Characterizing the physicochemical properties of membraneless condensates and its regulation by delta-9-tetrahydrocannabinol in HIV/SIV infection.

表征无膜冷凝物的物理化学性质及其在 HIV/SIV 感染中通过 delta-9-四氢大麻酚的调节。

• 批准号: 10220203
• 负责人: Mahesh Mohan
• 金额: $ 22.11万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220203
• 关键词: AIDS/HIV problem; Affect; Alzheimer' s Disease; Amyloid fibers; Anti-HIV Agents; Anti-Infective Agents; Anti-Retroviral Agents; Attenuated; Behavior; Biochemical; Biochemistry; Bioenergetics; Biological; Biological Assay; Biological Process; Biology; Blood; Body Fluids; Brain; CCR5 gene; Cell Nucleolus; Cell Nucleus; Cell physiology; Cells; Cellular biology; Centrosome; Cerebrospinal Fluid; Chemicals; Chronic; Classification; Clinical; Complex; Cytoplasm; Cytoplasmic Granules; Data; Data Set; Development; Disease; Dyes; Electron Microscope; Electron Microscopy; Environment; Exposure to; Genetic Transcription; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-associated neurocognitive disorder; Homeostasis; Human; Individual; Infection; Inflammation; Interruption; Intestinal Content; Knowledge; Lead; Link; Liquid Chromatography; Liquid substance; Macaca; Macaca mulatta; Malignant Neoplasms; Mediating; Membrane; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Target; Morphology; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Normal Range; Nucleic Acids; Outcome Study; Pathogenesis; Pathologic; Peripheral; Pharmaceutical Preparations; Phase; Physiological; Physiological Processes; Plasma; Play; Process; Property; Proteins; Proteomics; RNA; Regulation; Reporting; Resources; Retrieval; Roentgen Rays; Role; SIV; Saliva; Seminal fluid; Shapes; Stress; Structure; Symptoms; Testing; Tetrahydrocannabinol; Thermodynamics; Viral; Viral Pathogenesis; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; antiretroviral therapy; biological adaptation to stress; cytotoxic; data resource; design; electron energy; experimental study; extracellular vesicles; in vivo; live cell imaging; marijuana use; nanocompound; neuronal transport; new therapeutic target; novel; particle; physical property; protein aggregation; protein complex; protein function; recruit; response; scaffold; stress granule; tat Protein; tool; transmission process

Abstract The `central dogma' of molecular biology is that in cells, the functions of proteins and nucleic acids are primarily determined by their sequence. However, this dogma has been challenged by studies of secreted molecules including the membrane encased extracellular vesicles (EVs) and the membraneless structures known as BMCs, which indicate that cells create discrete chemical environments where they organize complex biomolecules. BMCs form in many compartments of the body, including the nucleus (nucleolus, centrosome, Cajal body, stress granules), the cytoplasm (P-bodies, stress granules), and even the neuronal cytoplasm (neuronal transport granules). While the details of BMC biology is still evolving, it is known that RNA and proteins with intrinsically disordered regions (IDRs) are key factors that influence intracellular composition, size, and morphology of BMCs. The size and composition of BMCs are also thought to be different. BMCs participate in the regulation of various physiological processes, such as modulation of host transcription and stress response. BMCs also play important roles in pathophysiological conditions, including cancer, neurodegenerative diseases, and viral pathogenesis, including modulation of HIV replication. Indeed, it is known that HIV infection results in peripheral and neuro inflammation, and that chronic exposure to THC in the context of HIV in humans or SIV infection in rhesus macaques results in amelioration of disease or symptoms. However, the regulators of HIV disease and THC response is not completely understood. Despite the reported role of BMCs in various conditions, whether or not BMCs regulate the pathogenesis of HIV infection and the intersection of HIV and THC use is unknown. It is also unknown whether BMCs assemble in vivo, where they may play relevant biological roles. There is paucity of information on the presence of BMCs in body fluids (in vivo) and their role in HIV pathogenesis. The reason for this scarcity of information is partly due to lack of tools for isolation of BMCs from body fluids. In preliminary studies, we developed a novel size-guided Particle Purification Liquid Chromatography (PPLC). We used PPLC to isolate and retrieve dye-free BMCs from human semen, rhesus macaque blood plasma and cerebrospinal fluid (CSF). Using standard electron microscopy and Energy Dispersive X-ray equipped transmission electron microscope (TEM-EDX) analyses, we obtained structural and elemental information of the BMCs. Proteomics analysis and RNA bioanalyzer study were used to show that the BMCs contain protein and RNA, while cell biology assay indicate that BMCs are cytotoxic to cells. These data provide compelling evidence that bioactive BMCs are present in vivo. Thus, in this proposal, we will use PPLC to isolate BMCs from rhesus macaque blood, CSF, saliva, and intestinal contents. We will then use various structural, biochemical, and cell biology assays to provide structural, molecular, and functional classification of BMCs from SIV infected and THC treated macaques, which will provide a framework to dissect BMC functions in HIV/AIDS and THC use.

摘要 分子生物学的“中心教条”是，在细胞中，蛋白质和核酸的功能主要是 由它们的顺序决定。然而，这一教条受到了对分泌分子的研究的挑战。 包括被膜包裹的细胞外小泡(EVS)和被称为BMCs的无膜结构， 这表明细胞创造了离散的化学环境，在那里它们组织复杂的生物分子。 骨髓细胞在人体的许多区域形成，包括核(核仁、中心体、卡哈尔小体、应力 颗粒)、细胞质(P小体、应激颗粒)，甚至神经元细胞质(神经元运输 颗粒)。虽然BMC生物学的细节仍在发展中，但已知rna和蛋白质具有内在的 无序区(IDR)是影响细胞内成分、大小和形态的关键因素。 BMC的大小和组成也被认为是不同的。BMC参与监管各种不同的 生理过程，如调节寄主转录和胁迫反应。BMC也扮演着重要的角色 在包括癌症、神经退行性疾病和病毒致病在内的病理生理条件中的作用， 包括对艾滋病毒复制的调节。事实上，众所周知，艾滋病毒感染会导致外周和神经病变。 炎症，以及在人类艾滋病毒或恒河猴SIV感染背景下长期暴露于THC 猕猴可以改善疾病或症状。然而，艾滋病毒疾病和THC的监管机构 人们还没有完全理解他们的反应。尽管据报道，骨髓基质细胞在不同的条件下发挥了作用，无论是否 骨髓细胞调节HIV感染的发病机制，而HIV和THC的交叉使用尚不清楚。它也是 尚不清楚骨髓细胞是否在体内组装，在那里它们可能发挥相关的生物学作用。缺乏…… 关于体液中存在骨髓细胞及其在艾滋病毒发病机制中作用的信息。其原因是 这种信息匮乏的部分原因是缺乏从体液中分离骨髓细胞的工具。在预赛中 在此基础上，我们建立了一种新型的尺寸导向颗粒纯化高效液相色谱(PPLC)。我们使用了PPLC 从人精液、恒河猴血浆和脑脊液中分离和回收无染料的骨髓细胞 液体(脑脊液)。使用标准电子显微镜和能量色散X射线装备的透射式电子 显微镜(TEM-EDX)分析，获得了BMCs的结构和元素信息。蛋白质组学 分析和RNA生物分析仪研究表明，BMCs含有蛋白质和RNA，而细胞 生物学实验表明，BMCs对细胞具有细胞毒作用。这些数据提供了令人信服的证据表明生物活性 骨髓细胞存在于体内。因此，在这个方案中，我们将使用PPLC从恒河猴血中分离出骨髓细胞， 脑脊液、唾液和肠道内容物。然后，我们将使用各种结构、生化和细胞生物学分析来 提供SIV感染和THC治疗的骨髓细胞的结构、分子和功能分类 猕猴，它将提供一个框架，剖析艾滋病毒/艾滋病和THC使用中的BMC功能。