Characterization of cocaine induced signaling pathways that enhances HIV transcription

 描述：辅因子是HIV-1感染、传播和发病的重要辅因子。辅酶A通过改变特定的细胞信号传导和表观遗传途径加速HIV-1基因表达，并促进HIV相关的神经认知障碍（HAND）。我们发表的数据表明，可卡因诱导的信号传导途径导致激酶的活化，例如随后催化NF-κ B的p65亚基在位置S276（P-p65 S276）和组蛋白H3在丝氨酸10（P-H3 S10）的磷酸化的MSK 1。除了促进转录活性染色质结构的建立外，P-H3 S10还促进P-TEFb在HIV LTR的募集。因此，可卡因通过分别激活NF-kB和P-TEFb加速HIV转录的起始和延伸阶段，这是产生完整HIV基因组转录物和新病毒子代的先决条件。然而，我们对可卡因利用以促进这些变化并诱导酶（包括MSK 1/2、RSK 1/2、Aurora-B、ELK 1和IKK-α）的信号通路的理解是非常模糊的。这些酶直接和间接地诱导不同的表观遗传修饰，这在HIV感染的背景下从未被研究过。因此，必须清楚地了解可卡因诱导的影响艾滋病毒复制的信号通路。在这篇文章中，我们提出了一个系统的分析。首先，我们将定义和表征可卡因诱导的信号通路，导致上述不同激酶的激活，它们的靶蛋白和它们的基因调控。稍后，我们将确定可卡因诱导的表观遗传修饰的作用和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-κ B和P-TEFb的信号通路。最后，我们将利用高通量方法来表征所涉及的表观遗传修饰和受可卡因暴露影响的基因，以找到可卡因使用的独特生物标志物。所有结果将在单核细胞/巨噬细胞和小胶质细胞系中重现。将使用来自使用可卡因的HIV患者的原代单核细胞衍生的巨噬细胞（MDM）确认关键发现。最后，为了验证可卡因的直接作用，我们将进行离体实验，将HIV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，并可能为吸毒者艾滋病毒患者的更好药物干预开辟新途径。

 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.