Chemistry and biology of protein histidine phosphorylation

蛋白质组氨酸磷酸化的化学和生物学

• 批准号: 8413055
• 负责人: Tom Muir
• 金额: $ 30.24万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413055
• 关键词: Address; Amino Acids; Antibodies; Area; Attention; Binding; Biochemical; Biological; Biology; Cell Cycle; Cell Fractionation; Cell Nucleus; Cell Proliferation; Cell Signaling Process; Cells; Cellular biology; Chemicals; Chemistry; Chromatin; Chronic; Cultured Cells; DNA Repository; Dependence; Disabled Persons; Electrostatics; Epigenetic Process; Epitopes; Eukaryota; Eukaryotic Cell; Family; Fiber; Foundations; Goals; Hand; Histidine; Histone H4; Histones; Human; Human Cell Line; Imidazole; In Vitro; Inflammation; Isomerism; Laboratories; Literature; Malignant Epithelial Cell; Malignant Neoplasms; Mammalian Cell; Methods; Modification; Monoclonal Antibodies; N-terminal; Nitrogen; Nucleoside-Diphosphate Kinase; Nucleosomes; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotransferases; Phosphotyrosine; Play; Post-Translational Protein Processing; Precipitation; Prokaryotic Cells; Protein Engineering; Proteins; Reagent; Regulation; Reporting; Research; Role; Science; Sequence Analysis; Series; Signal Pathway; Signal Transduction; Structure; Tail; Testing; Work; analog; arginyllysine; aspartylglutamate; cancer cell; computerized data processing; crosslink; design; handicapping condition; human disease; in vivo; interest; member; mimetics; novel; phosphohistidine; phosphoramidate; programs; protein-histidine kinase; public health relevance; tool

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.

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种氨基酸上，但可以说，磷酸磷酸，磷酸苏氨酸和磷酸酪氨酸在文献中捕获大多数注意力，至少与较高真核生物中的细胞信号有关。该提议使人们的聚光灯从该群体转向磷酸组织（PHIS）。组氨酸的磷酸化被广泛认为是原核生物和较低真核生物的信号传导过程至关重要。但是，PHI在哺乳动物信号通路中广泛报道，并与某些人类疾病状态（如癌症和炎症）有关。尽管如此，关于修饰在哺乳动物细胞生物学中的作用和程度仍然有待理解。实际上，研究PHI在体外或体内信号传导中的功能作用已被证明是艰难的，这主要是由于修饰的不稳定性。结果，我们目前受到慢性缺乏化学和生化工具来研究组氨酸磷酸化的方法。我们在此研究计划中寻求解决这个问题。我们最近合成了新型PHIS的新型稳定模拟物，并使用它们成功地提出了针对蛋白质修饰的首批抗体。这一突破是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。早就知道，组蛋白H4在组氨酸18和75上磷酸化。这种翻译后修饰（PTM）的确切作用尚不清楚，安装PTM的激酶也不是。组蛋白H4组氨酸激酶活性与细胞增殖之间存在相关性，实际上，在某些癌细胞中，特别是在人肝癌细胞中，该活性显着​​上调。我们将使用蛋白质工程方法与一系列生化和生物物理方法结合使用，以测试H4的组氨酸磷酸化以细胞周期依赖性方式改变染色质的压实状态。该提案的具体目的是：（i）开发一套针对Phis表位的抗体，并在染色质生物学及其他地区使用； （ii）表征H4组氨酸激酶并鉴定H4 PHIS结合因子，并且； （iii）阐明H4组氨酸磷酸化在染色质中的功能和结构作用。该研究计划旨在提供急需的研究工具，用于研究细胞信号中的PHI。借助这些，我们将迈出第一步，以阐明该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普遍忽视的PTM在表观遗传调节机制中的作用奠定了基础，这是该计划的长期目标。