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1.
Exp Mol Pathol ; 134: 104876, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37890651

RESUMO

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign, persistent skin lesions. MCV encodes a variety of immune evasion molecules to dampen host immune responses. Two of these proteins are the MC159 and MC160 proteins. Both MC159 and MC160 contain two tandem death effector domains and share homology to the cellular FLIPs, FADD, and procaspase-8. MC159 and MC160 dampen several innate immune responses such as NF-κB activation and mitochondrial antiviral signaling (MAVS)-mediated induction of type 1 interferon (IFN). The type 1 IFN response is also activated by the cytosolic DNA sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Both cGAS and STING play a vital role in sensing a poxvirus infection. In this study, we demonstrate that there are nuanced differences between both MC160 and MC159 in terms of how the viral proteins modulate the cGAS/STING and MAVS pathways. Specifically, MC160 expression, but not MC159 expression, dampens cGAS/STING-mediated induction of IFN in HEK 293 T cells. Further, MC160 expression prevented the K63-ubiquitination of both STING and TBK1, a kinase downstream of cGAS/STING. Ectopic expression of the MC160 protein, but not the MC159 protein, resulted in a measurable decrease in the TBK1 protein levels as detected via immunoblotting. Finally, using a panel of MC160 truncation mutants, we report that the MC160 protein requires both DEDs to inhibit cGAS/STING-induced activation of IFN-ß. Our model indicates MC160 likely alters the TBK1 signaling complex to decrease IFN-ß activation at the molecular intersection of the cGAS/STING and MAVS signaling pathways.


Assuntos
Vírus do Molusco Contagioso , Humanos , Vírus do Molusco Contagioso/genética , Vírus do Molusco Contagioso/metabolismo , Células HEK293 , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Imunidade Inata , Interferon beta/genética , Interferon beta/metabolismo , Interferons/metabolismo
2.
J Virol ; 89(9): 5002-11, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25717101

RESUMO

UNLABELLED: A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV(+)) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV(+) cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers. IMPORTANCE: Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV(+) cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies.


Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismo , Ativação Viral , Linhagem Celular , Imunoprecipitação da Cromatina , Humanos , Análise em Microsséries , Proteoma/análise
3.
J Virol ; 87(21): 11438-46, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23966384

RESUMO

Lytic activation of Epstein-Barr virus (EBV) is central to its life cycle and to most EBV-related diseases. However, not every EBV-infected B cell is susceptible to lytic activation. This lack of uniform susceptibility to lytic activation also directly impacts the success of viral oncolytic therapy for EBV cancers, yet determinants of susceptibility to lytic induction signals are not well understood. To determine if host factors influence susceptibility to EBV lytic activation, we developed a technique to separate lytic from refractory cells and reported that EBV lytic activation occurs preferentially in cells with lower levels of signal transducer and activator of transcription 3 (STAT3). Using this tool to detect single cells, we now extend the correlation between STAT3 and lytic versus refractory states to EBV-infected circulating B cells in patients with primary EBV infection, leading us to investigate whether STAT3 controls susceptibility to EBV lytic activation. In loss-of-function and gain-of-function studies in EBV-positive B lymphoma and lymphoblastoid cells, we found that the levels of functional STAT3 regulate susceptibility to EBV lytic activation. This prompted us to identify a pool of candidate cellular genes that might be regulated by STAT3 to limit EBV lytic activation. From this pool, we confirmed increases in transcript levels in refractory cells of a set of genes known to participate in transcription repression. Taken together, our findings place STAT3 at a critical crossroads between EBV latency and lytic activation, processes fundamental to EBV lymphomagenesis.


Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Ativação Viral , Adolescente , Linhagem Celular Tumoral , Criança , Humanos , Fator de Transcrição STAT3
4.
Hawaii J Health Soc Welf ; 80(7): 165-168, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34278324

RESUMO

The Pacific Tracker (PacTrac) is a web-based diet and physical activity assessment program created to analyze dietary recall or dietary record data from the Pacific region. Version 3.1 modifications make the tool available for public use (under check it out) to enter, analyze, view and print out data; and for research use, for saving and downloading of multiple entries in a research mode. PacTrac 3.1 (https://nappactrac31.ctahr.hawaii.edu/default.htm) is managed through the Children's Healthy Living Center of Excellence (CHL Center) at the College of Tropical Agriculture and Human Resources at the University of Hawai'i, in collaboration with the University of Hawai'i Cancer Center.


Assuntos
Dieta , Neoplasias , Criança , Exercício Físico , Humanos , Universidades
5.
Neuropharmacology ; 56(2): 399-404, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18824182

RESUMO

The transporters of dopamine, norepinephrine and serotonin are molecular targets of cocaine, amphetamine, and therapeutic antidepressants. The residues involved in binding these drugs are unknown. We have performed several rounds of random and site-directed mutagenesis in the mouse norepinephrine transporter and screened for mutants with altered sensitivity to cocaine inhibition of substrate uptake. We have identified a triple mutation that retains close to wild-type transport function but displays a 37-fold decrease in cocaine sensitivity and 24-fold decrease in desipramine sensitivity. In contrast, the mutant's sensitivities to amphetamine, methamphetamine, and methylphenidate are only slightly changed. Our data reveal critical residues contributing to the potent uptake inhibitions by these important drugs. Furthermore, this drug-resistant triple mutant can be used to generate a unique knock-in mouse line to study the role of norepinephrine transporter in the addictive effects of cocaine and the therapeutic effects of desipramine.


Assuntos
Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Mutação , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/genética , Animais , Biotinilação , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Camundongos , Mutagênese Sítio-Dirigida/métodos , Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/química , Ligação Proteica/genética , Transfecção/métodos , Trítio/metabolismo
6.
Neuropharmacology ; 79: 626-33, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24412674

RESUMO

Cocaine's main pharmacological actions are the inhibition of the dopamine, serotonin, and norepinephrine transporters. Its main behavioral effects are reward and locomotor stimulation, potentially leading to addiction. Using knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) we have shown previously that inhibition of the dopamine transporter (DAT) is necessary for both of these behaviors. In this study, we sought to determine brain regions in which DAT inhibition by cocaine stimulates locomotor activity and/or produces reward. We used adeno-associated viral vectors to re-introduce the cocaine-sensitive wild-type DAT in specific brain regions of DAT-CI mice, which otherwise only express a cocaine-insensitive DAT globally. Viral-mediated expression of wild-type DAT in the rostrolateral striatum restored cocaine-induced locomotor stimulation and sensitization in DAT-CI mice. In contrast, the expression of wild-type DAT in the dorsal striatum, or in the medial nucleus accumbens, did not restore cocaine-induced locomotor stimulation. These data help to determine cocaine's molecular actions and anatomical loci that cause hyperlocomotion. Interestingly, cocaine did not produce significant reward - as measured by conditioned place-preference - in any of the three cohorts of DAT-CI mice with the virus injections. Therefore, the locus or loci underlying cocaine-induced reward remain underdetermined. It is possible that multiple dopamine-related brain regions are involved in producing the robust rewarding effect of cocaine.


Assuntos
Cocaína/farmacologia , Condicionamento Clássico/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Agitação Psicomotora/fisiopatologia , Animais , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Condicionamento Clássico/fisiologia , Corpo Estriado/fisiopatologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Recompensa , Percepção Espacial/efeitos dos fármacos , Percepção Espacial/fisiologia
7.
J Vis Exp ; (57)2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-22090023

RESUMO

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis(1). LCL have been used to present antigens in a variety of immunologic assays(2, 3). In addition, LCL can be used to generate human monoclonal antibodies(4, 5) and provide a potentially unlimited source when access to primary biologic materials is limited(6, 7). A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide(8), and pokeweed mitogen(9) to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells(7, 10-12). The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23(hi)CD58(+) cells observed as early as three days post-infection indicates a successful outcome.


Assuntos
Linfócitos B/citologia , Linfócitos B/virologia , Transformação Celular Viral/fisiologia , Herpesvirus Humano 4/fisiologia , Linhagem Celular Transformada , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos
8.
Neuropharmacology ; 61(1-2): 112-20, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21420984

RESUMO

Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100× more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2ß position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2ß position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.


Assuntos
Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Animais , Células Cultivadas , Cocaína/química , Cocaína/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Camundongos , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Distribuição Aleatória
9.
PLoS One ; 4(10): e7578, 2009 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-19855831

RESUMO

Cocaine methiodide (CM), a charged cocaine analog, cannot pass the blood brain barrier. It has been assumed the effects of systemic CM represent cocaine actions in peripheral tissues. However, the IC(50) values of CM have not been clearly determined for the major cocaine targets: dopamine, norepinephrine, and serotonin transporters, and sodium channels. Using cells transfected with individual transporters from mice and synaptosomes from mouse striatum tissues, we observed that the inhibition IC(50) values for monoamine uptake by CM were 31-fold to 184-fold higher compared to cocaine at each of the transporters. In dorsal root ganglion neurons, cocaine inhibited sodium channels with an apparent IC(50) of 75 microM, while CM showed no observable effect at concentrations up to 3 mM. These results indicate that an equal dose of CM will not produce an equivalent peripheral effect of cocaine.


Assuntos
Cocaína/análogos & derivados , Cocaína/metabolismo , Neurônios/metabolismo , Sinaptossomos/metabolismo , Animais , Cocaína/farmacologia , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
10.
Mol Cell Biochem ; 298(1-2): 41-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17131045

RESUMO

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.


Assuntos
Cisteína/química , Dissulfetos/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Drosophila melanogaster/química , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Ditiotreitol/farmacologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Relação Estrutura-Atividade
11.
Proc Natl Acad Sci U S A ; 103(24): 9333-8, 2006 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-16754872

RESUMO

There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout (DAT-KO) mice and SERT or NET knockout mice still self-administer cocaine and/or display conditioned place preference (CPP) to cocaine, which led to the reevaluation of the DA hypothesis and the proposal of redundant reward pathways. To study the role of DAT in cocaine reward, we have generated a knockin mouse line carrying a functional DAT that is insensitive to cocaine. In these mice, cocaine suppressed locomotor activity, did not elevate extracellular DA in the nucleus accumbens, and did not produce reward as measured by CPP. This result suggests that blockade of DAT is necessary for cocaine reward in mice with a functional DAT. This mouse model is unique in that it is specifically designed to differentiate the role of DAT from the roles of NET and SERT in cocaine-induced biochemical and behavioral effects.


Assuntos
Cocaína/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Recompensa , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Cocaína/farmacologia , Condicionamento Psicológico , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Marcação de Genes , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Microdiálise , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Núcleo Accumbens/metabolismo , Técnicas de Patch-Clamp
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