Analysis of protein interactions in neurodegenerative disease

摘要/摘要 晚发性阿尔茨海默病（LOAD）是年龄相关性痴呆的最常见形式。 目前，美国有500万人患有LOAD，随着人口老龄化， 预计在未来几十年内，这一数字将翻一番。没有药物可以治愈或阻止 负载的进展。LOAD的两个病理标志是细胞外淀粉样斑块 由不溶性的A β肽和由过度磷酸化的 Tau蛋白。到目前为止，大多数临床候选药物都针对A β，但没有一个已经证明 有效改善AD的症状。需要新的药物靶点来对抗这一点 越来越常见和毁灭性的疾病。最近确定的LOAD风险因素 揭示了内体-溶酶体网络（ELN）中蛋白质的富集。这 证实了几十年来的证据，即ELN的中断是LOAD的早期事件 这表明ELN含有潜在的药物靶点。然而，缺乏 ELN的分子表征阻碍了合适候选物的发现。一旦 蛋白质-蛋白质相互作用（PPI）被认为是“不可药物治疗的”，正在成为有吸引力的靶点， 药物开发尚未对ELN中PPI的全球分析进行研究。我们建议使用 质谱法来定量不同的生化特征ELN蛋白复合物， 确定LOAD如何诱导ELN内PPI的改变。这些实验将 在人脑组织（AD与年龄匹配的对照）以及人AD和对照中进行 IPSC衍生的神经元和类器官。内源性ELN靶标将与 验证抗体并在条件之间定量以鉴定新的ELN相互作用物， 与疾病相关的药物靶点。将使用多重同量异位素标记进行定量 技术. ELN复合物的结构也将得到解决，差异也将得到解决。 使用我们的共价蛋白质绘画方法定量。此外，我们将采用一种新的 应用非规范氨基酸叠氮基高丙氨酸定量ELN的稳定性 蛋白质复合物我们的建议将产生三种不同的定量测量， LOAD发病机制对ELN蛋白复合物影响，并将提供替代药物 最常见的老年痴呆症的发展目标。

Summary/Abstract Late Onset Alzheimer's disease (LOAD) is the most common form of age-related dementia. Currently, 5 million people in the US are afflicted with LOAD and with the aging population this number is expected to double in the next decades. There are no drugs to cure or halt the progression of LOAD. The two pathological hallmarks of LOAD are extracellular amyloid plaques formed by the insoluble A peptide and neurofibrillary tangles consisting of hyperphosphorylated Tau protein. To date, most clinical drug candidates have targeted A, but none have proven effective at ameliorating the symptoms of AD. New drug targets are needed to combat this increasingly common and devastating disease. The recent identification of LOAD risk factors has revealed an enrichment of proteins in the endosomal-lysosomal network (ELN). This corroborates decades of evidence that the disruption of the ELN is an early event in LOAD pathogenesis and indicates that the ELN contains potential drug targets. However, a lack of molecular characterization of ELN has prevented the discovery of suitable candidates. Once considered “undruggable”, protein-protein interactions (PPI) are emerging as attractive targets for drug development. Global analysis of PPI in the ELN has not been studied. We propose to use mass spectrometry to quantitate different biochemical characteristics ELN protein complexes and determine how LOAD induces alterations in PPI within the ELN. These experiments will be performed in human brain tissues (AD vs. age-matched controls) and in human AD and control IPSC derived neurons and organoids. Endogenous ELN targets will be immunoprecipitated with validated antibodies and quantitated between conditions to identify novel ELN interactors and disease-relevant drug targets. Quantitation will be performed using multiplexing isobaric labeling technology. The structure of the ELN complexes will also be resolved, and differences will be quantitated with using our Covalent Protein Painting method. Additionally, we will employ a novel application of the non-canonical amino acid, azidohomoalanine, to quantitate the stability of ELN protein complexes. Our proposal will produce three different quantitative measurements of the influence of LOAD pathogenesis on ELN protein complexes and will provide alternative drug development targets for the most common form of age-related dementia.