The Invisible Phosphoproteome: New Tools to Study Histidine Phosphorylation

 描述（由申请人提供）：在过去的几十年中，通过蛋白磷酸化对蛋白功能的信号转导和调节的研究已经改变了分子和细胞生物学领域，并导致癌症治疗的突破。尽管9/20的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）的磷酸化上;这些羟基氨基酸形成酸稳定的磷酸酯（P-O）键。相反，His、Lys和Arg形成热和酸不稳定的氨基磷酸酯（P-N）键。磷酸特异性抗体（Abs）和磷酸酶抑制剂已经使得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经鉴定了> 200，000个非冗余的磷酸化位点。相比之下，缺乏特定的抗体和磷酸酶抑制剂来研究氨基磷酸酯蛋白磷酸化，以及在用于蛋白质组学的典型条件下P-N键的相对不稳定性，使得不可能确定这类蛋白磷酸化的患病率。目前的生物化学和蛋白质组学技术已经被优化用于磷酸酯氨基酸（pSer、pThr和pTyr）的保存和检测，并且需要类似的技术来研究磷酸化的碱性氨基酸，特别是磷酸组氨酸（pHis），其在很大程度上仍然是不可见的和未被充分认识的。开发特异性抗体和检测pHis的方法将提高科学界对这种翻译后修饰的认识，并提供一种可再生的资源，使蛋白质磷酸化的新位点的发现和功能分析成为可能。这项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点。为了使组氨酸磷酸化作为哺乳动物细胞中的调节过程的研究成为可能，将开发和使用以下工具和技术：1。将使用含有1-pHis和3-pHis的稳定类似物作为抗原的简并肽文库产生序列非依赖性抗1-pHis和3-pHis单克隆抗体。2.使用这些单克隆抗体研究His磷酸化的蛋白质组学技术将被优化，并用于通过质谱和其他方法（包括免疫印迹和免疫荧光）在正常和癌细胞系中调查His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结合结构域，并研究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组氨酸磷酸化水平。5.非天然氨基酸技术将用于在体内特定位点将稳定的pHis模拟物掺入蛋白质中 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（如癌症）中调节过程的重要性。

 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.