Nanobodies targeting stress granule components

对AD/ADRD的转录组学和蛋白质组学研究一致表明，AD/ADRD相关通路的改变， 免疫、脂质代谢、tau结合蛋白网络和RNA代谢。的最新进展 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。由RNA和RBP形成的SG 例如TDP-43、hnRNPA 2B 1和TIA 1是可形成单独液体生物分子缩合物（BMC 细胞中的阶段。RBPs向细胞质的错误定位增加了液-液相分离（LLPS） 倾向，导致SG形成增加。在慢性压力下，SG成熟为更固体或凝胶- 比如组装隔离SG组件在过去的几年里，我们和其他人已经确定了 RBP隔离作为额颞叶痴呆-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）积累有关。 hnRNPA 2B 1，当其寡聚化时优先与tau相互作用。由于hnRNPA 2B 1与m6 A结合， RNA甲基化，这项研究还揭示了RNA修饰在AD中的相关性。由于对限制性商业惯例的兴趣， 随着SGs的增长，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动力学而不改变SG组分的细胞内浓度。我们假设 内源性SG成分可以通过特异性， 单价粘合剂与SG组分的粘合。在这里，我们首次证明，纳米抗体（Nbs），单- 通过高通量筛选可以鉴定特异于RBP的结构域胞内结合蛋白 approach.我们的目标是使用Nbs检测SG组分，而不改变它们的细胞内浓度， 证明它们在AD的新型3D人诱导多能干细胞（iPSC）模型中的用途， tau相关的SG发病机制。我们还证明了使用Nb融合的靶向降解hnRNPA 2B 1 E3连接酶接头结构域，并旨在验证原代神经元和3D人类神经元中SGs的可逆性。 iPSC模型。最后，我们将筛选特异于m6 A RNA甲基化的Nbs，以实现m6 A RNA成像。 在SG中的甲基化。

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.