鉴定组蛋白赖氨酸琥珀酰化修饰结合蛋白及其在脑胶质瘤中的功能

The process of histone modification and its subsequent function are mainly conducted by three categories of proteins including writer, eraser, and reader proteins. Histone lysine succinylation (Ksucc) is critical for tumor development. However, reader proteins of this modification, which play instrumental roles for gene expression, remains unidentified. Our recent study published in Nature revealed for the first time that the nuclear α-ketoglutarate dehydrogenase (α-KGDH) complex is associated with KAT2A and that KAT2A functions as a succinyltransferase to catalyze α-KGDH–generated succinyl-CoA for succinylation of histone H3 at lysine 79 (H3K79). In this study, we will synthesize histone peptides bearing Ksucc and identify candidate reader proteins with high-throughput protein expression and purification technology. In addition, we will perform ChIP-seq and RNA-seq analysis and conduct cell culture-based and animal studies to reveal the cellular functions of the reader proteins in tumor cell proliferation and tumor development. Our studies, which aim to identify and characterize the function of histone Ksucc readers, will potentially provide a novel strategy to treat human cancer.

组蛋白修饰的发生及随后功能的发挥主要由三类蛋白组成：修饰酶、去修饰酶和结合蛋白。组蛋白琥珀酰化修饰对肿瘤的进展及基因表达调控至关重要，然而其结合蛋白至今还未被鉴定。最近我们发表在Nature的研究首次发现细胞核中的α-酮戊二酸脱氢酶复合物与KAT2A结合，KAT2A作为一个琥珀酰基转移酶以α-酮戊二酸脱氢酶合成的琥珀酰辅酶A为原料，催化组蛋白H3第79位赖氨酸发生琥珀酰化。本课题将首先从合成组蛋白赖氨酸琥珀酰化肽着手，通过高通量蛋白表达和纯化技术筛选出结合蛋白；接着通过ChIP及RNA测序、基于细胞培养及动物模型寻找结合蛋白在基因表达调控和肿瘤增殖及进程方面的功能。通过研究，鉴定出琥珀酰化修饰结合蛋白并寻找新的生物学功能，为肿瘤的临床治疗提供新策略。

琥珀酰化修饰是近几年发现的一种新型蛋白质翻译后修饰，其与肿瘤代谢调控息息相关。已有研究表明KAT2A是组蛋白H3的琥珀酰基转移酶，而对于组蛋白的去琥珀酰化酶目前还没有报道。本项目试图鉴定出琥珀酰化修饰结合蛋白并阐明其潜在的生物学功能。通过合成生物素化组蛋白H4 N端21个氨基酸的琥珀酰化修饰多肽，结合质谱寻找组蛋白琥珀酰化修饰结合蛋白，我们筛选到了琥珀酸辅酶A合成酶复合物。在确定琥珀酸辅酶A合成酶复合物在体外是否能够去琥珀酰化组蛋白H3 K79位琥珀酰化修饰实验中，由于在体外反应过程中可能会导致琥珀酰化修饰基团脱落，我们重复多次都很难检测到组蛋白H3的琥珀酰化修饰。对此，我们对本项目研究内容进行了部分微调，将研究精力集中在寻找关键代谢酶的琥珀酰化修饰及其功能。我们发现了磷酸果糖激酶2（PFK2）能发生琥珀酰化修饰，通过质谱及位点突变我们鉴定了PFK2的三个琥珀酰化位点，且发现SIRT5能结合并去琥珀酰化PFK2。针对PFK2特点位点琥珀酰化及其动态调控机制在肿瘤的恶性进程中的功能我们还在继续开展深入的研究。通过本项目持续深入的研究，将为肿瘤的靶向药物开发提供新的靶点。