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: 10842560
• 负责人: Mahesh Mohan
• 金额: $ 44.06万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 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 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10842560
• 关键词: Characterizing; physicochemical; properties; membraneless; condensates

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.

抽象的 分子生物学的“中心法则”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，这一教条受到了分泌分子研究的挑战 包括膜包裹的细胞外囊泡 (EV) 和称为 BMC 的无膜结构， 这表明细胞创造了离散的化学环境，在其中组织复杂的生物分子。 BMC 在身体的许多区室中形成，包括细胞核（核仁、中心体、卡哈尔体、应激体） 颗粒）、细胞质（P-体、应激颗粒），甚至神经元细胞质（神经元转运 颗粒）。虽然 BMC 生物学的细节仍在不断发展，但众所周知，RNA 和蛋白质本质上具有 无序区域（IDR）是影响 BMC 细胞内组成、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参与各种监管 生理过程，例如宿主转录和应激反应的调节。 BMC 也发挥着重要作用 在病理生理状况中的作用，包括癌症、神经退行性疾病和病毒发病机制， 包括调节 HIV 复制。事实上，众所周知，HIV 感染会导致外周和神经损伤 炎症，以及在人类 HIV 或恒河猴 SIV 感染的情况下长期接触 THC 猕猴可改善疾病或症状。然而，HIV 疾病和 THC 的监管者 响应尚未完全理解。尽管据报道 BMC 在各种条件下都发挥着作用，但无论是否 BMC 调节 HIV 感染的发病机制，而 HIV 和 THC 使用的交叉点尚不清楚。这也是 目前尚不清楚 BMC 是否在体内组装，以及它们可能发挥相关的生物学作用。存在着匮乏的 有关体液（体内）中 BMC 的存在及其在 HIV 发病机制中的作用的信息。原因是 信息匮乏的部分原因是缺乏从体液中分离 BMC 的工具。在初步 研究中，我们开发了一种新型的尺寸引导颗粒纯化液相色谱 (PPLC)。我们使用了PPLC 从人类精液、恒河猴血浆和脑脊液中分离和回收无染料的 BMC 液体（脑脊液）。使用标准电子显微镜和配备透射电子的能量色散 X 射线 通过显微镜（TEM-EDX）分析，我们获得了 BMC 的结构和元素信息。蛋白质组学 通过分析和RNA生物分析仪研究表明BMCs含有蛋白质和RNA，而细胞 生物学检测表明BMCs对细胞具有细胞毒性。这些数据提供了令人信服的证据表明生物活性 BMC 存在于体内。因此，在本提案中，我们将使用 PPLC 从恒河猴血液中分离 BMC， 脑脊液、唾液和肠内容物。然后，我们将使用各种结构、生化和细胞生物学测定来 提供 SIV 感染和 THC 治疗的 BMC 的结构、分子和功能分类 猕猴，这将提供一个框架来剖析 BMC 在艾滋病毒/艾滋病和 THC 使用中的功能。