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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显示参与的通路改变