Nanobodies targeting stress granule components

针对应激颗粒成分的纳米抗体

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos s disease model Alzheimer&apos 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

项目摘要

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 的转录组学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、脂质代谢、tau 结合蛋白网络和 RNA 代谢。最近的进展了解参与 RNA 代谢的蛋白质，包括参与 RNA 代谢的 RNA 结合蛋白 (RBP) 应激颗粒（SG）的形成为 AD 的发病机制提供了新的见解。 SG由RNA和RBP形成 TDP-43、hnRNPA2B1 和 TIA1 等生物分子缩合物 (BMC) 可以形成单独的液体细胞中的相。 RBP 错误定位到细胞质会增加液-液相分离 (LLPS) 倾向，导致 SG 形成增加。在慢性压力下，SGs 成熟为更坚固或凝胶状的结构。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 RBP 的隔离是额颞叶痴呆 -TDP-43、ALS 和 AD 功能障碍的关键机制。我们发现应激反应通过 RBP 与寡聚 tau (o-tau) 积累相关 hnRNPA2B1，在寡聚化时优先与 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修饰与AD的相关性。由于对 RBP 的兴趣和 SG 不断增长，越来越需要在体内验证这些组件。然而我们缺乏能力监测 SG 动态而不改变 SG 成分的细胞内浓度。我们假设通过特定的、 SG 组分的单价结合剂。在这里，我们首次证明了纳米抗体（Nbs），单可以通过高通量筛选来鉴定 RBP 特异的胞内结合蛋白结构域方法。我们的目标是使用 Nbs 来检测 SG 成分而不改变其细胞内浓度和展示它们在 AD 的新型 3D 人类诱导多能干细胞 (iPSC) 模型中的用途，该模型概括了 tau相关的SG发病机制。我们还证明了使用 Nb 融合对 hnRNPA2B1 进行靶向降解到 E3 连接酶适配器结构域，旨在验证原代神经元和 3D 人类中 SG 的可逆性 iPSC 模型。最后，我们将筛选 m6A RNA 甲基化特异性的 Nb，以实现 m6A RNA 成像 SG 中的甲基化。