Defining the acute global proteome effects of the unfolding and aggregation of a single protein

项目总结/摘要 蛋白质组稳态（proteostasis）是细胞生命所必需的，并由一系列细胞内的蛋白质组来维持。 蛋白质的合成、折叠、溶解性和降解 蛋白质组当这些蛋白质稳态网络（PN）组件工作时，可实现最佳细胞功能 共同校准正确折叠的水平，从而功能蛋白质，以满足细胞的需求 并防止有毒蛋白质聚集体的形成。许多遗传形式的构象疾病（CD）， 包括许多神经退行性疾病，都是由基因突变引起的， 构象（折叠）的一个单一的基础蛋白质，这增加了这些蛋白质的倾向， 骨料在这些构象受损的蛋白过度表达的情况下， 易聚集蛋白与蛋白质稳态损伤（PI）有关。然而， 导致CD中PI的细胞事件仍然难以捉摸。越来越多但有限的证据表明， 称为伴侣滴定（CT）的现象与CD中的PI机械相关并可引发CD中的PI。 分子伴侣是重要的PN组分，因为它们调节变性客户端的重新折叠或重折叠， 蛋白质，防止，解决或重塑客户聚集体和目标终端错误折叠或聚集的客户， 降解CT模型假定，随着伴侣对一个“妥协”客户端的关注增加， 导致这些分子伴侣远离它们的其他客户滴定，导致受损的折叠和/或 这些客户的降级。本提案的目标是确定CT的范围和时间， 构象受损的聚集倾向蛋白的积累。这个目标是无法实现的 迄今为止，由于以前缺乏生物化学工具来全面评估受损的伴侣蛋白的多个指标， 维持和缺乏模型，其中单一蛋白质的解折叠可以用蛋白质诱导。 精确测试这个模型所需的敏锐性和同步性。为此，拟议的研究将利用 蛋白质组学工具包，旨在检测与受损伴侣蛋白一致的多个蛋白质指标的变化 维持和一种新的细胞模型，其中单个蛋白质（DD）的解折叠和聚集可以 在细胞群中，在去除 稳定配体。在目标1中，蛋白质组将被筛选以鉴定出蛋白质组中假定的“正常”客户。 通过DD使用这个工具箱滴定掉的伴侣蛋白，该工具箱测量蛋白质泛素化的改变， 总水平、降解率和补充到DD聚集体中。在目标2中，将验证这些变更 使用正交生化分析。在目标3中，将通过实验阐明CT的范围 旨在明确识别被DD滴定的分子伴侣和这些分子伴侣的“正常”客户， 分子伴侣（目的1和2中鉴定的pueromide）受其滴定的影响。这项研究将推动我们的 了解CD的分子发病机制和伴侣-客户网络的相互联系。

Project Summary/Abstract Proteome homeostasis (proteostasis) is essential for cellular life and is maintained by an array of cellular components that regulate the synthesis, folding, solubility and degradation of proteins throughout the proteome. Optimal cellular function is achieved when these proteostasis network (PN) components work collectively to calibrate the levels of correctly folded, and thus functional proteins, to meet cellular demands and prevent the formation of toxic protein aggregates. Many inherited forms of conformational diseases (CD), including many neurodegenerative diseases, are caused by gene mutations that result in non-native conformations (folding) of a single underlying protein, which increases the propensity of these proteins to aggregate. Cellular dysfunction in the context of the overexpression of these conformationally compromised aggregation-prone proteins has been linked to proteostasis impairment (PI). However, the timing and nature of the cellular events that lead to PI in CD remain elusive. A growing, but limited body of evidence suggests that a phenomenon termed chaperone titration (CT) is mechanistically linked to and may initiate PI in CD. Chaperones are essential PN components as they regulate the de novo folding or refolding of denatured client proteins, prevent, resolve or remodel client aggregates and target terminally misfolded or aggregated clients for degradation. The CT model posits that increasing preoccupation of chaperones with one “compromised” client results in the titration of these chaperones away from their other clients leading to impaired folding and/or degradation of these clients. The goal of this proposal is to determine the extent and timing of CT in response to the accumulation of a conformationally compromised aggregation-prone protein. This goal was unattainable until now due to a previous lack of biochemical tools to globally assess multiple metrics of impaired chaperone maintenance and a lack of models in which the unfolding of a single protein could be induced with the acuteness and synchrony required to accurately test this model. To do this, the proposed studies will utilize a proteomics toolkit designed to detect changes in multiple protein metrics consistent with impaired chaperone maintenance and a novel cell model in which the unfolding and aggregation of a single protein (DD) can be induced with unprecedented acuteness and synchrony (within sec to min) in a cell population upon removal of a stabilizing ligand. In Aim 1, the proteome will be screened to identify the putative “normal” clients of the chaperones that are titrated away by DD using this toolkit which measures alterations in protein ubiquitination, total levels, degradation rates and recruitment to DD aggregates. In Aim 2, these alterations will be validated using orthogonal biochemical analyses. In Aim 3, the extent of CT will be elucidated through experiments designed to definitively identify chaperones that are titrated away by DD and the “normal” clients of these chaperones (putatively identified in Aims 1 and 2) affected by their titration. This research will advance our understanding of the molecular pathogenesis of CD and the interconnectedness of chaperone-client networks.