Uncovering cell factors with aggregate clearance activity by scalable induced proximity

通过可扩展的诱导接近来发现具有聚集清除活性的细胞因子

• 批准号: 10705215
• 负责人: Yevgeniy Vladimirovich Serebrenik
• 金额: $ 12.96万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10705215
• 关键词: Address; Alzheimer' s Disease; Alzheimer' s disease model; Binding; Biochemical; Cells; Cellular biology; Code; Complement; Computational Technique; Development; Disease; Disease model; Environment; Foundations; Frontotemporal Dementia; Functional disorder; Future; Genes; Goals; Human; Image; Image Analysis; In Vitro; Libraries; Ligand Binding; Ligand Binding Domain; Ligands; Mediating; Mentors; Methodology; Methods; Microscopy; Modeling; Molecular Chaperones; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Pathogenicity; Pathology; Pharmaceutical Preparations; Phase; Population; Postdoctoral Fellow; Process; Productivity; Protein Degradation Induction; Proteins; Quality Control; RNA; Reagent; Research; Research Personnel; Severity of illness; System; TDP-43 aggregation; Techniques; Therapeutic; Training; Transcript; Ubiquitin; Untranslated RNA; Visualization; Work; Writing; age related; age related neurodegeneration; aggregation factor; analysis pipeline; aptamer; career; computational pipelines; disease phenotype; drug development; improved; in situ sequencing; inhibitor; insight; learning biochemistry; loss of function; multicatalytic endopeptidase complex; mutant; neuron loss; neuroprotection; neurotoxic; novel; pharmacologic; prevent; protein TDP-43; protein aggregation; protein function; proteostasis; recruit; screening; skills; small molecule; symptom treatment; targeted treatment; transcriptome

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.

概括 蛋白质的毒性聚集是神经退行性疾病（ND）的致病机制。蛋白质 例如额颞叶痴呆 (FTD) 或阿尔茨海默病 (AD) 中的 TDP-43 错误折叠成聚集体， 两者都会破坏内源性蛋白质功能并赋予有毒的新功能，导致神经元细胞死亡。 AD 或 FTD 无法治愈，但目前的研究表明聚集清除是一种有前途的治疗方法 战略。 TDP-43 的聚集体受到蛋白质稳态网络的影响，该网络由分子伴侣和 降解机制，但哪些因素可以介导聚集体清除以及它们如何做到这一点尚不清楚。 先前的工作使用称为 PROTAC 的小分子配体来诱导靶蛋白与成分的接近 泛素-蛋白酶体系统的研究已被证明是清除异常蛋白质种类的有前途的方法。 类似地，该提案旨在系统地筛选蛋白质稳态的大部分未经询问的部分 网络因其能够通过配体诱导的接近来清除 TDP-43 聚集体。除了可能含有的蛋白质外， 作为降解剂或解聚酶，RNA 也将被筛选以进行聚集清除活性。这部作品 将揭示涉及总体清除的新质量控制因素和机制，并将提供 ND 多特异性药物的转化开发平台。 这项工作将通过三个目标来完成，为我过渡到 独立研究。在目标 1 中，我将开发一个实验工作流程来对易于聚集的 TDP-43 进行成像 在多重标签细胞库中表达。我将编写计算管道来表征机制 聚合清除率并将其与自动原位测序相结合，以揭示假定的身份 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选的分析方法， 对我进行 ND 模型和汇集图像分析方面的培训。在目标 2 中，我将重点筛选蛋白质稳态网络 通过可扩展诱导接近来验证成分，并验证介导神经元中聚集清除的因素和 在体外，获得细胞生物学和生化技术方面的培训，以表征降解剂和解聚酶。 这项工作将揭示导致总体清除的可招募因素。在目标 3 中，作为独立 研究员，我将开发转录本的汇集标签，并使用生成的多重细胞库来筛选 用于具有诱导的基于邻近的聚集体清除活性的RNA。这项工作的成果将系统地 表征 RNA 在调节聚集方面的蛋白抑制潜力，并极大地扩展了研究空间 具有潜在治疗益处的可招募效应器。我组建的专家指导团队，以及 CHOP和宾夕法尼亚大学优良的训练环境，将极大地促进我在校期间的研究和训练 指导该提案的阶段，并为我提供开始独立研究所需的技能 系统地表征了 ND 相关蛋白聚集中涉及的蛋白稳态机制。