Chemistry and biology of protein histidine phosphorylation

描述（由申请人提供）：将进行一项研究计划，以研究组氨酸磷酸化在真核细胞中的作用。虽然已知蛋白质磷酸化发生在几个氨基酸上，但可以公平地说，磷酸丝氨酸、磷酸苏氨酸和磷酸酪氨酸在文献中引起了大部分注意，至少因为它涉及高等真核生物中的细胞信号传导。这一提议将焦点从这一群体转向磷酸组氨酸（pHis）。组氨酸的磷酸化被认为是原核生物和低等真核生物中信号转导过程的关键。然而，pHis在哺乳动物信号通路中被广泛报道，并与某些人类疾病状态如癌症和炎症有关。尽管如此，仍有许多关于哺乳动物细胞生物学中的修饰的作用和程度有待了解。事实上，无论是在体外还是在体内，研究pHis在信号传导中的功能作用都非常困难，这主要是由于修饰的不稳定性。因此，我们目前长期缺乏研究组氨酸磷酸化的化学和生物化学工具。这正是我们在这个研究项目中试图解决的问题。我们最近合成了新的稳定的pHis模拟物，并使用这些成功地提高了第一个抗体对蛋白质中的这种修饰。这一突破是我们研究组氨酸磷酸化对组蛋白H4的功能作用的起点。人们早就知道组蛋白H4在组氨酸18和75上被磷酸化。这种翻译后修饰（PTM）的确切作用是未知的，也不是安装PTM的激酶。组蛋白H4组氨酸激酶活性与细胞增殖之间存在相关性，并且实际上，这种活性在某些癌细胞中，特别是在人肝癌细胞中显著上调。我们将使用蛋白质工程方法结合一系列的生物化学和生物物理方法来测试的假设，组氨酸磷酸化的H4改变染色质的压实状态，在细胞周期依赖的方式。该提案的具体目标是：（i）开发一套用于染色质生物学领域及其他领域的pHis表位抗体;（ii）表征H4组氨酸激酶并鉴定H4 pHis结合因子;（iii）阐明H4组氨酸磷酸化在染色质中的功能和结构作用。该研究计划旨在为研究细胞信号中的pHis提供急需的研究工具。有了这些在手，我们将采取第一步阐明这PTM在染色质生物学中的作用。我们希望这项工作为理解这种通常被忽视的PTM在表观遗传调控机制中的作用奠定基础，这是该计划的长期目标。

DESCRIPTION (provided by applicant): A research program will be undertaken to study the role of histidine phosphorylation in eukaryotic cells. While protein phosphorylation is known to occur on several amino acids, it is fair to say that phosphoserine, phosphothreonine and phosphotyrosine capture most of the attention in the literature, at least as it pertains to cellular signaling in higher eukaryotes. This proposal turns the spotlight away from this group towards phosphohistidine (pHis). Phosphorylation of histidine is well recognized as being critical to signaling processes in prokaryotes and lower eukaryotes. However, pHis is becoming widely reported in mammalian signaling pathways and implicated in certain human disease states such as cancer and inflammation. Nonetheless, much remains to be understood about the role and extent of the modification in mammalian cell biology. Indeed, studying the functional role of pHis in signaling, either in vitro or in vivo, has proven devilishly hard, largely due to the instability of the modification. As a consequence, we are currently handicapped by a chronic lack of chemical and biochemical tools with which to study histidine phosphorylation. It is this problem that we seek to address in this research program. We have recently synthesized novel stable mimetics of pHis and used these to successfully raise the first antibodies against this modification in proteins. This breakthrough serves as the starting point for the proposed research program in which we will investigate the functional role of histidine phosphorylation on histone H4. It has long been known that histone H4 is phosphorylated on histidines 18 and 75. The precise role of this post-translational modification (PTM) is unknown, nor is the kinase that installs the PTM. A correlation exists between histone H4 histidine kinase activity and cell proliferation and, indeed, this activity is dramatically upregulated in certain cancer cells, specifically, in human hepato-carcinoma cells. We will use protein engineering methods in conjunction with a series of biochemical and biophysical approaches to test the hypothesis that histidine phosphorylation of H4 alters the compaction state of chromatin in a cell cycle dependent manner. The specific aims of this proposal are: (i) To develop a suite of antibodies against pHis epitopes with uses in the area of chromatin biology and beyond; (ii) To characterize the H4 histidine kinase and to identify H4 pHis binding factors, and; (iii) To elucidate the functional and structural role of H4 histidine phosphorylation in chromatin. This research program is designed to provide much needed research tools for studying pHis in cellular signaling. With these in hand, we will take the first steps towards elucidating the role of this PTM in chromatin biology. We expect this work to lay the foundation for understanding the role of this generally overlooked PTM in epigenetic regulatory mechanisms, a long term goal of this program.