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

摘要 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 由它们的序列决定。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，在那里它们组织了复杂的生物分子。 BMC在身体的许多隔室中形成，包括细胞核（核仁、中心体、Cajal体、应力）。 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生物学的细节仍在发展中，但已知RNA和蛋白质具有内在的生物学特性。 无序区（IDR）是影响BMC的细胞内组成、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，如调节宿主转录和应激反应。BMC也很重要 在病理生理条件下的作用，包括癌症，神经退行性疾病和病毒发病机制， 包括HIV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系统疾病。 炎症，以及在人类HIV或恒河猴SIV感染情况下长期暴露于THC 猕猴导致疾病或症状的改善。然而，艾滋病毒疾病和四氢大麻酚的调节者 答案并不完全清楚。尽管报道了BMC在各种条件下的作用， BMC调节HIV感染的发病机制，HIV和THC使用的交叉点尚不清楚。也是 目前尚不清楚BMC是否在体内组装，在体内它们可能发挥相关的生物学作用。缺乏 关于体液（体内）中存在BMC及其在HIV发病机制中的作用的信息。的原因 这种信息的缺乏部分是由于缺乏从体液中分离BMC的工具。初步 研究中，我们开发了一种新的尺寸导向颗粒纯化液相色谱（PPLC）。我们使用PPLC 从人精液、恒河猴血浆和脑脊液中分离和回收无染料的BMCs， 脑脊液（CSF）。利用标准电子显微镜和能量色散X射线透射电子显微镜 通过透射电子显微镜（TEM-EDX）分析，我们获得了BMCs的结构和元素信息。Proteomics 结果表明，BMCs含有蛋白质和RNA，而细胞 生物学实验表明，BMCs对细胞具有细胞毒性。这些数据提供了令人信服的证据， BMC存在于体内。因此，在本建议中，我们将使用PPLC从恒河猴血液中分离BMCs， 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， 提供SIV感染和THC治疗的BMCs的结构、分子和功能分类 猕猴，这将提供一个框架，剖析BMC的功能，在艾滋病毒/艾滋病和四氢大麻酚的使用。

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.