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.

AD/ADRD的转录组和蛋白质组学研究一致表明，参与AD/ADRD的通路发生了变化 免疫、脂类代谢、tau结合蛋白网络和RNA代谢。的最新进展 了解参与RNA新陈代谢的蛋白质，包括参与 应激颗粒(SG)的形成为AD的发病机制提供了新的认识。由RNA和限制性商业惯例组成的SGS 例如TDP-43、hnRNPA2B1和TIA1是生物分子冷凝物(BMC)，它们可以形成单独液体 细胞中的相。限制性商业惯例在细胞质中的错误定位增加了液-液相分离(LLP) 倾向，导致更多的SG形成。在慢性压力下，SGS成熟为更固体或凝胶- 就像组装一样，隔离SG组件。在过去的几年里，我们和其他人已经确定了 RBPs的隔离是额颞叶痴呆患者功能障碍的关键机制--TDP-43、ALS和AD。 我们发现，应激反应与寡聚tau(o-tau)通过RBP积累有关。 HnRNPA2B1，当tau齐聚时，它优先与tau相互作用。由于hnRNPA2B1与M6A结合 RNA甲基化，这项研究也揭示了RNA修饰在AD中的相关性。作为对限制性商业惯例和 随着SGS的生长，在体内验证这些组件的需求越来越大。然而，我们缺乏能力 在不改变SG组分的胞内浓度的情况下监控SG的动态。我们假设 内源性SG成分可以通过特定的、 SG组分的单价粘结剂。在这里，我们第一次证明了纳米体(NBS)，单个- 可以通过高通量筛选鉴定针对限制性商业惯例的区域细胞内结合蛋白 接近。我们的目标是在不改变SG组分细胞内浓度的情况下使用NBS检测SG组分，并 展示了它们在一种新的3D人类诱导多能干细胞(IPSC)AD模型中的应用 Tau相关的SG发病机制。我们还展示了使用NB融合对hnRNPA2B1的靶向降解 目的是验证SGS在原代神经元和3D人类中的可逆性 IPSC模式。最后，我们将筛选m6A RNA甲基化的特异性NBS，以便对m6A RNA进行成像 SGS中的甲基化。