Chemistry and biology of protein histidine phosphorylation

描述(由申请人提供)：将开展一项研究计划，以研究组氨酸磷酸化在真核细胞中的作用。虽然已知的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占据了大部分的注意力，至少在与高等真核生物的细胞信号有关的方面。这项提议将聚光灯从这一组转移到磷酸组氨酸(PHIs)上。组氨酸的磷酸化在原核生物和低等真核生物的信号传递过程中起着至关重要的作用。然而，PHI在哺乳动物的信号通路中得到了广泛的报道，并与某些人类疾病状态有关，如癌症和炎症。尽管如此，关于这种修饰在哺乳动物细胞生物学中的作用和程度仍有许多需要了解的地方。事实上，研究PHI在信号传递中的功能作用，无论是在体外还是在体内，都被证明是极其困难的，这主要是由于修饰的不稳定性。因此，我们目前受制于长期缺乏研究组氨酸磷酸化的化学和生化工具。这就是我们在这个研究项目中寻求解决的问题。我们最近合成了新的稳定的PHIs模拟物，并利用这些模拟物成功地在蛋白质中产生了第一批针对这种修饰的抗体。这一突破作为拟议研究计划的起点，在该计划中，我们将研究组氨酸磷酸化在组蛋白H4上的功能作用。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)的确切作用尚不清楚，安装PTM的激酶也不清楚。组蛋白H4组氨酸激酶活性和细胞增殖之间存在相关性，确实，这种活性在某些癌细胞中显著上调，特别是在人类肝癌细胞中。我们将使用蛋白质工程方法结合一系列生化和生物物理方法来验证H4的组氨酸磷酸化以细胞周期依赖的方式改变染色质紧致状态的假设。这项建议的具体目的是：(I)开发一套用于染色质生物学领域和其他领域的针对PHIs表位的抗体；(Ii)表征H4组氨酸激酶并鉴定H4PHIs结合因子；以及(Iii)阐明H4组氨酸磷酸化在染色质中的功能和结构作用。这项研究计划旨在为研究细胞信号中的PHI提供急需的研究工具。有了这些，我们将采取第一步来阐明这种PTM在染色质生物学中的作用。我们期望这项工作为理解这种普遍被忽视的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.