Structural basis for cellular secretion and uptake of HIV-1 Tat

HIV-1 Tat 细胞分泌和摄取的结构基础

• 批准号: 9926634
• 负责人: Jamil Subhi Saad
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-06 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926634
• 关键词: Acids; Adverse effects; Alzheimer' s Disease; Automobile Driving; Binding; Biological; Biological Assay; Blood - brain barrier anatomy; Brain; CD4 Positive T Lymphocytes; Cell Culture Techniques; Cell Line; Cell membrane; Cell secretion; Cells; Cytosol; Data; Deposition; Development; Disease Progression; Docking; Drug usage; Electron Microscopy; Endocytosis; Endosomes; Genetic Transcription; HIV; HIV-1; HIV-associated neurocognitive disorder; Half-Life; Health; Hela Cells; Human; Incidence; Individual; Infection; Knowledge; Length; Link; Lipids; Luciferases; Membrane; Methodology; Methods; Microglia; Modeling; Molecular; Molecular Conformation; Mutate; Neuraxis; Neuroglia; Neuronal Injury; Neurons; Patients; Penetration; Pharmaceutical Preparations; Play; Preparation; Process; Proliferating; Proteins; Recombinants; Resistance; Resolution; Role; Senile Plaques; Severities; Severity of illness; Specificity; Spin Labels; Structure; T-Lymphocyte; Testing; Therapeutic Intervention; Toxic effect; Trans-Activators; Transactivation; Viral Cytopathogenic Effect; Virus; antiretroviral therapy; base; biophysical tools; brain tissue; design; dopamine transporter; drug development; drug discovery; drug of abuse; improved; inflammatory milieu; inhibitor/antagonist; innovation; insight; macrophage; molecular rearrangement; nanodisk; neuroblastoma cell; neurotoxic; neurotoxicity; novel; novel strategies; novel therapeutic intervention; prevent; promoter; success; therapeutic development; treatment strategy; uptake

Despite otherwise successful antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) is detected in ~50% of infected individuals. The presence of an HIV-1 reservoir in the brain is considered a contributor to an inflammatory milieu and to the neuronal injury in infected individuals. In the brain, HIV-1 infection is driven mostly by resident microglia cells and infiltrated macrophages. ART is less efficient in the brain tissue because of reduced drug penetration through the blood-brain barrier. Moreover, other than HIV-1 infected T cells that succumb to the cytopathic effect of the virus, microglia/macrophages are resistant to the viral cytopathic effect and can act as long-term producers. Given the long half-life of microglia and the ability of different lineages of macrophages and likely microglia to proliferate, the brain presents itself as a likely reservoir of HIV-1 infection despite ART. In the context of HAND, even if ART were to completely suppress HIV-1 replication, persistently infected microglia/macrophages can actively secrete the HIV-1 transactivator of transcription (Tat) protein, which is known to be particularly toxic for neurons. Tat is known to synergize with drugs of abuse and exacerbate disease progression and severity, further confounding the adverse effects on the central nervous system (CNS). There is mounting evidence that HIV-1 Tat exerts its neurotoxicity through interaction with human dopamine transporter (hDAT) in the CNS. There is also recent evidence that Tat interacts with amyloid-β plaques, a hallmark of Alzheimer's disease, that are deposited in the brain of HIV-infected patients. Tat secretion thus presents itself as an attractive target to improve HAND. However, our knowledge of how Tat is secreted by infected cells is limited. Conversely, Tat needs to cross membranes of target cells (such as neurons) to exert its neurotoxic effect. Therefore, inhibition of uptake is a second candidate mechanism to improve HAND. Preparation of a stable and functional recombinant full-length Tat has been a barrier to studying the secretion and uptake mechanisms, which also limits the development of drug discovery assays to target the “active” form of Tat needed to cross membranes. Leading to this application, we have devised new approaches to generate large amounts of stable and functional Tat and employed NMR and biophysical tools to confirm its interaction with membranes. We have also employed a functional assay to confirm Tat’s function in neuronal cell culture. Based on our preliminary data, we hypothesize that Tat undergoes a conformational change upon binding to membranes. Our two specific aims are: (i) identify the molecular rearrangements of Tat upon binding to membrane that facilitate secretion, and (ii) determine the molecular requirements for Tat insertion into endosomal membranes during uptake. The results generated here will provide novel insights into the molecular rearrangements of Tat upon binding to membranes prior to secretion and during uptake and will offer a new platform to target the membrane-bound form of Tat, which altogether will lead to the development of new therapeutic strategies for treatment of HAND.

尽管其他成功的抗逆转录病毒疗法，但仍检测到与HIV相关的神经认知障碍（手） 在约50％的感染者中。大脑中HIV-1储层的存在被认为是造成 感染个体的炎症环境和神经元损伤。在大脑中，HIV-1感染主要是驱动的 由居民小胶质细胞和浸润的巨噬细胞。由于ART在脑组织中的效率较低 通过血脑屏障降低了药物渗透。此外，除了HIV-1感染的T细胞外 屈服于病毒的细胞质效应，小胶质细胞/巨噬细胞具有对病毒细胞病毒作用的抗性 并可以充当长期生产者。鉴于小胶质细胞的半衰期和不同谱系的能力 巨噬细胞和可能要增殖的小胶质细胞，大脑可能是HIV-1感染的储层 尽管有艺术。在手背景下，即使艺术要完全抑制HIV-1复制， 感染的小胶质细胞/巨噬细胞可以主动分泌HIV-1转录剂（TAT）蛋白，该蛋白 已知对神经元特别有毒。众所周知，TAT会与滥用和加剧药物协同作用 疾病的进展和严重程度，进一步混淆了中枢神经系统（CNS）的不利影响。 有越来越多的证据表明，HIV-1 TAT通过与人多巴胺的相互作用来发挥其神经毒性 中枢神经系统中的转运蛋白（HDAT）。最近还有证据表明TAT与淀粉样蛋白β斑块相互作用，A 阿尔茨海默氏病的标志，沉积在艾滋病毒感染患者的大脑中。因此分泌 展示自己是改善手的有吸引力的目标。但是，我们对TAT如何分泌的了解 感染细胞受到限制。相反，TAT需要跨越靶细胞（例如神经元）的膜才能发挥其作用 神经毒性作用。因此，抑制摄取是改善手的第二种候选机制。 准备稳定且功能性重组全长TAT已成为研究秘密的障碍 和摄取机制，这也限制了药物发现法案的发展以瞄准“主动”形式 tat需要越过膜。导致此应用程序，我们设计了新的方法来生成 大量稳定且功能性的TAT以及使用NMR和生物物理工具来确认其相互作用 使用机制。我们还采用了功能评估来确认TAT在神经元细胞培养中的功能。 根据我们的初步数据，我们假设TAT在结合到与 膜。我们的两个具体目的是：（i）确定TAT结合后的分子重排 促进分泌的膜，（ii）确定TAT插入的分子要求 摄取过程中内体膜。这里产生的结果将为您提供新颖的见解 分泌前和摄取期间与膜结合后TAT的分子重排，并将提供 一个针对TAT膜结合形式的新平台，完全将导致新的发展 治疗手的治疗策略。