EZH2的巴豆酰化修饰在调控肺腺癌进展中的作用及机制研究

Post-translational modifications of EZH2 can affect its methyltransferase activity, and is essential for cell differentiation and tumor metastasis regulated by EZH2. However, there were no reports about whether EZH2 can be modified by crotonylation, and its function is unknown. In this study, using proteomics of post-translational modifications, for the first time we identified a large number of crotonylated proteins in human lung adenocarcinoma H1299 cells, which including EZH2. What is more, we found that acetyltransferase CBP can function as crotonyltransferase of EZH2. Accordingly, we propose that non-histones can also be modified by crotonylation, and crotonylation as a new post-translational modification type of EZH2, plays an important role in EZH2 promoting lung adenocarcinoma progression. This study will firstly verify EZH2 crotonylation and its modification site, then identify the structural basis of interaction between CBP and EZH2, finally investigative the functional effect of crotonylated EZH2 in regulating lung adenocarcinoma progression and its potential mechanism, from the molecular, cellular, animal and patient tissue level. We hope that this study will expand new mechanisms of EZH2 in regulating lung adenocarcinoma progression, and provide new ideas for the diagnosis, treatment and prognosis of lung adenocarcinoma.

EZH2蛋白翻译后修饰可以影响它的甲基转移酶活性，对于其调控细胞分化和肿瘤转移十分重要。但至今还没有EZH2存在巴豆酰化修饰的报道，该修饰能否影响EZH2的功能也是未知的。我们前期利用蛋白修饰组学的方法，在肺腺癌细胞系H1299中发现了大量非组蛋白的巴豆酰化修饰，这其中包括EZH2，并且我们证明乙酰化酶CBP也可以作为巴豆酰化酶催化EZH2发生巴豆酰化修饰。据此我们提出假设：非组蛋白同样可以存在巴豆酰化修饰，并且巴豆酰化作为EZH2的新型蛋白翻译后修饰，在EZH2促肺腺癌进展中发挥重要作用。本课题拟从分子、细胞、动物和病人组织标本等多方面验证EZH2的巴豆酰化修饰及其修饰位点，明确CBP催化EZH2的巴豆酰化修饰及二者相互作用的结构基础，深入探讨巴豆酰化修饰对EZH2促肺腺癌进展的影响及其潜在机制。希望通过该研究扩展EZH2调控肺腺癌进展的新机制，为肺腺癌的诊断、治疗和预后提供新思路。

本课题组在前期利用蛋白修饰组学方法筛选鉴定巴豆酰化修饰蛋白的基础上，进一步证实EZH2可以发生巴豆酰化修饰，并明确K735是其巴豆酰化修饰的氨基酸位点。接着我们筛选并证明CBP可以催化EZH2发生巴豆酰化修饰。并且，利用免疫荧光和免疫共沉淀实验，我们明确CBP和EZH2可以存在蛋白之间的相互作用。利用点突变技术，我们构建了EZH2巴豆酰化修饰不同突变体质粒。同时，我们利用细胞增殖和迁移实验阐明了EZH2巴豆酰化修饰可以调控肺腺癌细胞增殖、迁移和侵袭能力。通过对不同突变体细胞进行RNA测序分析，我们发现了自噬信号通路的多个基因表达发生变化。敲低自噬基因，EZH2巴豆酰化修饰调控细胞增殖迁移能力减弱，从而明确了自噬信号通路在EZH2巴豆酰化修饰调控肺癌功能中发挥重要作用。同时，我们初步发现EZH2巴豆酰化并不影响其磷酸化和乙酰化修饰水平。该研究初步阐明EZH2发生巴豆酰化修饰的分子机制，并明确了其调控肺癌进展的功能。后续研究我们将着重探讨EZH2巴豆酰化修饰如何调控自噬信号通路，进一步阐明其调控肺癌发生发展的分子机制。