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1.
J Biol Chem ; 296: 100787, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34015332

RESUMO

The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.


Assuntos
Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Animais , Transporte Biológico , Membrana Celular/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Expressão Gênica , Células HEK293 , Humanos , Neurônios/metabolismo , Optogenética , Multimerização Proteica
2.
Carcinogenesis ; 40(8): 998-1009, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30590459

RESUMO

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.


Assuntos
Anexina A6/genética , Lapatinib/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/efeitos adversos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
3.
Cell Physiol Biochem ; 47(2): 800-816, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29807365

RESUMO

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. METHODS: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. RESULTS: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. CONCLUSION: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.


Assuntos
Núcleo Celular/metabolismo , Proteínas Luminescentes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Ácidos Graxos Insaturados/farmacologia , Meia-Vida , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Hep G2 , Humanos , Leupeptinas/farmacologia , Luz , Proteínas Luminescentes/genética , Camundongos , Microscopia de Fluorescência , Fator 2 Relacionado a NF-E2/genética , Nordefrin/análogos & derivados , Nordefrin/farmacologia , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica/genética , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sumoilação
4.
Chemistry ; 24(36): 8985-8988, 2018 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-29679472

RESUMO

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays.


Assuntos
Esterases/metabolismo , Fígado/enzimologia , Rodaminas/metabolismo , Animais , Esterases/química , Esterases/genética , Células HEK293 , Humanos , Microscopia Confocal , Rodaminas/química , Espectrometria de Fluorescência , Suínos
5.
J Biol Chem ; 290(49): 29542-54, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26442590

RESUMO

The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , alfa-Sinucleína/metabolismo , Anfetamina/metabolismo , Animais , Biotinilação , Encéfalo/metabolismo , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Neurônios Dopaminérgicos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Microdomínios da Membrana/metabolismo , Doenças Neurodegenerativas/metabolismo , Transmissão Sináptica , Sinucleínas/metabolismo
6.
J Infect Dis ; 209(7): 1066-76, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24259524

RESUMO

BACKGROUND: Escherichia coli-bearing Dr-adhesins (Dr+ E. coli) cause chronic pyelonephritis in pregnant women and animal models. This chronic renal infection correlates with the capacity of bacteria to invade epithelial cells expressing CD55. The mechanism of infection remains unknown. METHODS: CD55 amino acids in the vicinity of binding pocket-Ser155 for Dr-adhesin were mutated to alanine and subjected to temporal gentamicin-invasion/gentamicin-survival assay in Chinese hamster ovary cells. CD55/microtubule (MT) responses were studied using confocal/electron microscopy, and 3-dimensional structure analysis. RESULTS: Mutant analysis revealed that complement-protective CD55-Ser165 and CD55-Phe154 epitopes control E. coli invasion by coregulating CD55-MT complex expression. Single-point CD55 mutations changed E. coli to either a minimally invasive (Ser165Ala) or a hypervirulent pathogen (Phe154Ala). Thus, single amino acid modifications with no impact on CD55 structure and bacterial attachment can have a profound impact on E. coli virulence. While CD55-Ser165Ala decreased E. coli invasion and led to dormant intracellular persistence, intracellular E. coli in CD55-Phe154Ala developed elongated forms (multiplying within vacuoles), upregulated CD55-MT complexes, acquired CD55 coat, and escaped phagolysosomal fusion. CONCLUSIONS: E. coli target complement-protective CD55 epitopes for invasion and exploit CD55-MT complexes to escape phagolysosomal fusion, leading to a nondestructive parasitism that allows bacteria to persist intracellularly.


Assuntos
Antígenos CD55/metabolismo , Proteínas do Sistema Complemento/imunologia , Endocitose , Microtúbulos/metabolismo , Escherichia coli Uropatogênica/imunologia , Escherichia coli Uropatogênica/fisiologia , Adesinas de Escherichia coli/imunologia , Adesinas de Escherichia coli/metabolismo , Animais , Antígenos CD55/genética , Células CHO , Cricetulus , Microscopia Confocal , Microscopia Eletrônica , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica
7.
J Biol Chem ; 288(20): 14569-14583, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23543742

RESUMO

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.


Assuntos
Leucemia Promielocítica Aguda/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Células Hep G2 , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Microscopia de Fluorescência , Estrutura Terciária de Proteína , Sumoilação , Fatores de Transcrição/metabolismo
8.
J Biol Chem ; 286(51): 43933-43943, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-21990355

RESUMO

Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-Synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. To elucidate the potential functional consequences of DAT/α-synuclein interaction, we explored α-synuclein modulation of DAT activity in midbrain dopaminergic neurons obtained from TH::RFP mice, immortalized DA neurons, and a heterologous system expressing DAT. We used dual pipette whole cell patch clamp recording to measure the DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons. Our data suggest that intracellular α-synuclein induces a Na+ independent but Cl--sensitive inward current in DAT-expressing cells. This current is blocked by DAT blocker GBR12935 and is absent when heat-inactivated α-synuclein is dialyzed into these cells. The functional consequence of this interaction on DAT activity was further examined with real-time monitoring of transport function using a fluorescent substrate of DAT, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Overexpression of α-synuclein in DAT-positive immortalized DA neurons and CHO cells expressing DAT decreased the magnitude and rate of DAT-mediated substrate uptake without a decrease in the initial binding of the substrate at the plasma membrane. Taken together our findings are consistent with the interpretation that DAT/α-synuclein interaction at the cell surface results in a DAT-dependent, Na+-insensitive, Cl-sensitive inward current with a decrease in substrate uptake, suggesting that DAT/α-synuclein interaction can modulate dopamine transmission and thus neuronal function.


Assuntos
Cloretos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Camundongos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp
9.
J Biol Chem ; 286(32): 28498-510, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21705339

RESUMO

HIV-1 Gag precursor directs virus particle assembly and release. In a search for Gag-interacting proteins that are involved in late stages of the HIV-1 replication cycle, we performed yeast two-hybrid screening against a human cDNA library and identified the non-muscle actin filament cross-linking protein filamin A as a novel Gag binding partner. The 280-kDa filamin A regulates cortical actin network dynamics and participates in the anchoring of membrane proteins to the actin cytoskeleton. Recent studies have shown that filamin A facilitates HIV-1 cell-to-cell transmission by binding to HIV receptors and coreceptors and regulating their clustering on the target cell surface. Here we report a novel role for filamin A in HIV-1 Gag intracellular trafficking. We demonstrate that filamin A interacts with the capsid domain of HIV-1 Gag and that this interaction is involved in particle release in a productive manner. Disruption of this interaction eliminated Gag localization at the plasma membrane and induced Gag accumulation within internal compartments. Moreover, blocking clathrin-dependent endocytic pathways did not relieve the restriction to particle release induced by filamin A depletion. These results suggest that filamin A is involved in the distinct step of the Gag trafficking pathway. The discovery of the Gag-filamin A interaction may provide a new therapeutic target for the treatment of HIV infection.


Assuntos
Proteínas Contráteis/metabolismo , Infecções por HIV/mortalidade , HIV-1/fisiologia , Proteínas dos Microfilamentos/metabolismo , Montagem de Vírus/fisiologia , Clatrina/genética , Clatrina/metabolismo , Proteínas Contráteis/genética , Endocitose/genética , Filaminas , Biblioteca Gênica , Infecções por HIV/genética , Infecções por HIV/transmissão , HIV-1/patogenicidade , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Transporte Proteico/genética , Saccharomyces cerevisiae , Técnicas do Sistema de Duplo-Híbrido , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana
10.
J Cell Biol ; 170(2): 261-72, 2005 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-16027222

RESUMO

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.


Assuntos
Complexo de Golgi/metabolismo , Palmitatos/metabolismo , Proteínas ras/fisiologia , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Cicloeximida/farmacologia , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mutação , Nocodazol/farmacologia , Transporte Proteico , Transdução de Sinais , Proteínas ras/genética , Proteínas ras/metabolismo
11.
J Neurosci ; 28(45): 11622-34, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-18987198

RESUMO

Recent studies have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to beta-amyloid (Abeta) accumulation and memory deficits in rodents. The aim of the present study was to evaluate the therapeutic effect of all-trans retinoic acid (ATRA), an active metabolite of vitamin A, on the neuropathology and deficits of spatial learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice, a well established AD mouse model. Here we report a robust decrease in brain Abeta deposition and tau phosphorylation in the blinded study of APP/PS1 transgenic mice treated intraperitoneally for 8 weeks with ATRA (20 mg/kg, three times weekly, initiated when the mice were 5 months old). This was accompanied by a significant decrease in the APP phosphorylation and processing. The activity of cyclin-dependent kinase 5, a major kinase involved in both APP and tau phosphorylation, was markedly downregulated by ATRA treatment. The ATRA-treated APP/PS1 mice showed decreased activation of microglia and astrocytes, attenuated neuronal degeneration, and improved spatial learning and memory compared with the vehicle-treated APP/PS1 mice. These results support ATRA as an effective therapeutic agent for the prevention and treatment of AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Antineoplásicos/uso terapêutico , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/metabolismo , Tretinoína/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Presenilina-1/genética , Tempo de Reação/efeitos dos fármacos
12.
J Pharmacol Exp Ther ; 326(2): 395-405, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18463320

RESUMO

We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E(-)/B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2alpha phosphorylation, on E(-)/B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E(-)/B48 lipoproteins enhanced phosphorylation of eIF-2alpha in macrophages. Incubation of MPMs with E(-)/B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E(-)/B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E(-)/B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2alpha mutant also attenuated the suppressive effect of E(-)/B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2alpha phosphorylation.


Assuntos
2-Aminopurina/farmacologia , Apolipoproteína B-48/metabolismo , Apolipoproteínas E/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Células Espumosas/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Animais , Apolipoproteína B-48/genética , Apolipoproteínas E/genética , Western Blotting , Células Cultivadas , Células Espumosas/citologia , Células Espumosas/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Transfecção
13.
Sci Rep ; 8(1): 8812, 2018 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-29891956

RESUMO

The association between mucosal microbiota and HIV-1 infection has garnered great attention in the field of HIV-1 research. Previously, we reported a receptor-independent HIV-1 entry into epithelial cells mediated by a Gram-negative invasive bacterium, Porphyromonas gingivalis. Here, we present evidence showing that P. gingivalis outer membrane vesicles (OMVs) promote mucosal transmission of HIV-1. We demonstrated, using the Dynabeads technology, a specific interaction between HIV-1 and P. gingivalis OMVs which led to an OMV-dependent viral entry into oral epithelial cells. HIV-1 was detected in human oral keratinocytes (HOKs) after a 20 minute exposure to the HIV-vesicle complexes. After entry, most of the complexes appeared to dissociate, HIV-1 was reverse-transcribed, and viral DNA was integrated into the genome of HOKs. Meanwhile, some of the complexes exited the original host and re-entered neighboring HOKs and permissive cells of HIV-1. Moreover, P. gingivalis vesicles enhanced HIV-1 infection of MT4 cells at low infecting doses that are not able to establish an efficient infection alone. These findings suggest that invasive bacteria and their OMVs with ability to interact with HIV-1 may serve as a vehicle to translocate HIV through the mucosa, establish mucosal transmission of HIV-1, and enhance HIV-1 infectivity.


Assuntos
Transmissão de Doença Infecciosa , Células Epiteliais/virologia , Vesículas Extracelulares/metabolismo , Infecções por HIV/transmissão , HIV-1/fisiologia , Porphyromonas gingivalis/metabolismo , Internalização do Vírus , Células Cultivadas , Humanos , Modelos Teóricos
14.
Neurotoxicology ; 28(5): 965-78, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17606297

RESUMO

The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children [Perera FP, Rauh V, Whyatt RM, Tsai WY, Tang D, Diaz D, et al. Effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children. Environ Health Perspect 2006;114:1287-92]. The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-d-aspartate receptor (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150mug/kg BW B(a)P on gestation days 14-17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in down-regulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: (1) disposition to the hippocampus and cortex, (2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and (3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes. These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.


Assuntos
Benzo(a)pireno/toxicidade , Poluentes Ambientais/toxicidade , Plasticidade Neuronal/efeitos dos fármacos , Receptores de Glutamato/biossíntese , Animais , Benzo(a)pireno/farmacocinética , Encéfalo/patologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Eletrofisiologia , Poluentes Ambientais/farmacocinética , Feminino , Imuno-Histoquímica , Deficiências da Aprendizagem/induzido quimicamente , Deficiências da Aprendizagem/psicologia , Plasticidade Neuronal/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Receptores de AMPA/biossíntese , Receptores de AMPA/genética , Receptores de Glutamato/genética , Receptores de N-Metil-D-Aspartato/biossíntese , Receptores de N-Metil-D-Aspartato/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Aging (Albany NY) ; 9(3): 627-649, 2017 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-28351997

RESUMO

Decreased energy production and increased oxidative stress are considered to be major contributors to aging and aging-associated pathologies. The role of mitochondrial calcium homeostasis has also been highlighted as an important factor affecting different pathological conditions. Here, we present evidence that loss of a small mitochondrial protein Fus1 that maintains mitochondrial homeostasis results in premature aging, aging-associated pathologies, and decreased survival. We showed that Fus1KO mice develop multiple early aging signs including lordokyphosis, lack of vigor, inability to accumulate fat, reduced ability to tolerate stress, and premature death. Other prominent pathological changes included low sperm counts, compromised ability of adult stem cells to repopulate tissues, and chronic inflammation. At the molecular level, we demonstrated that mitochondria of Fus1 KO cells have low reserve respiratory capacity (the ability to produce extra energy during sudden energy demanding situations), and show significantly altered dynamics of cellular calcium response.Our recent studies on early hearing and memory loss in Fus1 KO mice combined with the new data presented here suggest that calcium and energy homeostasis controlled by Fus1 may be at the core of its aging-regulating activities. Thus, Fus1 protein and Fus1-dependent pathways and processes may represent new tools and targets for anti-aging strategies.


Assuntos
Senilidade Prematura/metabolismo , Envelhecimento/metabolismo , Cálcio/metabolismo , Metabolismo Energético/genética , Proteínas Supressoras de Tumor/metabolismo , Adiposidade/genética , Envelhecimento/genética , Senilidade Prematura/genética , Animais , Sinalização do Cálcio , Homeostase/genética , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides/genética , Proteínas Supressoras de Tumor/genética
16.
PLoS One ; 12(8): e0179710, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28817680

RESUMO

Inability to distinguish Crohn's colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn's colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable differentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn's colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn's colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn's disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn's colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn's colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn's colitis.


Assuntos
Biomarcadores , Doenças Inflamatórias Intestinais/metabolismo , alfa-Defensinas/metabolismo , Biópsia , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/metabolismo , Doença de Crohn/diagnóstico , Doença de Crohn/metabolismo , Diagnóstico Diferencial , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/cirurgia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Muramidase/metabolismo , Proctocolectomia Restauradora , Estudos Retrospectivos
18.
Artigo em Inglês | MEDLINE | ID: mdl-27826542

RESUMO

Atherosclerosis, a chronic inflammatory disease of the blood vessels, is one of the most common causes of morbidity and mortality world-wide. Involvement of Porphyromonas gingivalis in atherosclerosis is supported by observations from epidemiological, clinical, immunological, and molecular studies. Previously we reported that P. gingivalis vesicles have a much higher invasive efficiency than their originating cells. Here, we further compare the role of P. gingivalis cells and their vesicles in expression of chemoattractant proteins including CXCL1, CXCL2, and CXCL8, and adhesive molecules such as E-selectin in human umbilical vein endothelial cells (HUVECs). Both P. gingivalis 33277 cells and vesicles were able to up-regulate expression of these molecules, while the vesicles acted as more potent inducers of the inflammatory response associated with the development of atherosclerosis, consequently resulting in significant monocyte adhesion to a monolayer of HUVECs. Interestingly, we found that elevated expression of CXCL8 and E-selectin in endothelial cells induced by P. gingivalis correlated with the invasive ability of P. gingivalis cells and vesicles. Non-invasive bacterial cells and vesicles had no effect on expression of these genes. This study highlights the potential risk of P. gingivalis cells and vesicles in initiation of atherosclerosis and provides a potential target for the development of novel therapeutics against bacteria-associated atherosclerosis.


Assuntos
Aterosclerose/imunologia , Aterosclerose/microbiologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Imunidade Inata , Porphyromonas gingivalis/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Vasos Sanguíneos , Adesão Celular , Células Cultivadas , Quimiocina CXCL1/biossíntese , Quimiocina CXCL1/genética , Quimiocina CXCL2/biossíntese , Quimiocina CXCL2/genética , Selectina E/biossíntese , Selectina E/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Monócitos/metabolismo , Porphyromonas gingivalis/patogenicidade , Regulação para Cima
19.
PLoS One ; 11(2): e0149618, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26894834

RESUMO

Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocytic epithelial cells and fibroblasts, which is known to facilitate P. gingivalis adaptation and survival in the gingival environment. In this study, we investigated two small compounds, Alop1 and dynasore, for their role in inhibition of P. gingivalis invasion. Using confocal microscopy, we showed that these two compounds significantly reduced invasion of P. gingivalis and its outer membrane vesicles into human oral keratinocytes in a dose-dependent manner. The inhibitory effects of dynasore, a dynamin inhibitor, on the bacterial entry is consistent with the notion that P. gingivalis invasion is mediated by a clathrin-mediated endocytic machinery. We also observed that microtubule arrangement, but not actin, was altered in the host cells treated with Alop1 or dynasore, suggesting an involvement of microtubule in this inhibitory activity. This work provides an opportunity to develop compounds against P. gingivalis infection.


Assuntos
Hidrazonas/farmacologia , Mucosa Bucal/microbiologia , Piperidinas/farmacologia , Porphyromonas gingivalis/efeitos dos fármacos , Células Cultivadas , Dinaminas/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/microbiologia , Microtúbulos/microbiologia , Porphyromonas gingivalis/fisiologia , Quinolizidinas
20.
PLoS One ; 11(9): e0162561, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27607467

RESUMO

We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H: quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1µM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites, and enhances the ratio of BaP phenolic metabolites versus diol/diones in endothelial microsomes.


Assuntos
Benzo(a)pireno/metabolismo , Catalase/metabolismo , Células Endoteliais/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Aorta/citologia , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Retículo Endoplasmático/metabolismo , Inativação Metabólica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Peróxidos/metabolismo , Frações Subcelulares/metabolismo , Xenobióticos/metabolismo
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