The Invisible Phosphoproteome: New Tools to Study Histidine Phosphorylation

看不见的磷酸化蛋白质组：研究组氨酸磷酸化的新工具

• 批准号: 9017975
• 负责人: TONY R. HUNTER
• 金额: $ 44.38万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9017975
• 关键词: Acids; Amino Acids; Antibodies; Antigens; Attention; Awareness; Bacteria; Basic Amino Acids; Binding; Biochemical; Biological Preservation; Cancer cell line; Cell physiology; Cells; Communities; Detection; Development; Diagnostics Research; Disease; Fluorescence; Future; Goals; Health; Heating; Histidine; Immunoblotting; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Methodology; Methods; Molecular; Molecular and Cellular Biology; Monoclonal Antibodies; Normal Cell; Peptide Library; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Prevalence; Process; Protein Phosphatase Inhibitor; Proteins; Proteomics; Regulation; Research; Resources; Role; Serine; Serine/Threonine Phosphorylation; Signal Transduction; Signal Transduction Pathway; Site; Surveys; System; Techniques; Technology; Therapeutic; Threonine; Tyrosine; Tyrosine Phosphorylation; analog; arginyllysine; base; carcinogenesis; clinical application; human disease; in vivo; inorganic phosphate; interest; mimetics; novel; phosphatase inhibitor; phospho-L-arginine; phosphohistidine; phosphoramidate; protein function; therapeutic target; tool; unnatural amino acids

 DESCRIPTION (provided by applicant): Over the past several decades research into signal transduction and regulation of protein function by protein phosphorylation has transformed the fields of molecular and cellular biology and led to breakthroughs in cancer therapeutics. Although 9/20 amino acids can be phosphorylated including histidine (His), arginine (Arg) and lysine (Lys), the majority of attention has been focused on phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr); these hydroxyamino acids form acid-stable, phosphoester (P-O) bonds. In contrast, His, Lys, and Arg form heat- and acid-labile phosphoramidate (P-N) bonds. Phosphospecific antibodies (Abs) and phosphatase inhibitors have enabled the routine study of phosphoester protein phosphorylation, and the use of MS-proteomics has identified >200,000 non-redundant sites of phosphorylation. In contrast, the lack of specific Abs and phosphatase inhibitors to study phosphoramidate protein phosphorylation and the relative instability of the P-N bond under typical conditions used for proteomics have made it impossible to determine the prevalence of this class of protein phosphorylation. Current biochemical and proteomic technologies have been optimized for preservation and detection of phosphoester amino acids (pSer, pThr and pTyr), and there is a need for analogous technology to study phosphorylated basic amino acids, and in particular phosphohistidine (pHis), which have remained largely invisible and underappreciated. The development of specific Abs and methods for detection of pHis will increase awareness of this posttranslational modification in the scientific community, and provide a renewable resource that will allow discovery and functional analysis of novel sites of protein phosphorylation. This technology has the potential to uncover new signal transduction pathways and identify therapeutic targets for human diseases. To enable the study of histidine phosphorylation as a regulatory process in mammalian cells, the following tools and technologies will be developed and employed: 1. Sequence-independent anti-1-pHis and 3-pHis monoclonal antibodies will be generated using degenerate peptide libraries containing stable analogues of 1-pHis and 3-pHis as antigens. 2. Proteomic techniques for the study of His phosphorylation using these MAbs will be optimized and used to survey His phosphorylation by mass spectrometry and other methods, including immunoblotting and immuno- fluorescence, in normal and cancer cell lines, to understand its role in normal cell physiology and in cancer. 3. pHis-binding domains will be identified, and their role in histidine phosphorylation signaling will be studied. 4. pHis protein phosphatase inhibitors will be developed to elevate histidine phosphorylation levels in cells. 5. Unnatural amino acid technology will be used to incorporate stable pHis mimetics into proteins at specific sites in vivo to study the consequences of histidine phosphorylation. The overarching goal is to develop tools needed to study histidine phosphorylation in mammalian cells and begin to determine its importance as a regulatory process in normal cells and in diseases, such as cancer.

 描述（由申请人提供）：尽管 9/20 的氨基酸可以被磷酸化，但在过去的几十年中，对信号转导和通过蛋白质磷酸化调节蛋白质功能的研究已经改变了分子和细胞生物学领域，并导致了癌症治疗的突破。包括组氨酸（His）、精氨酸（Arg）和赖氨酸（Lys），大多数注意力集中在丝氨酸（Ser）、苏氨酸的磷酸化上(Thr) 和酪氨酸 (Tyr)；这些羟基氨基酸形成酸稳定的磷酸酯 (P-O) 键，相反，His、Lys 和 Arg 形成热和酸不稳定的氨基磷酸酯 (P-N) 键。 ）和磷酸酶抑制剂使磷酸酯蛋白磷酸化的常规研究成为可能，并且 MS 蛋白质组学的使用已确定相比之下，由于缺乏用于研究氨基磷酸酯蛋白磷酸化的特异性抗体和磷酸酶抑制剂，以及蛋白质组学典型条件下 P-N 键的相对不稳定性，因此无法确定此类的普遍性。目前的生化和蛋白质组学技术已针对磷酸酯氨基酸（pSer、pThr 和 pThr）的保存和检测进行了优化。 pTyr），并且需要类似的技术来研究磷酸化碱性氨基酸，特别是磷酸组氨酸（pHis），其在很大程度上仍然不可见且未被充分重视。特定抗体和检测 pHis 的方法的开发将提高对此的认识。该技术有潜力发现新的信号转导途径并确定人类疾病的治疗靶点。为了能够研究组氨酸磷酸化作为哺乳动物细胞中的调节过程，将开发和采用以下工具和技术： 1. 将使用包含1-pHis 和 3-pHis 的稳定类似物作为抗原 2. 使用这些 MAb 研究 His 磷酸化的蛋白质组学技术将被优化并用于调查 His。通过质谱和其他方法（包括免疫印迹和免疫荧光）在正常细胞系和癌细胞系中进行磷酸化，以了解其在正常细胞生理学和癌症中的作用。 3. 将鉴定 pHis 结合域，及其在组氨酸磷酸化信号传导中的作用。 4. 将开发pHis 蛋白磷酸酶抑制剂以提高细胞中的组氨酸磷酸化水平。 5. 将使用非天然氨基酸技术来整合稳定的pHis。在体内特定位点模拟蛋白质 研究组氨酸磷酸化的后果，总体目标是开发研究哺乳动物细胞中组氨酸磷酸化所需的工具，并开始确定其作为正常细胞和疾病（例如癌症）中的调节过程的重要性。