Celular/efeitos dos fármacos , Cinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/toxicidade , Oxirredução , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia
2.
Int J Nanomedicine ; 14: 4353-4366, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354265

RESUMO

Purpose: Gene therapy has become a promising remedy to treat disease by modifying the person's genes. The therapeutic potential of related tools such as CRISPR-Cas9 depends on the efficiency of delivery to the targeted cells. Numerous transfection reagents have been designed and lots of efforts have been devoted to develop carriers for this purpose. Therefore, the aim of the present study was to develop novel cholesterol-rich lipid-based nanoparticles to enhance transfection efficiency and serum stability. Materials and methods: We constructed two-, three- and four-component cationic liposomes (CLs) to evaluate the combined effect of cholesterol domain and DOPE (dioleoyl phosphatidylethanolamine), a fusogenic lipid, and the PEG (polyethylene glycol) moiety location inside or outside of the cholesterol domain on transfection efficiency and other properties of the particle. Lipoplex formation and pDNA (plasmid DNA) entrapment were assessed by gel retardation assay at different N/P ratios (3, 5, 7). Physicochemical characteristics, cytotoxicity, serum stability and endosomal escape capability of the lipoplexes were studied and transfection potential was measured by firefly luciferase assay. Next, HEK293 cell line stably expressing GFP was utilized to demonstrate the editing of a reporter through Cas9 and sgRNA plasmids delivery by the selected CL formula, which showed the highest transfection efficiency. Results: Among the designed CLs, the four-component formula [DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane)/DOPE/cholesterol/Chol-PEG (cholesterol-polyethylene glycol)] showed the highest rate of transfection at N/P 3. Finally, transfection of Cas9/sgRNA by this formulation at N/P 3 resulted in 39% gene-editing efficiency to knockout GFP reporter. The results also show that this CL with no cytotoxicity effect can totally protect the plasmids from enzymatic degradation in serum. Conclusion: The novel PEGylated cholesterol domain lipoplex providing serum stability, higher transfection efficiency and endosomal release can be used for in vivo Cas9/sgRNA delivery and other future gene-therapy applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Colesterol/química , Edição de Genes , Nanopartículas/química , Transfecção/métodos , Cátions/química , Morte Celular , Colesterol/análogos & derivados , Ensaio de Desvio de Mobilidade Eletroforética , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos/metabolismo , Polietilenoglicóis/química , Compostos de Amônio Quaternário/química , RNA Guia/metabolismo , Eletricidade Estática
3.
J Nanobiotechnology ; 17(1): 77, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226993

RESUMO

BACKGROUND: The design of efficient drug delivery vectors requires versatile formulations able to simultaneously direct a multitude of molecular targets and to bypass the endosomal recycling pathway of cells. Liposomal-based vectors need the decoration of the lipid surface with specific peptides to fulfill the functional requirements. The unspecific binding of peptides to the lipid surface is often accompanied with uncontrolled formulations and thus preventing the molecular mechanisms of a successful therapy. RESULTS: We present a simple synthesis pathway to anchor cysteine-terminal peptides to thiol-reactive lipids for adequate and quantitative liposomal formulations. As a proof of concept, we have synthesized two different lipopeptides based on (a) the truncated Fibroblast Growth Factor (tbFGF) for cell targeting and (b) the pH sensitive and fusogenic GALA peptide for endosomal scape. CONCLUSIONS: The incorporation of these two lipopeptides in the liposomal formulation improves the fibroblast cell targeting and promotes the direct delivery of cargo molecules to the cytoplasm of the cell.


Assuntos
Dissulfetos/química , Lipídeos/química , Peptídeos/química , Piridinas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Cisteína/química , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/química , Camundongos , Estrutura Molecular , Imagem Óptica/métodos , Estudo de Prova de Conceito
4.
Nat Commun ; 10(1): 2702, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221991

RESUMO

Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.


Assuntos
Portadores de Fármacos/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Animais , Linhagem Celular Tumoral , Membrana Celular , Portadores de Fármacos/efeitos adversos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Receptores ErbB/genética , Feminino , Terapia Genética/métodos , Complexo de Golgi/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Nanopartículas/química , Neoplasias/genética , Neoplasias/terapia , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 10(1): 2340, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138794

RESUMO

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.


Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma de Células de Transição/genética , Membrana Celular/metabolismo , Endossomos/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Receptor ErbB-2/metabolismo , Neoplasias da Bexiga Urinária/genética , Animais , Neoplasias da Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma de Células de Transição/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Lisossomos/metabolismo , Células MCF-7 , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transporte Proteico , Neoplasias da Bexiga Urinária/metabolismo
6.
Nat Commun ; 10(1): 2341, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138801

RESUMO

Understanding nanoparticle uptake by biological cells is fundamentally important to wide-ranging fields from nanotoxicology to drug delivery. It is now accepted that the arrival of nanoparticles at the cell is an extremely complicated process, shaped by many factors including unique nanoparticle physico-chemical characteristics, protein-particle interactions and subsequent agglomeration, diffusion and sedimentation. Sequentially, the nanoparticle internalisation process itself is also complex, and controlled by multiple aspects of a cell's state. Despite this multitude of factors, here we demonstrate that the statistical distribution of the nanoparticle dose per endosome is independent of the initial administered dose and exposure duration. Rather, it is the number of nanoparticle containing endosomes that are dependent on these initial dosing conditions. These observations explain the heterogeneity of nanoparticle delivery at the cellular level and allow the derivation of simple, yet powerful probabilistic distributions that accurately predict the nanoparticle dose delivered to individual cells across a population.


Assuntos
Endossomos/metabolismo , Nanopartículas/metabolismo , Células A549 , Transporte Biológico , Linhagem Celular , Endossomos/ultraestrutura , Ensaios de Triagem em Larga Escala , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Nanopartículas/ultraestrutura
7.
Nat Commun ; 10(1): 2247, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113953

RESUMO

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Rejeição de Enxerto/patologia , NF-kappa B/metabolismo , Vasculite/patologia , Aloenxertos/patologia , Animais , Linhagem Celular , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Vasos Coronários/patologia , Vasos Coronários/transplante , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos SCID , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
8.
Cell Mol Life Sci ; 76(17): 3349-3361, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073744

RESUMO

The actin-related protein complex 2/3 (Arp2/3) generates branched actin networks important for many cellular processes such as motility, vesicular trafficking, cytokinesis, and intercellular junction formation and stabilization. Activation of Arp2/3 requires interaction with actin nucleation-promoting factors (NPFs). Regulation of Arp2/3 activity is achieved by endogenous inhibitory proteins through direct binding to Arp2/3 and competition with NPFs or by binding to Arp2/3-induced actin filaments and disassembly of branched actin networks. Arp2/3 inhibition has recently garnered more attention as it has been associated with attenuation of cancer progression, neurotoxic effects during drug abuse, and pathogen invasion of host cells. In this review, we summarize current knowledge on expression, inhibitory mechanisms and function of endogenous proteins able to inhibit Arp2/3 such as coronins, GMFs, PICK1, gadkin, and arpin. Moreover, we discuss cellular consequences of pharmacological Arp2/3 inhibition.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Ligação Competitiva , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Fator de Maturação da Glia/química , Fator de Maturação da Glia/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tiazolidinas/química , Tiazolidinas/metabolismo
9.
Nat Commun ; 10(1): 2193, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097705

RESUMO

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.


Assuntos
Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Morfogênese/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/diagnóstico por imagem , Epitélio/diagnóstico por imagem , Epitélio/crescimento & desenvolvimento , Fibroblastos , Microscopia Intravital , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Fosforilação/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Proteínas de Transporte Vesicular/genética
10.
Nat Commun ; 10(1): 1608, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962439

RESUMO

Membrane traffic operates by vesicles that bud from precursor organelles and are transported to their target compartment where they dock and fuse. Targeting requires tethering factors recruited by small GTPases and phosphoinositides whereas fusion is carried out by SNARE proteins. Here we report that vesicles containing the Q-SNAREs syntaxin 13 (Stx13) and syntaxin 6 (Stx6) together are targeted to a different endosomal compartment than vesicles containing only Stx6 using injection of artificial vesicles. Targeting by Stx6 requires Vps51, a component of the GARP/EARP tethering complexes. In contrast, targeting by both Stx6 and Stx13 is governed by Vps13B identified here as tethering factor functioning in transport from early endosomes to recycling endosomes. Vps13B specifically binds to Stx13/Stx6 as well as to Rab14, Rab6, and PtdIns(3)P. We conclude that SNAREs use a combinatorial code for recruiting tethering factors, revealing a key function in targeting that is independent of SNARE pairing during fusion.


Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Lipossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas Qa-SNARE/metabolismo , Animais , Cercopithecus aethiops , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Microscopia Intravital , Microscopia Confocal , Ligação Proteica/fisiologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem com Lapso de Tempo , Células Vero
11.
Nat Commun ; 10(1): 1833, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015428

RESUMO

In response to extracellular signals, many signalling proteins associated with the plasma membrane are sorted into endosomes. This involves endosomal fusion, which depends on the complexes HOPS and CORVET. Whether and how their subunits themselves modulate signal transduction is unknown. We show that Vps11 and Vps18 (Vps11/18), two common subunits of the HOPS/CORVET complexes, are E3 ubiquitin ligases. Upon overexpression of Vps11/Vps18, we find perturbations of ubiquitination in signal transduction pathways. We specifically demonstrate that Vps11/18 regulate several signalling factors and pathways, including Wnt, estrogen receptor α (ERα), and NFκB. For ERα, we demonstrate that the Vps11/18-mediated ubiquitination of the scaffold protein PELP1 impairs the activation of ERα by c-Src. Thus, proteins involved in membrane traffic, in addition to performing their well-described role in endosomal fusion, fine-tune signalling in several different ways, including through ubiquitination.


Assuntos
Proteínas Correpressoras/metabolismo , Endossomos/metabolismo , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Receptor alfa de Estrogênio/metabolismo , Células HEK293 , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Proteínas Wnt/metabolismo , Quinases da Família src/metabolismo
12.
Artif Cells Nanomed Biotechnol ; 47(1): 1312-1320, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30987439

RESUMO

Hearing loss is the most common neurosensory impairment worldwide. While conductive hearing loss can be managed by surgery, the management of sensorineural hearing loss (SNHL), related to the damage of sensory cells of the inner ear is more challenging to manage medically. Many causes of SNHL such as sudden idiopathic SNHL, Meniere's disease, noise-induced hearing loss, autoimmune hearing loss or hearing loss from exposure to ototoxic substances can benefit from delivery of otoprotective drugs to the inner ear. However, systemic drug delivery through oral, intravenous and intramuscular methods leads to undesirable side effects due to the inner ear's limited blood supply and the relatively poor penetration of the blood-inner ear barrier (BLB). Therefore, there has been an increased interest for the targeted drug delivery to the inner ear using nanoparticles. Drug delivery through nanoparticles offers several advantages including drug stabilization for controlled release and surface modification for specific targeting. Understanding the biocompatibility of nanoparticles with cochlea and developing novel non-invasive delivery methods will promote the translation of nanoparticle-mediated drug delivery for auditory disorders from bench to bedside.


Assuntos
Portadores de Fármacos , Orelha Interna/metabolismo , Nanopartículas , Portadores de Fármacos/metabolismo , Endossomos/metabolismo , Perda Auditiva/tratamento farmacológico , Perda Auditiva/metabolismo , Humanos
13.
Tohoku J Exp Med ; 247(4): 215-222, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30971638

RESUMO

Carbon monoxide (CO) and nitric oxide (NO) exhibit physiological properties that include the activation of guanylate cyclase. NO inhibits replication of rhinovirus (RV), a major cause of the common cold and exacerbation of bronchial asthma and chronic obstructive pulmonary disease. However, the anti-rhinoviral effects of CO remain unclear. This study investigated whether the exogenous application of low-dose CO could inhibit RV replication in human alveolar and airway epithelial cells. A549 human lung carcinoma cells with alveolar epithelial features and primary cultures of human tracheal epithelial (HTE) cells were pretreated with CO (100 ppm) and infected with a major group RV, type 14 RV (RV14). CO exposure reduced RV14 titers in the supernatants and RV RNA levels in A549 and HTE cells. The treatment with a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, reversed the inhibitory effects of CO exposure on RV14 replication in A549 cells. Pretreatment of A549 cells with 8-Br-cGMP, a cell-permeable cGMP analog, caused the decrease in RV14 replication, while CO exposure increased cGMP production. CO exposure also increased the expression levels of interferon (IFN)-γ mRNA and protein. In contrast, pretreatment with CO did not increase DNA fragmentation and did not reduce the expression of intercellular adhesion molecule-1, the RV14 receptor, or the number of acidic endosomes, through which RV RNA enters the cytoplasm. These findings suggest that low-dose CO may decrease RV14 replication in alveolar and airway epithelial cells. IFN-γ production, which is induced by CO exposure via guanylate cyclase activation-mediated cGMP production, may be involved in RV14 replication inhibition.


Assuntos
Monóxido de Carbono/farmacologia , Células Epiteliais/virologia , Alvéolos Pulmonares/virologia , Rhinovirus/fisiologia , Replicação Viral/efeitos dos fármacos , Células A549 , Ácidos , GMP Cíclico/antagonistas & inibidores , GMP Cíclico/biossíntese , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Células Epiteliais/efeitos dos fármacos , Guanilato Ciclase/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interferon gama/biossíntese , Alvéolos Pulmonares/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos
14.
Chem Commun (Camb) ; 55(31): 4562-4565, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30931453

RESUMO

In this report, we designed and synthesized a novel fluorescent single tailed surfactant (termed FEDS), which can disrupt endosomes, complex lipofectamine, and can also identify cells that have been transfected. FEDS was able to increase the gene editing efficiency of lipofectamine/Cas9 ribonucleoprotein by 300% via a combination of fluorescent based enrichment and endosomal disruption.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Animais , Linhagem Celular , Endossomos/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Coelhos
15.
Pancreas ; 48(4): 459-470, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973461

RESUMO

Acute pancreatitis (AP) is a potentially lethal inflammatory disease that lacks specific therapy. Damaged pancreatic acinar cells are believed to be the site of AP initiation. The primary function of these cells is the synthesis, storage, and export of digestive enzymes. Beginning in the endoplasmic reticulum and ending with secretion of proteins stored in zymogen granules, distinct pancreatic organelles use ATP produced by mitochondria to move and modify nascent proteins through sequential vesicular compartments. Compartment-specific accessory proteins concentrate cargo and promote vesicular budding, targeting, and fusion. The autophagy-lysosomal-endosomal pathways maintain acinar cell homeostasis by removing damaged/dysfunctional organelles and recycling cell constituents for substrate and energy. Here, we discuss studies in experimental and genetic AP models, primarily from our groups, which show that acinar cell injury is mediated by distinct mechanisms of organelle dysfunction involved in protein synthesis and trafficking, secretion, energy generation, and autophagy. These early AP events (often first manifest by abnormal cytosolic Ca signaling) in the acinar cell trigger the inflammatory and cell death responses of pancreatitis. Manifestations of acinar cell organelle disorders are also prominent in human pancreatitis. Our findings suggest that targeting specific mediators of organelle dysfunction could reduce disease severity.


Assuntos
Células Acinares/metabolismo , Homeostase , Pancreatite/metabolismo , Vesículas Secretórias/metabolismo , Doença Aguda , Autofagia , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Pancreatite/patologia
17.
Biomater Sci ; 7(5): 1888-1897, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30843539

RESUMO

Inefficient cytosolic delivery has limited the development of many promising biomacromolecular drugs, a long-standing challenge that has prompted extensive development of drug carriers that facilitate endosomal escape. Although many such carriers have shown considerable promise for cytosolic delivery of a diversity of therapeutics, the rupture or destabilization of endo/lysosomal membranes has also been associated with activation of the inflammasome with attendant risk of inflammation and toxicity. In this study, we investigated relationships between pH-dependent membrane destabilization, cytosolic drug delivery, and inflammasome activation using a series of well-defined poly[(ethylene glycol)-block-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] copolymers of variable second block composition and pH-responsive properties. We found that polymers that demonstrated the most potent membrane-destabilizing activity at early endosomal pH values in an erythrocyte hemolysis assay were most efficient at delivery of siRNA, yet tended to be associated with the least amount of NOD-like related protein 3 (NLRP3) inflammasome activation. By contrast, polymers that displayed minimal hemolysis activity and poor siRNA knockdown, and instead mediated lysosomal rupture likely due to a proton sponge mechanism, strongly induced NLPR3 inflammasome activation in a caspase- and cathepsin-dependent manner. Collectively, these findings reinforce the importance of early endosomal escape in minimizing inflammasome activation and also demonstrate the ability to tune the degree inflammasome activation via control of polymer structure with potential implications for design of vaccine adjuvants and immunotherapeutics.


Assuntos
Citosol/metabolismo , Portadores de Fármacos/química , Endossomos/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polímeros/química , Linhagem Celular , Membrana Celular/metabolismo , Portadores de Fármacos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Polímeros/metabolismo
18.
Nat Commun ; 10(1): 1387, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30918259

RESUMO

Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.


Assuntos
Anti-Infecciosos/farmacologia , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Receptor de Endotelina A/efeitos dos fármacos , Sulfisoxazol/farmacologia , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Células MCF-7 , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Biogênese de Organelas , Receptor de Endotelina A/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Nat Commun ; 10(1): 1454, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926795

RESUMO

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7-the logistical centerpiece of LE biology-as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.


Assuntos
Endocitose , Endossomos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Proteínas rab de Ligação ao GTP/metabolismo , Biocatálise , Linhagem Celular Tumoral , Humanos , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Transporte Proteico , Proteólise , Especificidade por Substrato
20.
Cell Struct Funct ; 44(1): 61-74, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30905923

RESUMO

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.


Assuntos
Endocitose/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/fisiologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinase/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Fragmentos de Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Proteínas ras/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA