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1.
Int J Nanomedicine ; 15: 4793-4810, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764921

RESUMO

Background: Platinum resistance is a major challenge in the management of ovarian cancer. Even low levels of acquired resistance at the cellular level lead to impaired response to cisplatin. In ovarian cancer intraperitoneal therapy, nanoparticle formulation can improve the cisplatin's pharmacokinetics and safety profile. Purpose: This work aimed to investigate the chemo-sensitivity of ovarian cancer SKOV3 cells upon short-term (72h) single treatment of cisplatin and cisplatin-loaded biodegradable nanoparticles (Cis-NP). The aim was then to determine the therapeutic properties of Cis-NP in vivo using a SKOV3-luc cells' xenograft model in mice. Methods: Cell cytotoxicity was assessed after the exposure of the cell culture to cisplatin or Cis-NP. The effect of treatments on EMT and CSC-like phenotype was studied by analyzing a panel of markers by flow cytometry. Intracellular platinum concentration was determined by inductively coupled plasma mass spectrometry (ICS-MS), and gene expression was evaluated by RNAseq analysis. The efficacy of intraperitoneal chemotherapy was evaluated in a SKOV3-luc cells' xenograft model in mice, through a combination of bioluminescence imaging, histological, and immunohistochemical analyses. Results: We observed in vitro that short-term treatment of cisplatin has a critical role in determining the potential induction of chemoresistance, and a nanotechnology-based drug delivery system can modulate it. The RNAseq analysis underlines a protective effect of nanoparticle system according to their ability to down-regulate several genes involved in chemoresistance, cell proliferation, and apoptosis. The highest intracellular platinum concentration obtained with Cis-NP treatment significantly improved the efficacy. Consistent with in vitro results, we found that Cis-NP treatment in vivo can significantly reduce tumor burden and aggressiveness compared to the free drug. Conclusion: Nanoparticle-mediated cisplatin delivery may serve as an intracellular depot impacting the cisplatin pharmacodynamic performance at cellular levels. These features may contribute to improving the drawbacks of conventional intraperitoneal therapy, and therefore will require further investigations in vivo.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Portadores de Fármacos/química , Espaço Intracelular/metabolismo , Nanomedicina/métodos , Nanopartículas/química , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Cisplatino/metabolismo , Cisplatino/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Commun ; 11(1): 4268, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848159

RESUMO

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton's Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.


Assuntos
Tirosina Quinase da Agamaglobulinemia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acrilamidas/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Linhagem Celular , Sobrevivência Celular , Corantes Fluorescentes , Meia-Vida , Humanos , Espaço Intracelular/metabolismo , Ligantes , Simulação de Dinâmica Molecular , Mutação , Fenômenos de Química Orgânica , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise
3.
PLoS One ; 15(7): e0235546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609743

RESUMO

Resistin and resistin-like molecules are pleiotropic cytokines that are involved in inflammatory diseases. Our previous work suggested that resistin has the potential to be used as a biomarker and therapeutic target for human pulmonary arterial hypertension. However, data are limited on the distribution of resistin in healthy human organs. In this study, we used our newly developed anti-human resistin (hResistin) antibody to immunohistochemically detect the expression, localization, and intracellular/extracellular compartmentalization of hResistin in a full human tissue panel from healthy individuals. The potential cross reactivity of this monoclonal anti-hResistin IgG1 with normal human tissues also was verified. Results showed that hResistin is broadly distributed and principally localized in the cytoplasmic granules of macrophages scattered in the interstitium of most human tissues. Bone marrow hematopoietic precursor cells also exhibited hResistin signals in their cytoplasmic granules. Additionally, hResistin labeling was observed in the cytoplasm of nervous system cells. Notably, the cytokine activity of hResistin was illustrated by positively stained extracellular material in most human tissues. These data indicate that our generated antibody binds to the secreted hResistin and support its potential use for immunotherapy to reduce circulating hResistin levels in human disease. Our findings comprehensively document the basal expression patterns of hResistin protein in normal human tissues, suggest a critical role of this cytokine in normal and pathophysiologic inflammatory processes, and offer key insights for using our antibody in future pharmacokinetic studies and immunotherapeutic strategies.


Assuntos
Anticorpos Monoclonais/imunologia , Regulação da Expressão Gênica , Resistina/imunologia , Resistina/metabolismo , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Espaço Intracelular/metabolismo , Especificidade de Órgãos , Transporte Proteico
4.
Cancer Sci ; 111(7): 2508-2525, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32415868

RESUMO

Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3- /HER4+ cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 Ab C6, which binds to a conformational epitope located on a.a. 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptor ErbB-4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Monoclonais/imunologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Mapeamento de Epitopos , Humanos , Espaço Intracelular/metabolismo , Camundongos , Mitocôndrias/metabolismo , Neuregulina-1/farmacologia , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-4/imunologia
5.
Nucleic Acids Res ; 48(10): 5235-5253, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356888

RESUMO

Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.


Assuntos
Oligonucleotídeos Fosforotioatos/química , Proteínas/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Ligantes , Oligonucleotídeos Fosforotioatos/metabolismo , Oligonucleotídeos Fosforotioatos/farmacologia , Oligonucleotídeos Fosforotioatos/toxicidade , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteínas/toxicidade , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
6.
DNA Cell Biol ; 39(7): 1228-1242, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32429692

RESUMO

Heat shock protein (HSP) is a family of highly conserved protein, which exists widely in various organisms and has a variety of important physiological functions. Currently, there is no systematic analysis of HSPs in human glioma. The aim of this study was to investigate the characteristics of HSPs through constructing protein-protein interaction network (PPIN) considering the expression level of HSPs in glioma. After the identification of the differentially expressed HSPs in glioma tissues, a specific PPIN was constructed and found that there were many interactions between the differentially expressed HSPs in glioma. Subcellular localization analysis shows that HSPs and their interacting proteins distribute from the cell membrane to the nucleus in a multilayer structure. By functional enrichment analysis, gene ontology analysis, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, the potential function of HSPs and two meaningful enrichment pathways was revealed. In addition, nine HSPs (DNAJA4, DNAJC6, DNAJC12, HSPA6, HSP90B1, DNAJB1, DNAJB6, DNAJC10, and SERPINH1) are prognostic markers for human brain glioma. These analyses provide a full view of HSPs about their expression, biological process, as well as clinical significance in glioma.


Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Glioma/genética , Proteínas de Choque Térmico/genética , Biomarcadores Tumorais/genética , Biologia Computacional , Glioma/diagnóstico , Glioma/metabolismo , Glioma/patologia , Proteínas de Choque Térmico/metabolismo , Humanos , Espaço Intracelular/metabolismo , Prognóstico , Mapas de Interação de Proteínas
7.
Nature ; 581(7807): 209-214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405004

RESUMO

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.


Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Organelas/química , Organelas/metabolismo , Termodinâmica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Corpos Enovelados/química , Corpos Enovelados/metabolismo , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/deficiência , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Transição de Fase , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , RNA Helicases/deficiência , Proteínas com Motivo de Reconhecimento de RNA/deficiência , RNA Ribossômico/química , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA , Ribossomos/química , Ribossomos/metabolismo
8.
PLoS One ; 15(4): e0231977, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352982

RESUMO

Poxviruses are large enveloped viruses that replicate exclusively in the cytoplasm. Like all viruses, their replication cycle begins with virion adsorption to the cell surface. Unlike most other viral families, however, no unique poxviral receptor has ever been identified. In the absence of a unique receptor, poxviruses are instead thought to adhere to the cell surface primarily through electrostatic interactions between the positively charged viral envelope proteins and the negatively charged sulfate groups on cellular glycosaminoglycans (GAGs). While these negatively charged GAGs are an integral part of all eukaryotic membranes, their specific expression and sulfation patterns differ between cell types. Critically, while poxviral binding has been extensively studied using virally centered genetic strategies, the impact of cell-intrinsic changes to GAG charge has never been examined. Here we show that loss of heparin sulfation, accomplished by deleting the enzyme N-Deacetylase and N-Sulfotransferase-1 (NDST1) which is essential for GAG sulfation, significantly reduces the binding affinity of both vaccinia and myxoma viruses to the cell surface. Strikingly, however, while this lowered binding affinity inhibits the subsequent spread of myxoma virus, it actually enhances the overall spread of vaccinia by generating more diffuse regions of infection. These data indicate that cell-intrinsic GAG sulfation plays a major role in poxviral infection, however, this role varies significantly between different members of the poxviridae.


Assuntos
Poxviridae/fisiologia , Replicação Viral , Animais , Linhagem Celular , Heparina/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Poxviridae/metabolismo , Sulfotransferases/deficiência
9.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1145-L1157, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267731

RESUMO

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.


Assuntos
Fibrose Cística/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Espaço Intracelular/metabolismo , Resveratrol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Acetilação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carbazóis/farmacologia , Células Cultivadas , Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nariz/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Resorcinóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuínas/metabolismo , Estilbenos/farmacologia , Tubulina (Proteína)/metabolismo
10.
J Cancer Res Clin Oncol ; 146(7): 1647-1658, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32335720

RESUMO

BACKGROUND: Additional prognostic factors and personalized therapeutic alternatives for vulvar squamous cell carcinoma (VSCC), especially for advanced stages with poor prognosis, are urgently needed. OBJECTIVES: To review and assess literature regarding underlying molecular mechanisms of VSCC target therapeutic and prognostic approaches. METHODS: We performed a narrative literature review from the inception of the database up to January 2020 limited to English language, organizing knowledge in five main fields: extracellular and intracellular cell cycle deregulation, tumor immune microenvironment, tumor angiogenesis and hormones. RESULTS: EGFR immunohistochemical overexpression/gene amplification, representing early events in VSCC carcinogenesis, have been correlated with a worse prognosis and led to inclusion of erlotinib in cancer guidelines. p16 expression and HPV positivity are linked to a better prognosis, while p53 overexpression is linked to a worse prognosis; thus, biomarkers could help tailoring conventional treatment and follow-up. The implications of PD-L1 positivity in reference to HPV status and prognosis are still not clear, even though pembrolizumab is part of available systemic therapies. The role of tumor angiogenesis emerges through data on microvessel density, immunohistochemical VEGF staining and evaluation of serum VEGF concentrations. Few data exist on hormonal receptor expression, even though hormonal therapy showed great manageability. CONCLUSIONS: We suggest adding p16, p53 and HPV status to routine hystopathological examination of vulvar biopsies or surgical specimens. Predictive biomarkers for anti-EGFR and anti-PD-1/PD-L1 drugs are needed. Enough preclinical data supporting anti-angiogenic target therapies in clinical trials are existing. Hormonal receptor expression deserves further investigation.


Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Neoplasias Vulvares/etiologia , Neoplasias Vulvares/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Ciclo Celular/genética , Tomada de Decisão Clínica , Suscetibilidade a Doenças , Espaço Extracelular/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Terapia de Alvo Molecular , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Neoplasias Vulvares/tratamento farmacológico , Neoplasias Vulvares/patologia
11.
PLoS Comput Biol ; 16(3): e1007717, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32210422

RESUMO

Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.


Assuntos
Modelos Biológicos , Processamento de RNA/fisiologia , RNA Mensageiro/metabolismo , Spliceossomos , Biologia Computacional , Simulação por Computador , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Cinética , RNA Mensageiro/química , Spliceossomos/química , Spliceossomos/metabolismo , Spliceossomos/fisiologia
12.
Nat Methods ; 17(5): 524-530, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32203387

RESUMO

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus is spatially heterogeneous at the nanoscale, and that variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the organization of the genome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the rate is determined by the specific subcellular environments. We thus unveil rich heterogeneities and charge effects in intracellular diffusion at the nanoscale.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Espaço Intracelular/metabolismo , Modelos Teóricos , Nanopartículas/metabolismo , Proteínas/metabolismo , Imagem Individual de Molécula/métodos , Células Cultivadas , Difusão , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos
13.
Int J Nanomedicine ; 15: 1643-1659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210558

RESUMO

Purpose: Aseptic loosening is a major complication after total joint replacement. Reactive oxygen species generated by local tissue cells and liberated from implant surfaces have been suggested to cause implant failures. Surface modification of titanium (Ti)-based implants with proanthocyanidins (PAC) is a promising approach for the development of anti-oxidant defense mechanism to supplement the mechanical functions of Ti implants. In this study, a controlled PAC release system was fabricated on the surface of Ti substrates using the layer-by-layer (LBL) assembly. Materials and Methods: Polyethyleneimine (PEI) base layer was fabricated to enable layer-by-layer (LBL) deposition of hyaluronic acid/chitosan (HA/CS) multi-layers without or with the PAC. Surface topography and wettability of the fabricated HA/CS-PAC substrates were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and contact angle measurement. PAC release profiles were investigated using drug release assays. MC3T3-E1 pre-osteoblast cells were used to assess the osteo-inductive effects of HA/CS-PAC substrates under conditions H2O2-induced oxidative stress in vitro. A rat model of femoral intramedullary implantation evaluated the osseo-integration and osteo-inductive potential of the HA/CS-PAC coated Ti implants in vivo. Results: SEM, AFM, FTIR and contact angle measurements verified the successful fabrication of Ti surfaces with multi-layered HA/CS-PAC coating. Drug release assays revealed controlled and sustained release of PAC over 14 days. In vitro, cell-based assays showed high tolerability and enhanced the osteogenic potential of MC3T3-E1 cells on HA/CS-PAC substrates when under conditions of H2O2-induced oxidative stress. In vivo evaluation of femoral bone 14 days after femoral intramedullary implantation confirmed the enhanced osteo-inductive potential of the HA/CS-PAC coated Ti implants. Conclusion: Multi-layering of HA/CS-PAC coating onto Ti-based surfaces by the LBL deposition significantly enhances implant osseo-integration and promotes osteogenesis under conditions of oxidative stress. This study provides new insights for future applications in the field of joint arthroplasty.


Assuntos
Antioxidantes/farmacologia , Osteogênese/efeitos dos fármacos , Proantocianidinas/farmacologia , Próteses e Implantes , Titânio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quitosana/química , Liberação Controlada de Fármacos , Feminino , Ácido Hialurônico/química , Peróxido de Hidrogênio/farmacologia , Espaço Intracelular/metabolismo , Camundongos , Osseointegração/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Polietilenoimina/química , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Molhabilidade , Microtomografia por Raio-X
14.
Genes Dev ; 34(5-6): 254-262, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32029457

RESUMO

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for redox enzymes, but also moonlights as a substrate for signaling enzymes. When used as a substrate by signaling enzymes, it is consumed, necessitating the recycling of NAD+ consumption products (i.e., nicotinamide) via a salvage pathway in order to maintain NAD+ homeostasis. A major family of NAD+ consumers in mammalian cells are poly-ADP-ribose-polymerases (PARPs). PARPs comprise a family of 17 enzymes in humans, 16 of which catalyze the transfer of ADP-ribose from NAD+ to macromolecular targets (namely, proteins, but also DNA and RNA). Because PARPs and the NAD+ biosynthetic enzymes are subcellularly localized, an emerging concept is that the activity of PARPs and other NAD+ consumers are regulated in a compartmentalized manner. In this review, I discuss NAD+ metabolism, how different subcellular pools of NAD+ are established and regulated, and how free NAD+ levels can control signaling by PARPs and redox metabolism.


Assuntos
Espaço Intracelular/metabolismo , NAD/biossíntese , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Espaço Intracelular/enzimologia , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Oxirredução
15.
EBioMedicine ; 52: 102658, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32058943

RESUMO

BACKGROUND: Naturally occurring variation in Membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), encoding for an enzyme involved in phosphatidylinositol acyl-chain remodelling, has been associated with fatty liver and hepatic disorders. Here, we examined the relationship between hepatic Mboat7 down-regulation and fat accumulation. METHODS: Hepatic MBOAT7 expression was surveyed in 119 obese individuals and in experimental models. MBOAT7 was acutely silenced by antisense oligonucleotides in C57Bl/6 mice, and by CRISPR/Cas9 in HepG2 hepatocytes. FINDINGS: In obese individuals, hepatic MBOAT7 mRNA decreased from normal liver to steatohepatitis, independently of diabetes, inflammation and MBOAT7 genotype. Hepatic MBOAT7 levels were reduced in murine models of fatty liver, and by hyper-insulinemia. In wild-type mice, Mboat7 was down-regulated by refeeding and insulin, concomitantly with insulin signalling activation. Acute hepatic Mboat7 silencing promoted hepatic steatosis in vivo and enhanced expression of fatty acid transporter Fatp1. MBOAT7 deletion in hepatocytes reduced the incorporation of arachidonic acid into phosphatidylinositol, consistently with decreased enzymatic activity, determining the accumulation of saturated triglycerides, enhanced lipogenesis and FATP1 expression, while FATP1 deletion rescued the phenotype. INTERPRETATION: MBOAT7 down-regulation by hyper-insulinemia contributes to hepatic fat accumulation, impairing phosphatidylinositol remodelling and up-regulating FATP1. FUNDING: LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute RF-2016-02,364,358, Ricerca corrente Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico; LV and AG received funding from the European Union Programme Horizon 2020 (No. 777,377) for the project LITMUS-"Liver Investigation: Testing Marker Utility in Steatohepatitis". MM was supported by Fondazione Italiana per lo Studio del Fegato (AISF) 'Mario Coppo' fellowship.


Assuntos
Aciltransferases/genética , Hepatócitos/metabolismo , Hiperinsulinismo/genética , Hiperinsulinismo/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Animais , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Hiperinsulinismo/diagnóstico , Resistência à Insulina , Espaço Intracelular/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/diagnóstico , Obesidade/etiologia , Obesidade/metabolismo , Transdução de Sinais
16.
Mol Med Rep ; 21(2): 851-857, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31974625

RESUMO

Hexavalent chromium [Cr(VI)], is a well­known toxic form of the heavy metal chromium in the natural environment. Clinical evidence has indicated that exposure to Cr(VI) can cause severe renal damage. The production of reactive oxygen species (ROS) due to intracellular reduction of Cr(VI) is the main mechanism underlying the induction of cellular dysfunction and apoptosis. The present study aimed to investigate in detail the apoptotic pathways induced by Cr(VI)­exposure in a human immortalized proximal tubular epithelial cell line HK­2, in order to understand the mechanism involved therein. Exposure to 10 µM potassium dichromate (K2Cr2O7), a toxic compound of Cr(VI), significantly decreased cell viability after 24 and 48 h of incubation and induced intracellular ROS generation. The expression levels of markers that activate the apoptotic pathway including cleaved caspase­3 and poly (ADP­ribose) polymerase were significantly upregulated in K2Cr2O7­exposed HK­2 cells. In addition, the induction of intrinsic and extrinsic apoptotic markers was detected in K2Cr2O7­exposed HK­2 cells. In summary, the present study described for the first time the novel apoptotic mechanism of Cr(VI)­toxicity in human renal cells which may be beneficial in designing optimal clinical treatment for renal damage caused by acute Cr(VI) toxicity.


Assuntos
Apoptose/efeitos dos fármacos , Cromo/toxicidade , Rim/patologia , Adulto , Caspases/metabolismo , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo
17.
Biochem Biophys Res Commun ; 523(4): 841-846, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31954514

RESUMO

Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.


Assuntos
Diferenciação Celular , Polpa Dentária/patologia , Nanismo/genética , Mutação com Ganho de Função/genética , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocondrodisplasias/genética , Células-Tronco/metabolismo , Canais de Cátion TRPV/genética , Adolescente , Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais
18.
Am J Physiol Endocrinol Metab ; 318(3): E405-E416, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935112

RESUMO

The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform (PFKP) and G6PDH mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.


Assuntos
Proteínas de Transporte/metabolismo , Glucose/metabolismo , Oócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Azasserina/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Bovinos , Feminino , Técnicas de Silenciamento de Genes , Glicólise , Técnicas de Maturação in Vitro de Oócitos , Infertilidade Feminina/genética , Infertilidade Feminina/metabolismo , Espaço Intracelular/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Interferência de RNA , RNA Interferente Pequeno/farmacologia
19.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936671

RESUMO

(1) Background: Zinc oxide (ZnO) particles are widely used as zinc (Zn) fortifiers, because Zn is essential for various cellular functions. Nanotechnology developments may lead to production of nano-sized ZnO, although nanoparticles (NPs) are not intended to be used as food additives. Current regulations do not specify the size distribution of NPs. Moreover, ZnO is easily dissolved into Zn ions under acidic conditions. However, the fate of ZnO in commercial foods or during intestinal transit is still poorly understood. (2) Methods: We established surfactant-based cloud point extraction (CPE) for ZnO NP detection as intact particle forms using pristine ZnO-NP-spiked powdered or liquid foods. The fate determination and dissolution characterization of ZnO were carried out in commercial foods and human intestinal cells using in vitro intestinal transport and ex vivo small intestine absorption models. (3) Results: The results demonstrated that the CPE can effectively separate ZnO particles and Zn ions in food matrices and cells. The major fate of ZnO in powdered foods was in particle form, in contrast to its ionic fate in liquid beverages. The fate of ZnO was closely related to the extent of its dissolution in food or biomatrices. ZnO NPs were internalized into cells in both particle and ion form, but dissolved into ions with time, probably forming a Zn-ligand complex. ZnO was transported through intestinal barriers and absorbed in the small intestine primarily as Zn ions, but a small amount of ZnO was absorbed as particles. (4) Conclusion: The fate of ZnO is highly dependent on food matrix type, showing particle and ionic fates in powdered foods and liquid beverages, respectively. The major intracellular and intestinal absorption fates of ZnO NPs were Zn ions, but a small portion of ZnO particle fate was also observed after intestinal transit. These findings suggest that the toxicity of ZnO is mainly related to the Zn ion, but potential toxicity resulting from ZnO particles cannot be completely excluded.


Assuntos
Contaminação de Alimentos/análise , Intestinos/citologia , Óxido de Zinco/análise , Transporte Biológico , Células CACO-2 , Humanos , Absorção Intestinal , Espaço Intracelular/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Espectrometria por Raios X
20.
J Biol Chem ; 295(9): 2650-2663, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31974164

RESUMO

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.


Assuntos
Vesículas Extracelulares/química , Espaço Intracelular/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Fúngicas/metabolismo , Periplasma/metabolismo , Biossíntese de Proteínas , Transporte Proteico , Proteômica/métodos , Saccharomyces cerevisiae/citologia , Proteínas de Transporte Vesicular/biossíntese
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