Biochemistry of lysine crotonylation pathway

描述(申请人提供)：翻译后修饰(PTM)可以显著调节蛋白质的结构和功能，并已知调节多种细胞途径。PTM通路的失调已被证明会导致多种人类疾病，如癌症、糖尿病和神经退行性疾病。组蛋白中的PTM，如赖氨酸乙酰化(KAc)和赖氨酸甲基化(KME)，是主要的表观遗传标记，有助于表观遗传程序决定不同的DNA模板生物输出和疾病。除了磷酸化以外，调节组蛋白PTM的酶(如KAc、KME和赖氨酸泛素化)已成为治疗疾病的重要药物靶点。我们最近报告了一种新的、进化上保守的赖氨酸修饰，赖氨酸 巴豆化(KCR)。我们发现KCR在核心组蛋白中含量丰富，并在哺乳动物细胞中确定了28个组蛋白KCR位点。在减数分裂后的雄性生殖细胞中，组蛋白KCR，而不是组蛋白KAc，富含在性染色体上，并专门标记睾丸特异基因。组蛋白Kcr的独特结构和基因组定位表明，它与组蛋白Kac具有不同的动态和功能，组蛋白Kac是一种具有不同功能的PTM。尽管有这些初步研究，但九广铁路途径中的主要参与者仍然未知。我们假设，KCR途径有一套独特的调节酶、底物和直接结合剂，这决定了它与KAc的不同。因此，我们建议使用酶学、化学生物学和蛋白质组学的方法来表征KCR途径。由于我们的专业知识和我们在过去几年中开展的大量相关研究，我们的团队能够很好地开展这一项目。在这个项目中，我们将首先使用各种化学生物学和酶学方法来鉴定和表征KCR调节酶、巴豆酰转移酶和去氯化酶。然后，我们将鉴定KCR的底物，并通过定量蛋白质组学方法研究它们对KCR调节酶的反应以及在精子发生过程中的动态。最后，我们将鉴定和确认组蛋白KCR多肽的直接结合。组蛋白KCR的新的酶和结合剂可能会为组蛋白生物学及其在疾病中的作用开辟完全意想不到的丰硕途径。因此，这项拟议的研究应该会通过揭示新的表观遗传机制来加速当前染色质生物学的研究，可能类似于发现组蛋白赖氨酸乙酰化和组蛋白赖氨酸甲基化的KAc和Kme调节酶。

DESCRIPTION (provided by applicant): Post-translational modifications (PTMs) can significantly modulate the structure and function of proteins and are known to regulate a wide variety of cellular pathways. Dysregulation of PTM pathways have been shown to lead to diverse human diseases, such as cancer, diabetics, and neurodegenerative diseases. PTMs in histones, such as lysine acetylation (Kac) and lysine methylation (Kme), are major epigenetic marks, contributing to the epigenetic program that dictates diverse DNA-templated biological outputs and diseases. Enzymes that regulate histone PTMs other than phosphorylation (such as Kac, Kme, and lysine ubiquitination) have emerged as important drug targets for diseases. We recently reported the identification of a new, evolutionarily-conserved lysine modification, lysine crotonylation (Kcr). We showed that Kcr is abundant in core histones and identified 28 histone Kcr sites in mammalian cells. In male germinal cells immediately following meiosis, histone Kcr, but not histone Kac, is enriched on sex chromosomes and specifically marks testis-specific genes. The unique structure and genomic localization of histone Kcr suggest that it is dynamic and functionally different from histone Kac, a previously-described PTM with diverse functions. Despite of these preliminary studies, major players in the Kcr pathway remain unknown. We hypothesize that Kcr pathway has a unique set of regulatory enzymes, substrates and direct binders, which determines its difference from Kac. We therefore propose to use enzymology, chemical biology, and proteomics approaches to characterize Kcr pathway. Our team is well positioned to carry out this project because of our expertise and the tremendous relevant studies we have carried out in the past few years. In this project, we will first identify and characterize Kcr-regulatory enzymes, crotonyltransferase and decrotonylases, using a variety of chemical biology and enzymology approaches. We will then identify substrates of Kcr and study their dynamics in response to Kcr- regulatory enzymes and during spermatogenesis, by a quantitative proteomics approach. Finally, we will identify and confirm the direct binders for histone Kcr peptides. The new enzymes and binders for histone Kcr are likely to open entirely unforeseen fruitful avenues for histone biology and their roles in disease. Thus, the proposed study should accelerate current research in chromatin biology by revealing novel epigenetic mechanisms, perhaps analogous to the discovery of Kac- and Kme-regulatory enzymes for histone lysine acetylation and histone lysine methylation.