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: 9228357
• 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/9228357
• 关键词: Acids; Amino Acids; Antibodies; Antigens; Arginine; Attention; Awareness; Bacteria; Basic Amino Acids; Binding; Biochemical; Biological Preservation; Cancer cell line; Cell physiology; Cells; Communities; Detection; Development; Diagnostic; Disease; Future; Goals; Histidine; Immunoblotting; Immunofluorescence Immunologic; Lysine; 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; Signal Transduction; Signal Transduction Pathway; Site; Surveys; System; Techniques; Technology; Therapeutic; Threonine; Tyrosine; Tyrosine Phosphorylation; analog; antibody inhibitor; arginyllysine; base; carcinogenesis; clinical application; human disease; in vivo; inorganic phosphate; interest; mimetics; novel; phosphatase inhibitor; phospho-L-arginine; phosphohistidine; phosphoramidate; protein function; public health relevance; therapeutic target; tool; unnatural amino acids; virtual

 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（由应用提供）：在过去的几十年中，蛋白质磷酸化对蛋白质功能的信号翻译和调节的研究已经改变了分子和细胞生物学的领域，并导致了癌症治疗的突破。尽管9/20氨基酸可以被磷酸化，包括组氨酸（HIS），精氨酸（ARG）和赖氨酸（LYS），但大多数注意力集中在丝氨酸（SER），苏氨酸（THR）和酪氨酸（Tyr）（Tyr）的磷酸化上。这些羟基氨基酸形成酸稳定的磷酸酯（P-O）键。相比之下，他的，LYS和ARG形成热和酸性磷光酰胺（P-N）键。磷酸蛋白蛋白磷酸化的常规研究已经确定了磷酸蛋白磷酸化的常规研究，并且使用MS-蛋白质学已经确定了磷酸化> 200,000个非冗余位点。相反，在用于蛋白质磷酸化的典型条件下，缺乏研究磷酰胺蛋白磷酸化的特异性ABS和磷酸酶抑制剂。当前的生化和蛋白质组学技术已被优化，用于保存和检测磷酸氨基酸（PSER，PTHR和PTYR），并且需要类似的技术来研究磷酸化的碱性氨基酸，尤其是磷酸化氨基酸（PHIS）（PHIS），这些氨基酸（PHIS）均保持了很大的印象和不足。特定的ABS的开发和检测PHI的方法将提高科学界对这种翻译后修饰的认识，并提供可再生资源，以发现和对蛋白质磷酸化新颖位点的发现和功能分析。该技术有可能发现新的信号转导途径并确定人类疾病的治疗靶标。为了使组氨酸磷酸化作为哺乳动物细胞中的调节过程，将开发和使用以下工具和技术：1。使用含有稳定类型的稳定类似物的稳定类似物和3-Phiss ant ant ant ant ans 3- pphis as astig as ans As ans 3-phisigigigies，将生成不序列无关的抗1 PHIS和3-PHIS单克隆抗体。 2.使用这些mAB进行磷酸化研究的蛋白质组学技术将得到优化，并用于通过质谱和其他方法来调查其磷酸化，包括在正常和癌细胞系中的免疫印迹和免疫荧光，以了解其在正常细胞生理学和癌症中的作用。 3。将确定PHIS结合结构域，并将研究它们在组氨酸磷酸化信号中的作用。 4。将开发Phis蛋白磷酸酶抑制剂以升高细胞中组氨酸的磷酸化水平。 5。非天然氨基酸技术将用于将稳定的Phis Mimetics纳入体内特定部位的蛋白质 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）的调节过程的重要性。