APEX2-mediated Identification of factors involved in seeding by tau protein

APEX2 介导的 tau 蛋白播种相关因子的鉴定

基本信息

批准号：
9896356
负责人：
MARC I DIAMOND
金额：
$ 44.93万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9896356
关键词：
Affinity Affinity Chromatography Alzheimer&apos s Disease Amino Acids Amyloid Biological Biological Assay Biotin Biotinylation Brain CRISPR interference CRISPR/Cas technology Candidate Disease Gene Cell Culture Techniques Cells Clustered Regularly Interspaced Short Palindromic Repeats Cultured Cells Data Development Disease Enzymes Genes Genetic Heparan Sulfate Proteoglycan Holoenzymes Human Hydrogen Peroxide Individual Knock-out Label Length Mass Spectrum Analysis Measures Mediating Mediator of activation protein Methods Modeling Modern Medicine Molecular Molecular Conformation Molecular Sieve Chromatography Mus Neurodegenerative Disorders Neuroglia Neurons Pathologic Pathology Peptides Peroxidases Phenols Prions Process Proteins Quantitative Reverse Transcriptase PCR Recombinants Resolution Seeds Set protein Signal Transduction Stable Isotope Labeling Streptavidin Structure System Tauopathies Testing Time Transgenic Mice Work amyloid formation ascorbate base follow-up genetic approach improved insertion/deletion mutation neuropathology novel strategies novel therapeutic intervention novel therapeutics overexpression prion hypothesis progressive neurodegeneration reconstitution recruit small hairpin RNA tau Proteins tau aggregation therapeutic target tool uptake

项目摘要

Propagation of tau pathology in the manner of prions has been hypothesized to underlie the progressive accumulation of tau aggregates in tauopathies. In experimental systems it has been easy to observe propagation of tau aggregation from the outside to the inside of the cell, and within brain networks in transgenic mice. However, it remains unknown whether this phenomenon is the cause of progressive neurodegeneration in human tauopathies. We do not know the molecular mechanisms by which tau aggregates taken into a cell can replicate unique structures. The identification of factors that are involved will help test the prion hypothesis of progressive neurodegeneration. Furthermore, understanding these mechanisms could help with the development of new therapies. We propose to use a newly developed tool to identify and characterize factors that are involved in tau aggregation. Ascorbate peroxidase (APEX2)-dependent proximity labeling is an enzymatic method for time-resolved biotinylation of proteins within a cell. We will identify peptides that have been labeled based on affinity purification and mass spectrometry. This will let us identify factors in close proximity to tau as the aggregation process begins, after macropinocytosis or direct delivery of aggregates to the cell interior. We will use standard genetics to follow up hits. This work could have strong impact on our understanding of the propagation of intracellular amyloids, and could form the basis for new therapeutic strategies.

tau 病理学以朊病毒的方式传播已被假设为渐进性的基础 tau蛋白病中tau蛋白聚集体的积累。在实验系统中很容易观察到 tau 蛋白聚集从细胞外部传播到细胞内部，并在转基因脑网络内传播老鼠。然而，目前尚不清楚这种现象是否是进行性神经退行性变的原因在人类tau蛋白病中。我们不知道 tau 蛋白聚集进入细胞的分子机制可以复制独特的结构。确定相关因素将有助于检验朊病毒假说进行性神经变性。此外，了解这些机制有助于新疗法的开发。我们建议使用新开发的工具来识别和表征因素参与 tau 聚集。抗坏血酸过氧化物酶 (APEX2) 依赖性邻近标记是一种用于细胞内蛋白质的时间分辨生物素化的酶法。我们将鉴定具有以下特征的肽：基于亲和纯化和质谱法进行标记。这将使我们能够确定密切相关的因素当聚集过程开始时，在巨胞饮作用或直接将聚集体递送至细胞内部。我们将使用标准遗传学来追踪命中。这项工作可能会对我们产生重大影响了解细胞内淀粉样蛋白的传播，并可以为新的治疗方法奠定基础策略。