Molecular recognition of pathological tau fibril conformations

病理性 tau 原纤维构象的分子识别

• 批准号: 10389134
• 负责人: Emma C Carroll
• 金额: $ 6.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389134
• 关键词: Adopted; Alzheimer' s Disease; Alzheimer' s disease patient; Amyloid Fibrils; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biology; Biophysics; Chemicals; Circular Dichroism; Cryoelectron Microscopy; Development; Diagnostic; Dictionary; Disease; Dyes; Environmental Risk Factor; Epitope Mapping; Event; Fluorescence-Activated Cell Sorting; Foundations; Goals; Heparin; Immunotherapeutic agent; In Vitro; Individual; Kinetics; Label; Libraries; Light; Lysine; Maps; Methods; Microtubule Stabilization; Microtubules; Modification; Molecular; Molecular Conformation; Morphology; Neurodegenerative Disorders; Neurons; Onset of illness; Pathologic; Patients; Positron-Emission Tomography; Post-Translational Protein Processing; Process; Property; Proteins; Recombinants; Research; Resistance; Resources; Running; Sampling; Site; Structure; System; Tauopathies; Testing; Therapeutic; Ubiquitin; Ubiquitination; Work; Yeasts; amyloid fibril formation; base; biophysical properties; biophysical techniques; disease phenotype; fluorophore; high throughput screening; in vitro Model; insight; instrumentation; molecular recognition; monomer; nanobodies; novel therapeutics; polyanion; precision medicine; prevent; prion-like; protein aggregation; protein protein interaction; reconstitution; recruit; scaffold; screening; small molecule; success; tau Proteins; tau aggregation; tau conformation; tau phosphorylation; tool; training opportunity; undergraduate student

ABSTRACT Tau is an intrinsically disordered protein that binds microtubules in healthy neurons but forms filamentous aggregates that drive neurodegenerative diseases known as tauopathies. Cryo-EM structures from patient samples revealed that these amyloid fibrils adopt unique tauopathy-dependent conformations that differ from those of heparin-induced recombinant tau fibrils in vitro. These observations suggest that tau fibril morphology begets disease phenotype, and that these aggregates propagate from neuron to neuron, recruiting tau monomer in a “prion-like” mechanism. However, the mechanistic basis for transformation of the healthy intracellular tau pool to pathological aggregation remains largely undefined, and there are no in vitro methods for generating tauopathy-like fibril conformations with recombinant tau. Recently characterized alternative inducers of tau fibrillization in vitro may faithfully recapitulate tauopathy-associated fibril morphologies; however, an understanding of the cellular and environmental factors driving conformation-specific fibrillization is hampered by the lack of tools for readily determining fibril conformation and the incomplete understanding of molecular crosstalk with tau posttranslational modifications (PTMs). Here, I propose to leverage two unique molecular recognition platforms to develop conformationally selective tools for rapid identification of tau fibril conformations. My hypothesis is that cellular factors modulate the tau conformational landscape to promote amyloid fibril formation in neurodegenerative disease and that new tools for detecting and inhibiting this process will be useful therapeutics. For my first aim, I will utilize a small molecule fluorogenic probe library to identify molecules that recognize disease-associated fibril conformations by comparing dye binding profiles of Alzheimer’s disease patient-derived fibrils and fibrils generated in vitro with diverse polyanionic inducers. For my second aim, I will use a yeast-displayed humanized nanobody library system to identify tau conformation-selective nanobodies that will and serve as candidates for immunotherapeutics that inhibit the addition of monomers to tau fibrils and expand and multiplex molecular recognition of fibrils. For my third aim, I will use chemical biology approaches to reconstitute disease-associated tau ubiquitinations and, in combination with newly identified, conformationally unique in vitro-induced tau fibrils, systematically examine the effect of ubiquitination on fibrillization propensity, kinetics, and conformation and changes in fibril protein-protein interaction networks. Upon the successful completion of this proposal, I envision a new toolkit for neurodegenerative disease precision medicine in which small molecule fluorogenic probes detect tau fibril conformation/PTM status, and then fibril propagation is specifically inhibited using nanobody immunotherapeutics.

摘要 Tau是一种本质上无序的蛋白质，它与健康神经元中的微管结合， 导致神经退行性疾病的聚集体称为tau蛋白病。来自患者的Cryo-EM结构 样品显示，这些淀粉样蛋白原纤维采用独特的tau蛋白依赖性构象， 体外肝素诱导的重组tau原纤维的那些。这些观察结果表明，tau纤维形态 引起疾病表型，并且这些聚集体在神经元之间传播，募集tau单体 在一种“朊病毒样”机制中。然而，健康细胞内tau蛋白转化的机制基础 合并到病理性聚集仍然在很大程度上不确定，并且没有体外方法产生 重组tau蛋白的tau蛋白病样原纤维构象。最近表征的tau替代诱导剂 体外纤维化可以忠实地再现tau蛋白病相关的纤维形态;然而， 阻碍了对驱动构象特异性纤维化的细胞和环境因素的理解 由于缺乏容易确定原纤维构象的工具和对分子构象的不完全理解， 与tau翻译后修饰（PTM）的串扰。在这里，我建议利用两个独特的分子 识别平台，以开发用于快速鉴定tau原纤维构象的构象选择性工具。 我的假设是，细胞因子调节tau蛋白构象景观，以促进淀粉样纤维 在神经退行性疾病中的形成，并且用于检测和抑制该过程的新工具将是有用的。 治疗学对于我的第一个目标，我将利用一个小分子荧光探针库来识别分子， 通过比较阿尔茨海默病的染料结合谱来识别疾病相关的原纤维构象 患者来源的原纤维和用不同的聚阴离子诱导剂在体外产生的原纤维。我的第二个目标， 使用酵母展示的人源化纳米抗体文库系统鉴定tau构象选择性纳米抗体 其将用作抑制单体添加到tau原纤维的免疫治疗剂的候选物， 扩展和多重化原纤维的分子识别。对于我的第三个目标，我将使用化学生物学方法， 重组疾病相关的tau泛素化，并结合新发现的构象 独特的体外诱导的tau原纤维，系统地检查泛素化对纤维化倾向的影响， 动力学，以及原纤维蛋白质-蛋白质相互作用网络中的构象和变化。一旦成功 完成这项建议后，我设想了一个新的神经退行性疾病精准医学工具包， 小分子荧光探针检测tau原纤维构象/PTM状态，然后检测原纤维增殖。 使用纳米抗体免疫治疗剂特异性抑制。