Nanobodies targeting stress granule components

针对应激颗粒成分的纳米抗体

• 批准号: 10739370
• 负责人: Yongku Peter Cho
• 金额: $ 46.08万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739370
• 关键词: 3-Dimensional; Address; Affinity; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease related dementia; Amyloidosis; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Astrocytes; Binding; Binding Proteins; Biological; Brain; Cells; Chronic stress; Cytoplasm; DNA; Detection; Directed Molecular Evolution; Disease; Environment; Exhibits; Frontotemporal Dementia; Functional disorder; Gel; Goals; Hippocampus; Human; Image; Immunity; Immunoglobulin Fragments; In Vitro; Induced pluripotent stem cell derived neurons; Investigation; Kinetics; Knowledge; Link; Liquid substance; Methods; Microglia; Modification; Molecular Profiling; Monitor; Mus; Mutation; Nature; Nerve Degeneration; Nervous System; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathologic; Pathology; Pathway interactions; Pattern; Phase; Physical condensation; Plasmid Cloning Vector; Play; Property; Proteins; Proteomics; RNA; RNA Degradation; RNA metabolism; RNA methylation; RNA-Binding Proteins; Reagent; Role; Solid; Specificity; Tauopathies; Testing; Time; Tissues; Validation; Viral; Viral Vector; Virus; biological adaptation to stress; cell type; complex biological systems; high throughput screening; human pluripotent stem cell; improved; in vivo; in vivo imaging; induced pluripotent stem cell; insight; interest; lipid metabolism; nanobodies; novel; protein TDP-43; response; screening; stem cell model; stress granule; stress reduction; success; tau Proteins; tau aggregation; three-dimensional modeling; tool; transcriptomics; ubiquitin-protein ligase

Transcriptomic and proteomic studies on AD/ADRD consistently show alterations of pathways involved in immunity, lipid metabolism, tau-binding protein network, and RNA metabolism. Recent advances in understanding the proteins involved in RNA metabolism, including RNA-binding proteins (RBPs) involved in stress granule (SG) formation provided new insights into the pathogenesis of AD. SGs formed of RNA and RBPs such as TDP-43, hnRNPA2B1, and TIA1 are biomolecular condensates (BMCs) that can form a separate liquid phase in cells. Mislocalization of RBPs to the cytoplasm increases the liquid-liquid phase separation (LLPS) propensity, leading to increased SG formation. Under chronic stress, the SGs mature into a more solid or gel- like assembly, sequestering the SG components. Over the past years, we and others have identified the sequestration of RBPs as a critical mechanism of dysfunction in frontotemporal dementia-TDP-43, ALS, and AD. We discovered that stress response is linked to oligomeric tau (o-tau) accumulation through the RBP hnRNPA2B1, which preferentially interacts with tau when it is oligomerized. Since hnRNPA2B1 binds to the m6A RNA methylation, this study also revealed the relevance of RNA modification in AD. As the interest in RBPs and SGs grows, there is an increasing need to validate these assemblies in vivo. However, we lack the ability to monitor SG dynamics without altering the intracellular concentration of SG components. We hypothesize that endogenous SG components can be detected without perturbing their LLPS propensity through specific, monovalent binders to SG components. Here we demonstrate for the first time that nanobodies (Nbs), single- domain intracellular binding proteins, specific to RBPs can be identified through a high-throughput screen approach. We aim to use the Nbs to detect SG components without altering their intracellular concentration and demonstrate their use in a novel 3D human induced pluripotent stem cell (iPSC) model of AD that recapitulates the tau-associated SG pathogenesis. We also demonstrate targeted degradation of hnRNPA2B1 using Nb fused to an E3 ligase adaptor domain, and aim to validate the reversibility of SGs in primary neurons and the 3D human iPSC model. Finally, we will screen Nbs specific to the m6A RNA methylation to enable imaging m6A RNA methylation in SGs.

关于AD/ADRD的转录组和蛋白质组学研究始终显示出涉及的途径的改变 免疫，脂质代谢，tau结合蛋白网络和RNA代谢。最近的进步 了解参与RNA代谢的蛋白质，包括参与的RNA结合蛋白（RBP） 应力颗粒（SG）形成为AD发病提供了新的见解。由RNA和RBP形成的SGS 例如TDP-43，HNRNPA2B1和TIA1是生物分子冷凝物（BMC），可以形成单独的液体 细胞中的相位。 RBP对细胞质的错误定位增加了液态液相分离（LLP） 倾向，导致SG形成增加。在慢性应激下，SGS成熟成固体或凝胶 像组装一样，隔离SG组件。在过去的几年中，我们和其他人都确定了 RBP作为额颞痴呆 - -TDP-43，ALS和AD中功能障碍的关键机制的隔离。 我们发现压力反应与通过RBP的低聚tau（O-TAU）相关 HNRNPA2B1，当将其寡聚时优先与Tau相互作用。由于HNRNPA2B1与M6A结合 RNA甲基化，这项研究还揭示了RNA修饰在AD中的相关性。作为RBP的兴趣和 SGS的生长，越来越需要在体内验证这些组件。但是，我们缺乏能力 监测SG动力学，而无需改变细胞内浓度的SG成分。我们假设这一点 可以检测到内源性SG组件，而不会通过特定的特异性扰动其LLP倾向 单价粘合剂到SG组件。在这里，我们首次证明了纳米体（NB），单个 可以通过高通量屏幕来识别域内的细胞内结合蛋白，特有的RBP 方法。我们旨在使用NB检测SG组件而不改变其细胞内浓度和 证明它们在新型的3D人类诱导多能干细胞（IPSC）AD模型中的使用 与Tau相关的SG发病机理。我们还使用NB融合来证明HNRNPA2B1的靶向降解 到E3连接酶适配器结构域，旨在验证SGS在原发性神经元和3D人类中的可逆性 IPSC模型。最后，我们将筛选特定于M6A RNA甲基化的NB，以启用成像M6A RNA SGS中的甲基化。