新型荧光探针应用于巴豆酰化修饰调控酶和阅读器的研究

Proteins are important components of living organisms. Through posttranslational modification, the structures and functions of proteins can be modulated in a precise manner, thereby affecting the cell signaling pathways and determining the fate of the cell. Lysine modification is one of the most prevalent posttranslational modifications, and it plays many important roles. Lysine acetylation, for example, is closely linked to the regulation of many vital cellular functions, including chromatin remodeling, DNA damage and cell-cycle control. In 2011, lysine crotonylation was discovered. Biological studies showed that lysine crotonylation is associated with gene transcription and related to cancer. However, its detailed biological mechanism remains unclear. In this project, we seek to develop new chemical tools for investigating enzymes and proteins related to lysine crotonylation. We will first develop fluorescent probes to detect the enzymatic activity of decrotonylase. Subsequently we will develop isoform-selective fluorescent probes for a given decrotonylase by employing the microarray approach. Finally, we will introduce photocrosslinker to the probe to develop dual-function probes. Through fluorimeter method and proteomic approach, we will identify new enzymes and proteins that can interact with the crotonylated probes. The newly identified enzymes and proteins can yield novel therapeutic targets. The proposed research will help to provide valuable insight into the biological roles of crotonylation and further advance the field of epigenetic research.

蛋白质是生命体系的重要组成成分，蛋白翻译后修饰通过特定氨基酸的共价修饰，对蛋白质的结构与功能进行精细调节，从而影响信号通路并决定细胞命运。赖氨酸的修饰因其独特的反应活性，在细胞中最为丰富广泛，并发挥重要的生物功能，例如赖氨酸的乙酰化与染色质重组及细胞周期调控密切相关。2011年，赖氨酸的巴豆酰化修饰被首次鉴定，此后研究显示巴豆酰化可激活基因转录，与癌症相关，但其具体生物机制并不清楚。在本项目中，我们将发展新型化学工具研究巴豆酰化修饰相关的酶与蛋白。我们将重点研究组蛋白巴豆酰化修饰的消除及识别机制，通过发展新型多肽荧光探针，我们将系统地研究不同去巴豆酰化酶的活性，在此基础上，我们还将引入光交联基团发展双功能荧光探针。通过结合荧光手段和化学蛋白质组学，我们有望发现新的去巴豆酰化酶或读者蛋白，这些酶和蛋白可能成为新的药物靶点。以上研究将有助于阐述巴豆酰化修饰的生物学功能，并推动表观遗传学的发展。

本课题完成情况良好，我们利用分子内0-NBD和N-NBD交换反应，建立了可用于去巴豆酰化酶活性检测和抑制剂筛选的荧光探针平台。同时，我们应用多肽芯片技术，研究巴豆酰化修饰多肽与Bromodomain的相互作用，发现一些Bromodomain能够结合巴豆酰化修饰多肽，初步晶体结构解析证明Bromodomain与巴豆酰化修饰多肽的结合机理基于π-π堆积作用。我们亦合成了赖氨酸硫辛酸化荧光探针，发现了Sirt2去硫辛酸化修饰的新功能及在细胞代谢中的生物学意义。此外我们设计了多肽探针用于检测Sirt5的去琥珀酰化活性，我们发现Sirt5催化引起的多肽自组装能够显著增强三种不同抗癌药物杀伤肿瘤细胞的能力，这种方法或为癌症治疗提供了新途径，通过降低药物剂量从而降低化疗过程中的副作用。我们期待以上成果将有助于进一步研究不同赖氨酸去修饰酶的生物功能，并促进生物标志物的鉴定和药物发现。