发展化学蛋白组学方法以研究组蛋白精氨酸甲基化

In the nuclei of all eukaryotic cells, genomic DNA is highly compacted into chromatin by wrapping around ‘spools’, which are comprised of histone proteins. Diverse posttranslational modifications (e.g. acetylation, methylation and phosphorylation) of histones are known to regulate DNA-templated processes, such as gene transcription, DNA replication and DNA damage repair. Histone modifications can serve as a signaling platform that would be recognized (or ‘read’) by specific binding proteins, which would then, in turn, exert effects on chromatin structure and function. Emerging evidence suggests that improper ‘reading’ of histone modifications contributes to a variety of human diseases such as cancer and developmental abnormalities. Proteins involved in recognizing histone modifications have therefore become potential drug targets. .While various modifications have been detected at more than 70 different sites of histones, the progress on finding proteins that recognize these modifications have largely lagged behind. As a result, it remains poorly understood how histone modifications are recognized and translated into meaningful biological processes. This is, in large part, due to the lack of a robust method to comprehensively identify ‘reader’ proteins that specifically recognize histone modifications. To fill this knowledge gap, the proposed research, which combines a photo-cross-linking strategy with state-of-the-art mass spectrometry, aims at developing a quantitative chemical proteomics approach to identify ‘readers’ of histone modifications and examining the structural and mechanistic basis of these posttranslational modification-mediated protein-protein interactions. In the long term, our research, at the interface of chemistry and biology, will improve our understanding on human diseases (e.g. cancer) associated with errors in ‘reading’ histone modifications and may lead to the discovery of new drug targets.

组蛋白的翻译后修饰参与调节了诸如基因转录、DNA复制以及受损修复等一系列重要的生物过程。组蛋白修饰是通过生物酶在组蛋白表面特定的氨基酸侧链上面进行化学修饰来完成的。同时，细胞内也存在着一些特有的蛋白。它们可以识别组蛋白表面的特定化学修饰，并将这些修饰“解读”并“翻译”成调节细胞正常生理功能信号。正因为如此，对组蛋白修饰的错误“解读”往往可能导致严重的人类疾病，例如：癌症和发育畸形。因此，找到那些能够识别组蛋白修饰的蛋白分子，并全面理解它们“解读”组蛋白密码的细胞调控机制，不仅对基础科学研究也对人类疾病治疗有着深远的作用。然而，到目前为止，仍然缺乏一种有效并可靠的方法，能在像细胞这样拥有大量的、种类繁多的蛋白质的复杂体系中，选择性地找出并鉴定那些能够识别特定组蛋白修饰的蛋白分子。因此本项目旨在发展一种以化学光交联与定量蛋白组学相结合方法来系统全面的寻找并鉴定组蛋白修饰的识别蛋白。

组蛋白翻译后修饰在诸如DNA复制，基因转录以及DNA损伤修复等细胞活动中扮演者至关重要的角色，组蛋白修饰的正常功能受到其“书写器”，“擦除器”和“阅读器”的紧密调节。组蛋白的精氨酸甲基化是一种非常普遍的组蛋白修饰，其调节包括mRNA剪接、翻译和细胞信号传导在内的一系列表观遗传学进程。近年来对精氨酸甲基化的“书写器”和“擦除器”研究颇多，而对其“阅读器”的了解还比较有限，这也限制了对其生物学功能的进一步探究。.在本项目中，我们构建了带有以下三种功能基团的多肽探针：（1）甲基化精氨酸，以捕获 “阅读器”；（2）光交联基团，以固定弱或者瞬时的相互作用；（3）端基炔基团，以富集捕获的蛋白质用于质谱鉴定。结合基于同位素标记的定量蛋白质组学，我们在全蛋白质组水平上对两种组蛋白精氨酸甲基化的“阅读器”进行了鉴定。.我们成功合成了两种芴甲氧羰基保护的含有光交联基团双吖丙啶的氨基酸，光亮氨酸和光甲硫氨酸。这两种氨基酸可以用来合成所需的多肽探针。我们还成功开发了多种具有不同程度精氨酸甲基化及不用光交联基团的多肽探针及相应的对照探针用以鉴定精氨酸甲基化的“阅读器”。这些探针不仅可以用于本课题，也可以作为公共科研资源提供给其他研究人员用来研究组蛋白精氨酸甲基化。同时，这种化学探针的设计原则及实验方法也可以被借鉴并用于研究其他组蛋白转录后修饰。利用开发的探针我们成功鉴定出了两个潜在的H3R2me1的“阅读器”蛋白CUL4A/B和NRDC，并利用大肠杆菌表达系统成功表达纯化了这两种蛋白。这是第一次在全蛋白质组水平上对特异性识别H3R2me1的蛋白进行鉴定，虽然后续的实验发现，CUL4A/B和NRDC在单蛋白水平上并不能特异性地的识别H3R2me1，但我们推测这是因为两个蛋白质在细胞内是通过形成蛋白质复合体来实现对H3R2me1的特异性识别。接下来我们会继续对这两种蛋白和H3R2me1之间的相互作用进行探究。我们还鉴定出一个潜在的H3R8me2a识别蛋白SPIN3并会继续对其进行探究。