蛋白磷酸化和泛素化在ox-LDL致血管内皮损伤中的动态变化和交互调控网络

Ox-LDL is the key factor leading to endothelial cell damage, vascular dysfunctions and vascular remodeling. Many amino acid residues can be oxidized by substances in ox-LDL. These oxidations result in the disruption of many important post-translational modifications, such as phosphorylation and ubiquitination, and cell signaling and function. This is one of the most important mechanisms of endothelial injury induced by oxidative stress. In addition to play regulatory roles individually, just like the "histone code", phosphorylation and ubiquitination can form complex interaction networks in one protein and/or among many proteins. In order to reveal the effects of these networks on the endothelial injury and vascular remodeling induced by ox-LDL, it is necessary to study them dynamically using omic technologies in the viewpoint of integration and systems at multiple time point. However, such studies have not been reported until now. So, based on our previous works, we plan to construct the networks of phosphorylation, ubiquitination and their interactions based on Boolean and Dynamic Bayesian networks in human aortic endothelial cell regulated by ox-LDL using affinity purification based on TiO2 and "antibody cocktail", SILAC quantitative proteomics and bioinformatics tools, such as Cytoscape. Then, the key sub-networks and node proteins/modifications would be found and some significant nodes, such as HDAC1/3/9 and DICER1, would be selected to be studied in function. Furthermore, the mechanisms of effects of key sub-networks and node proteins/modifications on endothelial injury induced by ox-LDL would be revealed. The proposal will help us to understand the interactions of different protein modifications and reveal the mechanisms of endothelial injury induced by ox-LDL resulting in vascular remodeling and the development of severe cardio-cerebrovascular diseases, such as atherosclerosis.

Ox-LDL能干扰很多重要蛋白翻译后修饰酶的功能，使蛋白翻译后修饰异常，这是其导致内皮损伤、血管功能失衡和异常重构的重要机制。蛋白磷酸化和泛素化修饰在这其中不但单独发挥重要作用，还能形成复杂的相互调控网络协同作用。然而，这两种修饰都变化迅速，要准确揭示它们的改变和意义，需要利用组学技术在多时间点系统研究它们的动态变化，但目前这类研究仍未见报道。本申请将在前期工作的基础上，采用TiO2/抗体亲和纯化、SILAC/iTRAQ组合蛋白组定量以及布尔和动态贝叶斯模型, 构建ox-LDL对人主动脉内皮细胞蛋白磷酸化和泛素化及其交互作用的动态调控网络，结合前期结果，寻找其中关键节点蛋白/修饰，重点研究HDAC1/3/9和DICER1，揭示其在ox-LDL致内皮损伤中的意义。这对深入理解蛋白修饰间的相互调控，阐明其在ox-LDL致内皮损伤和血管重构及动脉粥样硬化等心脑血管疾病发生发展中的作用有重要意义

血管内皮损伤是引起血管功能稳态失衡和异常重构的关键始动环节，而血管异常重构则是动脉粥样硬化(AS)、冠心病、高血压和脑卒中等重要心脑血管疾病的病理学基础。由正常低密度脂蛋白氧化而成的氧化低密度脂蛋白(ox-LDL) 是引起血管内皮损伤和功能障碍的关键因素之一。Ox-LDL含有大量脂质过氧化物和反应性醛类物质，具有强氧化性，能改变很多生物大分子的结构，影响多种蛋白翻译后修饰(PTM)。Ox-LDL 能破坏多种蛋白激酶、磷酸酶和泛素连接酶的正常功能，干扰细胞内各种蛋白的磷酸化和泛素化修饰，导致细胞信号传导和调控异常。而且磷酸化和泛素化修饰都变化迅速，需要在多时间点观察它们的动态变化才能准确揭示它们的改变和意义。在本研究中，我们利用亲和纯化及组合定量蛋白组方法，获得了ox-LDL 作用后多时间点血管内皮细胞在组学水平磷酸化和泛素化的表达差异数据；鉴定了多个磷酸化与泛素化修饰蛋白和修饰位点及其在不同时间点的变化特征；并利用WGCNA和动态网络生物标志物（dynamical network biomarkers，DNB）算法，构建了ox-LDL 调控的蛋白磷酸化动态变化网络，发现了TP53BP1及其磷酸化位点等多个在ox-LDL处理后HUVECs从正常到凋亡这一细胞状态改变过程中的关键蛋白及其磷酸化位点，研究了它们在ox-LDL 致内皮细胞损伤中的作用。另外，我们利用这些动态变化数据并结合已有蛋白相互作用数据库，在改良Apriori 算法的基础上，建立了一种新的预测蛋白翻译后修饰位点间相互调控关系的算法（protein/PTM interaction prediction algorithm Based on time-series data，Pipbots）。通过本项目，我们获得了ox-LDL影响内皮细胞磷酸化和泛素化修饰的动态变化数据，为今后类似研究提供了有益的借鉴和参考；同时，对于从时间和空间上深入理解蛋白修饰间复杂的相互调控，揭示ox-LDL 等氧化应激因素致内皮损伤和血管异常重构的机制也有重要意义。

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