RESUMEN
The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , VIH/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteína Quinasa C/metabolismo , Latencia del Virus/fisiología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Transducción de Señal/fisiología , Activación Viral/fisiologíaRESUMEN
Cells harboring latent HIV-1 pose a major obstacle to eradication of the virus. The 'shock and kill' strategy has been broadly explored to purge the latent reservoir; however, none of the current latency-reversing agents (LRAs) can safely and effectively activate the latent virus in patients. In this study, we report an ingenol derivative called EK-16A, isolated from the traditional Chinese medicinal herb Euphorbia kansui, which displays great potential in reactivating latent HIV-1. A comparison of the doses used to measure the potency indicated EK-16A to be 200-fold more potent than prostratin in reactivating HIV-1 from latently infected cell lines. EK-16A also outperformed prostratin in ex vivo studies on cells from HIV-1-infected individuals, while maintaining minimal cytotoxicity effects on cell viability and T cell activation. Furthermore, EK-16A exhibited synergy with other LRAs in reactivating latent HIV-1. Mechanistic studies indicated EK-16A to be a PKCγ activator, which promoted both HIV-1 transcription initiation by NF-κB and elongation by P-TEFb signal pathways. Further investigations aimed to add this compound to the therapeutic arsenal for HIV-1 eradication are in the pipeline.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Diterpenos/uso terapéutico , Infecciones por VIH/virología , VIH-1/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Muerte Celular , Supervivencia Celular , Células Cultivadas , Diterpenos/química , Sinergismo Farmacológico , Quimioterapia Combinada , Euphorbia/virología , Humanos , FN-kappa B/metabolismo , Ésteres del Forbol/uso terapéutico , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Linfocitos T/virología , Activación TranscripcionalRESUMEN
We used Drosophila melanogaster as a model system to study the absorption, metabolism and potential health benefits of plant bioactives derived from radish sprouts (Raphanus sativus cv. Rambo), a Brassicaceae species rich in glucosinolates and other phytochemicals. Flies were subjected to a diet supplemented with lyophilized radish sprouts (10.6 g/L) for 10 days, containing high amounts of glucoraphenin and glucoraphasatin, which can be hydrolyzed by myrosinase to the isothiocyanates sulforaphene and raphasatin, respectively. We demonstrate that Drosophila melanogaster takes up and metabolizes isothiocyanates from radish sprouts through the detection of the metabolite sulforaphane-cysteine in fly homogenates. Moreover, we report a decrease in the glucose content of flies, an upregulation of spargel expression, the Drosophila homolog of the mammalian PPARγ-coactivator 1 α, as well as the inhibition of α-amylase and α-glucosidase in vitro. Overall, we show that the consumption of radish sprouts affects energy metabolism in Drosophila melanogaster which is reflected by lower glucose levels and an increased expression of spargel, a central player in mitochondrial biogenesis. These processes are often affected in chronic diseases associated with aging, including type II diabetes mellitus.
Asunto(s)
Drosophila melanogaster/metabolismo , Metabolismo Energético/efectos de los fármacos , Isotiocianatos/administración & dosificación , Raphanus/química , Plantones/química , Animales , Cisteína/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Isotiocianatos/química , Isotiocianatos/metabolismo , Isotiocianatos/farmacología , Modelos Animales , Extractos Vegetales/administración & dosificación , Extractos Vegetales/química , Extractos Vegetales/farmacología , Factor B de Elongación Transcripcional Positiva/metabolismoRESUMEN
The latent reservoirs of HIV represent a major impediment to eradication of HIV/AIDS. To overcome this problem, agents that can activate latent HIV proviruses have been actively sought after, as they can potentially be used in combination with the highly active antiretroviral therapy (HAART) to eliminate the latent reservoirs. Although several chemical compounds have been shown to activate latency, they are of limited use due to high toxicity and poor clinical outcomes. In an attempt to identify natural products as effective latency activators from traditional Chinese medicinal herbs that have long been widely used in human population, we have isolated procyanidin C-13,3',3"-tri-O-gallate (named as REJ-C1G3) from Polygonum cuspidatum Sieb. et Zucc., that can activate HIV in latently infected Jurkat T cells. REJ-C1G3 preferentially stimulates HIV transcription in a process that depends on the viral encoded Tat protein and acts synergistically with prostratin (an activator of the NF-κB pathway) or JQ1 (an inhibitor of Brd4) to activate HIV latency. Our mechanistic analyses further show that REJ-C1G3 accomplishes these tasks by inducing the release of P-TEFb, a host cofactor essential for Tat-activation of HIV transcription, from the cellular P-TEFb reservoir 7SK snRNP.
Asunto(s)
Productos Biológicos/farmacología , Fallopia japonica/química , VIH-1/fisiología , Factor B de Elongación Transcripcional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Latencia del Virus/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Azepinas/farmacología , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Proteínas Fluorescentes Verdes/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Células Jurkat , Ésteres del Forbol/farmacología , Proantocianidinas/farmacología , Secuencias Repetidas Terminales/genética , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Triazoles/farmacologíaRESUMEN
Triptolide (TPL), a key biologically active component of the Chinese medicinal herb Tripterygium wilfordii Hook. f., has potent anti-inflammation and anti-cancer activities. Its anti-proliferative and pro-apoptotic effects have been reported to be related to the inhibition of Nuclear Factor κB (NF-κB) and Nuclear Factor of Activated T-cells (NFAT) mediated transcription and suppression of HSP70 expression. The direct targets and precise mechanisms that are responsible for the gene expression inhibition, however, remain unknown. Here, we report that TPL inhibits global gene transcription by inducing proteasome-dependent degradation of the largest subunit of RNA polymerase II (Rpb1) in cancer cells. In the presence of proteosome inhibitor MG132, TPL treatment causes hyperphosphorylation of Rpb1 by activation of upstream protein kinases such as Positive Transcription Elongation Factor b (P-TEFb) in a time and dose dependent manner. Also, we observe that short time incubation of TPL with cancer cells induces DNA damage. In conclusion, we propose a new mechanism of how TPL works in killing cancer. TPL inhibits global transcription in cancer cells by induction of phosphorylation and subsequent proteasome-dependent degradation of Rpb1 resulting in global gene transcription, which may explain the high potency of TPL in killing cancer.
Asunto(s)
Diterpenos/farmacología , Medicamentos Herbarios Chinos/farmacología , Fenantrenos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , ARN Polimerasa II/metabolismo , Transcripción Genética/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Daño del ADN , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Compuestos Epoxi/farmacología , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Factor B de Elongación Transcripcional Positiva/metabolismo , Factores de TiempoRESUMEN
The protective effect of berberine against antioxidant, antilipid peroxidation in serum and liver tissue, and positive transcription elongation factor b (P-TEFb) expression in liver tissue of type 2 diabetic rats was investigated. Overnight fasted rats were intraperitoneally injected 35 mg/kg streptozotocin. Diabetic rats were admitted after 2 weeks and given a high-carbohydrate/high-fat diet to induce hyperlipidemias. From week 16, diabetic rats were treated with 75, 150, 300 mg/kg berberine, 100mg/kg fenofibrate or 4 mg/kg rosiglitazone for another 16 weeks. P-TEFb (composed of cyclin-dependent kinase 9 and cyclin T1) mRNA and protein expression in liver tissue were detected by real time PCR and immunohistochemistry, respectively. Berberine significantly up-regulated the declined cyclin-dependent kinase 9, cyclin T1 mRNA and protein expression in diabetic rat liver. Berberine obviously decreased malondialdehyde level and increased catalase, superoxide dismutase, glutathione peroxidase, and glutathione activities in liver tissue and serum of diabetic rats. These results suggest that the effects of berberine on up-regulation of P-TEFb expression, antioxidant and antilipid peroxidation may be related to its protective potential on diabetes.
Asunto(s)
Antioxidantes/farmacología , Berberina/farmacología , Coptis/química , Diabetes Mellitus Experimental/metabolismo , Hígado/metabolismo , Fitoterapia , Factor B de Elongación Transcripcional Positiva/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , Berberina/uso terapéutico , Ciclina T/genética , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Dieta , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Enzimas/metabolismo , Fenofibrato/farmacología , Hiperlipidemias/complicaciones , Hiperlipidemias/tratamiento farmacológico , Hiperlipidemias/metabolismo , Hipoglucemiantes/farmacología , Hipolipemiantes/farmacología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Malondialdehído/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Rizoma , Rosiglitazona , Tiazolidinedionas/farmacología , Regulación hacia ArribaRESUMEN
Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat.P-TEFb complex containing HIV-1 Tat, human Cdk9 (also known as CDK9), and human cyclin T1 (also known as CCNT1). Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat.P-TEFb complex and block HIV replication.
Asunto(s)
VIH-1/química , Factor B de Elongación Transcripcional Positiva/química , Factor B de Elongación Transcripcional Positiva/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Ciclina T/química , Ciclina T/metabolismo , Quinasa 9 Dependiente de la Ciclina/química , Quinasa 9 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The positive transcription elongation factor (P-TEFb; CDK9/cyclin T1) regulates RNA polymerase II-dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat transactivation, and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription; this indicates that P-TEFb could be a potential target for developing anti-HIV-1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics.
Asunto(s)
Fármacos Anti-VIH/farmacología , Flavonoides/farmacología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Piperidinas/farmacología , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Flavonoides/síntesis química , Flavonoides/química , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Factor B de Elongación Transcripcional Positiva/metabolismoRESUMEN
Cleavage and polyadenylation define the 3' ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5' portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5' capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.