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

描述（由申请人提供）：HIV-1相关神经认知障碍（HAND）在有效抗逆转录病毒治疗的时代仍然非常普遍。中枢神经系统内HIV-1感染在HAND的发展中起核心作用。滥用药物，如可卡因，已被证明通过增强病毒复制来增加HAND的发病率并加重其严重程度。然而，可卡因与HAND进展之间的机制联系仍不明确。虽然许多神经递质系统的变化可能导致HAND，但中枢多巴胺（DA）系统在HAND患者神经认知功能障碍的发展和可卡因的精神兴奋作用的控制中起着至关重要的作用。HIV-1 Tat蛋白与可卡因的相互作用增加了突触DA水平和多巴胺能脑区域内Tat的释放。长期暴露于DA和Tat升高，最终导致DA缺失，从而加剧HAND的严重程度并加速其进展。抗逆转录病毒药物不能阻止含有原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感染的DA系统受损影响并发可卡因滥用者HAND进展的分子机制。突触前DA转运蛋白（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中的变构结合位点，增强了抑制作用

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.