The Invisible Phosphoproteome: New Tools to Study Histidine Phosphorylation

 描述(申请人提供)：在过去的几十年里，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，并导致了癌症治疗方面的突破。虽然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形成耐热和耐酸的磷酰胺(P-N)键。磷酸特异性抗体(Abs)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，MS蛋白质组学的使用已经确定了&gt；20万个非冗余的磷酸化位点。相比之下，缺乏专门的抗体和磷酸酶抑制剂来研究磷酰化蛋白的磷酸化，以及在蛋白质组学的典型条件下P-N键的相对不稳定，使得无法确定这类蛋白的磷酸化的流行程度。目前的生化和蛋白质组学技术已经在保存和检测磷酸酯氨基酸(pSer、pThr和pTyr)方面进行了优化，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)，它们在很大程度上仍然是不可见的和被低估的。特异性抗体和检测PHIs的方法的发展将提高科学界对这种翻译后修饰的认识，并提供一种可再生资源，使发现和功能分析新的蛋白质磷酸化位点成为可能。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。为了能够研究哺乳动物细胞中组氨酸磷酸化的调控过程，将开发和使用以下工具和技术：1.利用含有稳定的1-PHIs和3-PHIs类似物的简并肽库作为抗原，制备序列无关的抗1-PHIs和3-PHIs单抗。2.优化利用这些单抗研究His磷酸化的蛋白质组学技术，并利用质谱仪和其他方法，包括免疫印迹和免疫荧光，观察正常细胞和癌细胞中His的磷酸化，以了解其在正常细胞生理和癌症中的作用。3.确定PHI结合结构域，并研究其在组氨酸磷酸化信号转导中的作用。4.将开发PHIS蛋白磷酸酶抑制剂，以提高细胞内组氨酸的磷酸化水平。5.非天然氨基酸技术将在体内特定位置将稳定的PHIs模拟物整合到蛋白质中 研究组氨酸磷酸化的后果。首要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，并开始确定其在正常细胞和癌症等疾病中作为调节过程的重要性。

 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.