JMJD5的组蛋白N端域水解活性在非小细胞肺癌形成和转移中的功能与机制研究

Lung cancer is the cancer with the highest morbidity and mortality in the world. A sudden increase of lung cancer incidence in China over the recent years strongly indicates that in addition to the genetic factors, epigenetic alteration may serve as one of the major mechanisms responsible for lung cancer development. The recently discovered jomojiC domain (JMJD) bearing family belongs to histone demethylase superfamily. Some JMJD family members have been reported for histone N-tail demethylation. Our preliminary data revealed that JMJD5 removes H3 N-tail post-translational modifications via H3 N-tail degradation induction. In lung cancer cells, H3 N-tail post-translational modifications including methylation, acetylation, and phosphoryklation were all elevated, correlating with a reduction of JMJD5 expression. This indicates that JMJD5 may affect H3 N-tail post-translational modifications all in together. H3 N-tail peptide analysis in vitro with mass spectrometry reveals that JMJD clips H3 N-tail at the carboxyl side of monomethyl-lysine residues. Therefore, we propose that JMJD5 is a histone H3 N-tail specific protease, responsible for epigenetic regulation via H3 N-tail specific cleavage. In this project, we intend to apply in vivo and in vitro enzymatic activity assays, mass spectrometry analysis, and jmjd5-/- mouse model to investigate the mechanism by which JMJD5 cleaves histone H3 N-tail in non small lung cancer cell development; uncover the epigenetic regulation by JMJD5 in non small lung cancer cells; and provides potential target for lung cancer prevention and therapy.

肺癌是全球发病率和死亡率最高的恶性肿瘤，其中非小细胞肺癌占80%。我国肺癌发病率在近年内急剧上升，提示除遗传因素外，表观遗传的异常可能是导致肺癌发生的主要机制之一。新发现的JMJD家族是一类组蛋白去甲基化酶,该家族部分成员被报道为组蛋白的N端域去甲基化酶。我们前期实验惊讶地发现，该家族成员JMJD5具有组蛋白N端域水解酶功能，通过组蛋白H3-N端域剪切来调控表观遗传修饰。在非小细胞肺癌细胞中JMJD5的表达下降，伴随着组蛋白H3-N端域不同位点的多种翻译后修饰水平升高，提示JMJD5可能通过影响整个H3-N端域修饰参与非小细胞肺癌的发生发展。本课题拟通过运用肺癌临床样本、高分辨质谱分析及基因敲除小鼠模型等研究JMJD5对H3-N端域剪切修饰的确切机制，JMJD5的剪切功能在非小细胞肺癌发病中的作用和机制，为非小细胞肺癌的诊治提供表观遗传调控的重要机理和线索。

背景：在非小细胞肺癌细胞中，我们发现组蛋白H3表达一个比野生型略短的异构体。随后的研究发现，这个H3异构体是蛋白酶水解造成的。.研究内容、结果、数据：在寻找这个H3蛋白水解酶过程中，我们发现JMJD5虽然带有Jomoj区域，但却是一个地地道道的蛋白水解酶，负责H3N-端的剪切。利用蛋白质谱，我们反复仔细分析了JMJD5对组蛋白H3的水解位点。JMJD5切割H3蛋白的K9-S10肽键， 产生S10末端的H3。其实，Jomoj域具有同源二聚体形成、结合H3、以及蛋白水解功能。蛋白质一维和二维结构分析表明JMJD5和经典蛋白水解酶CathepsinL1 十分相像。而且，CathepsinL1特异抑制物E46可以非常有效地抑制JMJD5对H3的水解作用。这在体内体外实验都得到了验证。我们还发现酵母也表达一个类似大小的H3异构体，而酵母也表达JMJD5，但没有CathepsinL1基因。在许多肺癌细胞或组织，JMJD5表达下降，伴随H3N端水解的异构体出现减少，H3N末端不同位点的多种翻译后修饰水平（K4，K9的乙酰化和甲基化）的升高。提示JMJD5可能通过剪切H3N末端K9-S10肽键，影响表观遗传学、从而参与非小细胞肺癌的发生和发展。在小鼠，JMJD5敲除导致胚胎致死。说明JMJD5对动物胚胎发育的重要性。.科学意义：揭示了Jomoj域的全新蛋白水解酶功能、发现了H3的N-末端水解型翻译后修饰的意义、为非小细胞肺癌的诊治提供表观遗传体调控的靶标。