Uncovering cell factors with aggregate clearance activity by scalable induced proximity

摘要 蛋白质的毒性聚集是神经退行性疾病的致病机制之一。蛋白质 例如额颞叶痴呆(FTD)或阿尔茨海默病(AD)中TDP-43错误折叠成聚集体，该聚集体 两者都会扰乱内源性蛋白质的功能，并赋予有毒的新功能，导致神经细胞死亡。 目前尚无治疗AD或FTD的方法，但目前的研究表明，集中清除是一种很有前途的治疗方法 策略。TDP-43的聚集体受伴侣和蛋白平衡网络的影响 然而，目前尚不清楚哪些因素可以调节总清除量，以及它们是如何进行调节的。 以前的工作是使用称为PROTACs的小分子配体来诱导目标蛋白质与组分的接近 泛素-蛋白酶体系统的研究已被证明是清除异常蛋白质物种的一种很有前途的方法。 类似地，这项提议旨在系统地筛选蛋白质平衡中未被询问的大部分 网络具有通过配体诱导的接近清除TDP-43聚集体的能力。除了可能存在的蛋白质 作为降解物或解聚体，还将对RNA进行筛选，以确定聚合清除活性。这部作品 将揭示涉及总清关的新的质量控制因素和机制，并将提供 新城疫多特效药翻译开发平台。 这项工作将通过三个目标完成，为我提供过渡到 独立研究。在目标1中，我将开发一个实验性的工作流程来成像易于聚集的TDP-43 在多路传输标签单元库中表达。我将编写计算管道来描述 汇总清除并将其与自动原位测序相结合，以揭示推定的身份 每个细胞中的效应器。这一目标将优化试剂并开发诱导邻近筛查的分析方法， 对我进行ND模型和集合图像分析的培训。在目标2中，我将重点筛选蛋白质平衡网络 通过可扩展的诱导邻近度和验证因素来调节神经元和 在体外，接受细胞生物学和生化技术方面的培训，以确定降解物和解聚酶的特征。 这项工作将揭示导致总体清场的可招募因素。在《目标3》中，作为一个独立的 调查员，我将开发转录本的池标记，并使用产生的多路复用细胞库进行筛选 对于具有诱导的基于邻近的聚集清除活动的RNA。这项工作的成果将系统地 表征RNA在调节聚集中的蛋白恒定潜力，并极大地扩展 具有潜在治疗效益的可招募效应器。我组建的专家指导团队，以及 在CHOP和宾夕法尼亚大学，良好的培训环境将极大地促进我在 此建议的指导阶段，并为我提供开始独立研究所需的技能 系统地描述了参与ND相关蛋白聚集的蛋白稳定机制。

Summary Toxic aggregation of proteins is a pathogenic mechanism in neurodegenerative disease (ND). Proteins such as TDP-43 in frontotemporal dementia (FTD) or Alzheimer’s disease (AD) misfold into aggregates, which both disrupts endogenous protein functions and confers toxic new functions that lead to neuronal cell death. There are no cures for AD or FTD, but current research suggests aggregate clearance is a promising therapeutic strategy. Aggregates of TDP-43 are influenced by the proteostasis network consisting of chaperones and degradation machinery, though which factors can mediate aggregate clearance and how they do so is not known. Prior work using small molecule ligands called PROTACs to induce proximity of target proteins to components of the ubiquitin-proteasome system has proven a promising approach for clearing aberrant protein species. Analogously, this proposal aims to systematically screen the large, uninterrogated portion of the proteostasis network for its ability to clear aggregates of TDP-43 by ligand-induced proximity. In addition to proteins that may function as degraders or disaggregases, RNAs will also be screened for aggregate clearance activity. This work will reveal new quality control factors and mechanisms involved in aggregate clearance and will provide a platform for translational development of multispecific drugs for ND. This work will be accomplished in three Aims, providing me with critical training for transition into independent research. In Aim 1, I will develop an experimental workflow to image aggregate-prone TDP-43 expressed in a multiplexed tag cell library. I will write computational pipelines to characterize mechanisms of aggregate clearance and integrate them with automated in-situ sequencing to reveal the identity of the putative effector in each cell. This aim will optimize reagents and develop analysis methods for induced proximity screens, training me in models of ND and pooled image analysis. In Aim 2, I will focus on screening proteostasis network components by scalable induced proximity and validate factors mediating aggregate clearance in neurons and in vitro, gaining training in cell biology and biochemical techniques to characterize degraders and disaggregases. This work will uncover recruitable factors inducing aggregate clearance. In Aim 3, as an independent investigator, I will develop pooled tagging of transcripts and use the resulting multiplexed cell libraries to screen for RNAs with induced proximity-based aggregate clearance activity. The outcome of this work will systematically characterize the proteostatic potential of RNAs in modulating aggregation and greatly expand the space of recruitable effectors with potential therapeutic benefit. The expert mentoring team I have assembled, as well as the excellent training environment at CHOP and Penn, will greatly facilitate my research and training during the mentored phase of this proposal and provide me with the skills necessary to begin independent research systematically characterizing proteostasis mechanisms involved in ND-associated protein aggregation.