Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
J Cell Sci ; 133(24)2020 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-33376154

RESUMO

The centrosome, which consists of centrioles and pericentriolar material (PCM), becomes mature and assembles mitotic spindles by increasing the number of microtubules (MTs) emanating from the PCM. Among the molecules involved in centrosome maturation, Cep192 and Aurora A (AurA, also known as AURKA) are primarily responsible for recruitment of γ-tubulin and MT nucleators, whereas pericentrin (PCNT) is required for PCM organization. However, the role of Cep215 (also known as CDK5RAP2) in centrosome maturation remains elusive. Cep215 possesses binding domains for γ-tubulin, PCNT and MT motors that transport acentrosomal MTs towards the centrosome. We identify a mitosis-specific centrosome-targeting domain of Cep215 (215N) that interacts with Cep192 and phosphorylated AurA (pAurA). Cep192 is essential for targeting 215N to centrosomes, and centrosomal localization of 215N and pAurA is mutually dependent. Cep215 has a relatively minor role in γ-tubulin recruitment to the mitotic centrosome. However, it has been shown previously that this protein is important for connecting mitotic centrosomes to spindle poles. Based on the results of rescue experiments using versions of Cep215 with different domain deletions, we conclude that Cep215 plays a role in maintaining the structural integrity of the spindle pole by providing a platform for the molecules involved in centrosome maturation.

2.
Diagn Microbiol Infect Dis ; 97(3): 115040, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32354459

RESUMO

Staphylococci are the most common causes of periprosthetic joint infection (PJI). TNP-2092 is an investigational hybrid drug composed of rifamycin and quinolizinone pharmacophores conjugated via a covalent linker. We determined minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm bactericidal concentration (MBBC) values of TNP-2092 against 80 PJI-associated Staphylococcus aureus and Staphylococcus epidermidis isolates compared to ciprofloxacin and rifampin alone and in combination, alongside daptomycin and vancomycin. TNP-2092 exhibited the following activity against S. aureus: MIC50/MIC90, ≤0.0075/0.015 µg/mL; MBC50/MBC90, 0.5/4 µg/mL; and MBBC50/MBBC90, 0.5/2 µg/mL, and the following activity against S. epidermidis: MIC50/MIC90, ≤0.0075/0.015 µg/mL; MBC50/MBC90, 0.015/0.125 µg/mL; and MBBC50/MBBC90, 0.06/0.25 µg/mL. TNP-2092 MIC, MBC, and MBBC values were >8 µg/mL for 1 isolate, while MIC values were ≤0.25 µg/mL and MBC and MBBC values were ≤4 µg/mL for all other isolates. Results of this study show that TNP-2092 has promising in vitro activity against PJI-associated staphylococci.

3.
PLoS One ; 15(4): e0231212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32275682

RESUMO

Two major proteolytic systems, the proteasome and the autophagy pathway, are key components of the proteostasis network. The immunoproteasome, a proteasome subtype, and autophagy are upregulated under stress conditions, forming a coordinated unit designed to minimize the effect of cell stress. We investigated how genetic ablation of the LMP2 immunoproteasome subunit affects autophagy in retinal pigment epithelium (RPE) from WT and LMP2 knockout mice. We monitored autophagy regulation by measuring LC3, phosphorylation of AKT (S473), and phosphorylation of S6, a downstream readout of AKT (mTOR) pathway activation. We also evaluated transcription factor EB (TFEB) nuclear translocation, a transcription factor that controls expression of autophagy and lysosome genes. WT and LMP2 KO cells were monitored after treatment with EBSS to stimulate autophagy, insulin to stimulate AKT, or an AKT inhibitor (trehalose or MK-2206). Under basal conditions, we observed hyper-phosphorylation of AKT and S6, as well as lower nuclear-TFEB content in LMP2 KO RPE compared with WT. AKT inhibitors MK-2206 and trehalose significantly inhibited AKT phosphorylation and stimulated nuclear translocation of TFEB. Starvation and AKT inhibition upregulated autophagy, albeit to a lesser extent in LMP2 KO RPE. These data support the idea that AKT hyper-activation is an underlying cause of defective autophagy regulation in LMP2 KO RPE, revealing a unique link between two proteolytic systems and a previously unknown function in autophagy regulation by the immunoproteasome.


Assuntos
Autofagia , Complexo de Endopeptidases do Proteassoma/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/citologia , Animais , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Insulina/farmacologia , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais/efeitos dos fármacos
4.
Trends Mol Med ; 26(1): 105-118, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31771932

RESUMO

Mitochondrial dysfunction is involved in the pathology of two major blinding retinal diseases, diabetic retinopathy (DR) and age-related macular degeneration (AMD). These diseases accumulate mitochondrial defects in distinct retinal subcellular structures, the vascular/neural network in DR and the retinal pigment epithelium (RPE) in AMD. These mitochondrial defects cause a metabolic crisis that drives disease. With no treatments to stop these diseases, coupled with an increasing population suffering from AMD and DR, there is an urgent need to develop new therapeutics targeting the mitochondria to prevent or reverse disease-specific pathology.

5.
Oxid Med Cell Longev ; 2019: 5174957, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31485293

RESUMO

Age-related macular degeneration (AMD) involves the loss of retinal pigment epithelium (RPE) and photoreceptors and is one of the leading causes of blindness in the elderly. Oxidative damage to proteins, lipids, and DNA has been associated with RPE dysfunction and AMD. In this study, we evaluated oxidative stress in AMD and the efficacy of antioxidant, N-acetyl-L-cysteine (NAC), in protecting RPE from oxidative damage. To test this idea, primary cultures of RPE from human donors with AMD (n = 32) or without AMD (No AMD, n = 21) were examined for expression of NADPH oxidase (NOX) genes, a source of reactive oxygen species (ROS). Additionally, the cells were pretreated with NAC for 2 hours and then treated with either hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (t-BHP) to induce cellular oxidation. Twenty-four hours after treatment, ROS production, cell survival, the content of glutathione (GSH) and adenosine triphosphate (ATP), and cellular bioenergetics were measured. We found increased expression of p22phox, a NOX regulator, in AMD cells compared to No AMD cells (p = 0.02). In both AMD and No AMD cells, NAC pretreatment reduced t-BHP-induced ROS production and protected from H2O2-induced cell death and ATP depletion. In the absence of oxidation, NAC treatment improved mitochondrial function in both groups (p < 0.01). Conversely, the protective response exhibited by NAC was disease-dependent for some parameters. In the absence of oxidation, NAC significantly reduced ROS production (p < 0.001) and increased GSH content (p = 0.02) only in RPE from AMD donors. Additionally, NAC-mediated protection from H2O2-induced GSH depletion (p = 0.04) and mitochondrial dysfunction (p < 0.05) was more pronounced in AMD cells compared with No AMD cells. These results demonstrate the therapeutic benefit of NAC by mitigating oxidative damage in RPE. Additionally, the favorable outcomes observed for AMD RPE support NAC's relevance and the potential therapeutic value in treating AMD.


Assuntos
Acetilcisteína/uso terapêutico , Células Epiteliais/metabolismo , Degeneração Macular/genética , Epitélio Pigmentado da Retina/metabolismo , Acetilcisteína/farmacologia , Humanos , Degeneração Macular/patologia
6.
Invest Ophthalmol Vis Sci ; 59(4): AMD41-AMD47, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30025108

RESUMO

AMD is the leading cause of blindness in developed countries. The dry form of AMD, also known as atrophic AMD, is characterized by the death of RPE and photoreceptors. Currently, there are no treatments for this form of the disease due in part to our incomplete understanding of the mechanism causing AMD. Strong experimental evidence from studies of human donors with AMD supports the emerging hypothesis that defects in RPE mitochondria drive AMD pathology. These studies, using different experimental methods, have shown disrupted RPE mitochondrial architecture and decreased mitochondrial number and mass, altered content of multiple mitochondrial proteins, increased mitochondrial DNA damage that correlates with disease severity, and defects in bioenergetics for primary RPE cultures from AMD donors. Herein, we discuss a model of metabolic uncoupling that alters bioenergetics in the diseased retina and drives AMD pathology. These data provide the rationale for targeting the mitochondria in the RPE as the most efficacious intervention strategy if administered early, before vision loss and cell death.


Assuntos
Metabolismo Energético/fisiologia , Atrofia Geográfica/fisiopatologia , Células Fotorreceptoras de Vertebrados/fisiologia , Epitélio Pigmentado da Retina/fisiopatologia , Atrofia , DNA Mitocondrial/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Epitélio Pigmentado da Retina/metabolismo
7.
Redox Biol ; 13: 255-265, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28600982

RESUMO

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults. It has been suggested that mitochondrial defects in the retinal pigment epithelium (RPE) underlies AMD pathology. To test this idea, we developed primary cultures of RPE to ask whether RPE from donors with AMD differ in their metabolic profile compared with healthy age-matched donors. Analysis of gene expression, protein content, and RPE function showed that these cultured cells replicated many of the cardinal features of RPE in vivo. Using the Seahorse Extracellular Flux Analyzer to measure bioenergetics, we observed RPE from donors with AMD exhibited reduced mitochondrial and glycolytic function compared with healthy donors. RPE from AMD donors were also more resistant to oxidative inactivation of these two energy-producing pathways and were less susceptible to oxidation-induced cell death compared with cells from healthy donors. Investigation of the potential mechanism responsible for differences in bioenergetics and resistance to oxidative stress showed RPE from AMD donors had increased PGC1α protein as well as differential expression of multiple genes in response to an oxidative challenge. Based on our data, we propose that cultured RPE from donors phenotyped for the presence or absence of AMD provides an excellent model system for studying "AMD in a dish". Our results are consistent with the ideas that (i) a bioenergetics crisis in the RPE contributes to AMD pathology, and (ii) the diseased environment in vivo causes changes in the cellular profile that are retained in vitro.


Assuntos
Degeneração Macular/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Glicólise , Humanos , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Epitélio Pigmentado da Retina/citologia
8.
Protein Sci ; 24(9): 1495-507, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26130403

RESUMO

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes.


Assuntos
Biologia Computacional/métodos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Transporte de Elétrons , Heme/química , Heme/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA