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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.

已经假设以朊病毒的方式传播tau病理学是进行性免疫缺陷综合征的基础。 tau蛋白病中tau聚集体的积累。在实验系统中，在转基因小鼠中，tau聚集从细胞外传播到细胞内，以及在脑网络内传播。小鼠然而，这种现象是否是进行性神经退行性变的原因仍然未知在人类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。对相关因素的识别将有助于检验朊病毒假说进行性神经退化此外，了解这些机制有助于开发新的疗法。我们建议使用一种新开发的工具来识别和表征因素与tau蛋白聚集有关抗坏血酸过氧化物酶（APEX2）依赖性邻近标记是一种新的抗坏血酸过氧化物酶标记方法。用于细胞内蛋白质的时间分辨生物素化的酶促方法。我们将鉴定出基于亲和纯化和质谱进行标记。这将使我们能够确定密切相关的因素，当聚集过程开始时，在巨胞饮或聚集体直接递送至细胞内部。我们将使用标准遗传学来跟踪命中。这项工作可能会对我们的了解细胞内淀粉样蛋白的传播，并可能成为新的治疗方法的基础。战略布局