Characterization of cocaine induced signaling pathways that enhances HIV transcription

 描述：可卡因是HIV-1感染、传播和发病率的重要辅助因素。可卡因通过改变特定的细胞信号和表观遗传途径加速艾滋病毒-1基因的表达，并促进艾滋病毒相关的神经认知障碍(HAND)。我们已发表的数据表明，可卡因诱导的信号转导通路导致蛋白激酶的激活，如MSK1，从而催化位于S276位的NF-ĸB的p65亚单位(P-p65S276)和位于丝氨酸10位的组蛋白H3(P-H3S10)的磷酸化。P-H3S10除了促进转录活性染色质结构的建立外，还促进P-TEFb在HIV LTR上的招募。因此，可卡因通过分别激活NF-kB和P-TEFb来加速HIV转录的起始期和延伸期，这是产生完整的HIV基因组转录本和新的病毒后代的先决条件。然而，我们对可卡因用来促进这些变化和诱导酶的信号通路的了解非常模糊，包括MSK1/2、RSK1/2、Aurora-B、Elk1和IKK-α。这些酶直接和间接地诱导不同的表观遗传修饰，这在艾滋病毒感染的背景下从未被研究过。因此，必须对影响HIV复制的可卡因诱导的信号通路有清楚的了解。在这项拨款中，我们提出了一项系统的分析。首先，我们将定义和表征可卡因诱导的导致上述不同激酶激活的信号通路、它们的目标蛋白及其基因调控。稍后，我们将确定可卡因诱导的表观遗传修饰的作用，以及相关酶的确切作用及其对HIV基因表达和复制的影响。随后，我们将确定可卡因诱导激活NF-kB和P-TEFb的信号通路。最后，我们将利用高通量方法来表征涉及的表观遗传修饰和受可卡因暴露影响的基因，以寻找可卡因使用的独特生物标志物。所有结果都将在单核/巨噬细胞和小胶质细胞系中重现。关键的发现将通过使用艾滋病毒患者的可卡因原代单核细胞来源的巨噬细胞(MDM)来证实。最后，为了验证可卡因的直接作用，我们将进行体外实验，将艾滋病毒感染患者的PBMC暴露于可卡因。更广泛的影响：这些研究将确定可卡因的许多新作用、涉及的途径和酶，可用于开发改进的诊断测试，并可能为更好地对吸毒者艾滋病毒患者进行药物干预开辟新的途径。

 DESCRIPTION: Cocaine is a significant cofactor for HIV-1 infection, transmission and morbidity. Cocaine accelerates HIV-1 gene expression by altering specific cell-signaling and epigenetic pathways and promotes HIV-associated neurocognitive disorders (HAND). Our published data demonstrates that cocaine-induced signaling pathways lead to the activation of kinases, such as MSK1 which subsequently catalyzes the phosphorylation of p65 subunit of NF- ĸB at position S276 (P-p65S276) and histone H3 at Serine 10 (P-H3S10). P-H3S10, besides facilitating the establishment of transcriptionally active chromatin structures, promotes the recruitment of P-TEFb at HIV LTR. Consequently, cocaine accelerates both the initiation and the elongation phases of HIV transcription by activating NF-kB and P-TEFb, respectively, a prerequisite to generate complete HIV genomic transcript and new viral progeny. However, our understanding of the signaling pathways that cocaine utilizes in order to promote these changes and induce enzymes, including MSK1/2, RSK 1/2, Aurora-B, ELK1 and IKK-α is highly obscure. These enzymes directly and indirectly induce different epigenetic modifications, which never been investigated in the context of HIV infection. Thus, it is imperative to have clear knowledge about the cocaine induced signaling pathways that affect HIV replication. In this grant we have proposed a systematic analysis. First, we will define and characterize the cocaine induced signaling pathways that lead to the activation of above mentioned different kinases, their target proteins and their gene regulation. Later, we will define the role of cocaine induced epigenetic modifications and the precise role of involved enzymes and their effect on HIV gene expression and replication. Subsequently, we will define the signaling pathways which cocaine induces to activate NF-kB and P-TEFb. Finally, we will utilize high throughput approaches to characterize involved epigenetic modifications and the genes which are influenced by cocaine exposure; in order to find unique biomarkers of cocaine use. All the results will be reproduced in monocytic/macrophage and microglial cell lines. Crucial findings will be confirmed using primary Monocyte derived macrophages (MDMs) from cocaine using HIV patients. Finally, to validate the direct effect of cocaine, we will perform ex vivo experiments where we will expose PBMCs from HIV infected patients to cocaine. Broader Impact: These studies will identify many novel actions of cocaine, involved pathways and enzymes which can be exploited for the development of improved diagnostic tests and may open up new avenues for better pharmaceutical interventions in drug addict HIV patients.