Impact of HIV-1 Tat protein on cocaine-dopamine transporter interaction

描述(由申请人提供)：在有效的抗逆转录病毒治疗时代，HIV-1相关的神经认知障碍(HAND)仍然非常普遍。中枢神经系统内的HIV-1感染在HAND的发生发展中起着核心作用。滥用药物，如可卡因，已被证明通过促进病毒复制而增加发病率并加剧手部疾病的严重程度。然而，可卡因和手的进展之间的机制联系仍然不确定。虽然许多神经递质系统的改变可能与手部疾病有关，但中枢多巴胺(DA)系统在手部患者神经认知功能障碍的发生和可卡因的精神刺激作用的控制中起着至关重要的作用。HIV-1Tat蛋白与可卡因的相互作用增加了多巴胺能脑区内突触DA水平和Tat释放。长期暴露于升高的DA和TAT最终会导致DA缺乏症，从而加剧严重程度并加速手部疾病的发展。抗逆转录病毒药物不能阻止含有前病毒的脑细胞中产生HIV-1病毒蛋白，如Tat蛋白。目前尚不清楚HIV-1阳性可卡因滥用者的DA系统是如何改变的。因此，迫切需要确定HIV-1感染所致DA系统受损影响并发可卡因滥用者手部进展的分子机制(S)。突触前DA转运体(DAT)对神经认知功能至关重要，是TAT和可卡因影响DA系统的主要分子靶点。在这个应用中，我们假设TAT通过DAT中的变构结合部位，增强了 可卡因对DA转运的影响是手部患者发生DA系统功能障碍的关键。我们的实验将研究TAT和可卡因如何通过它们在人DAT上的识别结合部位与人DAT相互作用，从而导致DA系统的功能障碍。我们的策略包括创建一个动态3D计算模型来预测人DAT的潜在TAT和可卡因结合口袋残基，通过定点突变验证这些残基，并分析神经细胞和原代神经元中TAT和DAT相互作用的后续功能变化。这一应用的完成将在DAT上识别分子靶点，以开发特异性阻断DAT中TAT结合位点(S)的化合物，并稳定生理多巴胺能张力，这将有利于同时吸食可卡因的手部患者的神经认知功能的保护。

DESCRIPTION (provided by applicant): HIV-1 associated neurocognitive disorders (HAND) remain highly prevalent in the era of effective antiretroviral therapy. HIV-1 infection within CNS system plays a central role in the development of HAND. Drugs of abuse, such as cocaine, have been shown to increase the incidence and exacerbate the severity of HAND by enhancing viral replication. However, the mechanistic links between cocaine and HAND progression remain undefined. Although changes in many neurotransmitter systems may contribute to HAND, the central dopamine (DA) system plays a crucial role in the development of neurocognitive dysfunction in HAND patients and in the control of psychostimulant action of cocaine. The interplay of HIV-1 Tat protein with cocaine augments synaptic DA level and Tat release within dopaminergic brain regions. Long lasting exposure to elevated DA and Tat eventually lead to DA deficit that potentiates severity and accelerates progression of HAND. Antiretroviral agents cannot prevent the production of HIV-1 viral proteins, such as Tat protein, in proviral-containing brain cells. It is unclear how the DA system is altered in HIV-1 positive cocaine abusers. Therefore, there is a pressing need to define the molecular mechanism(s) by which the impaired DA system by HIV-1 infection affects the progression of HAND in concurrent cocaine abusers. Presynaptic DA transporter (DAT), which is critical for neurocognitive function, is a major molecular target for both Tat and cocaine to impact the DA system. In this application, we hypothesize that Tat, via allosteric binding sites in the DAT, potentiates inhibitory effects of cocaine on DA transport, which is the key to DA system dysfunction occurred in HAND patients. Our proposed experiments will investigate how Tat and cocaine interact with the human DAT through their recognition binding sites on human DAT, thereby leading to dysfunction of the DA system. Our strategy encompasses creating a dynamic 3D computational model to predict potential Tat and cocaine binding pocket residues of human DAT, validating these residues via site-directed mutagenesis, and analyzing the consequent functional changes of the Tat and DAT interaction in neuronal cells and primary neurons. The completion of this application will identify molecular targets on the DAT for developing compounds that specifically block Tat binding site(s) in DAT and stabilize physiological dopaminergic tone, which should be beneficial to the preservation of neurocognitive function in patients with HAND in concurrent cocaine abusers.