Histidine phosphorylation as a new target for cancer therapy

组氨酸磷酸化作为癌症治疗的新靶点

• 批准号: 10680390
• 负责人: TONY R. HUNTER
• 金额: $ 113.13万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-18 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680390
• 关键词: Acids; Affinity; Amino Acids; Antibodies; Bacteria; Binding; Cell Line; Cell physiology; Cells; Childhood; Comparative Study; Enzymes; Fab Immunoglobulins; Histidine; Histone H4; Human; Immunofluorescence Immunologic; Individual; Malignant Neoplasms; Malignant neoplasm of pancreas; Mammals; Methods; Molecular; Monoclonal Antibodies; Mus; NME1 gene; Neuroblastoma; Normal Cell; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Post-Translational Protein Processing; Potassium Channel; Primary carcinoma of the liver cells; Protein Isoforms; Proteins; Protocols documentation; Reagent; Recording of previous events; Role; Series; Serine; Signal Transduction; Site; Site-Directed Mutagenesis; Stains; Structure; Surface; System; Threonine; Tumor Cell Line; Tumor Suppressor Proteins; Tumor Tissue; Tyrosine; Western Blotting; detection of nutrient; experimental study; human disease; improved; inorganic phosphate; novel therapeutic intervention; phosphohistidine; receptor; targeted cancer therapy; therapeutic target; tool; tumor

Project Summary/Abstract Phosphorylation of histidine (His) in proteins has a 60-year history, initially identified as a P-enzyme intermediate, but subsequently as a regulatory mechanism in bacteria essential for signal transduction by surface receptors that sense nutrients. Such signaling systems are lacking in mammals, but phosphohistidine (pHis) is not only a key P-enzyme intermediate in mammalian enzymes (e.g. NME1/2, ACLY), but also occurs as a reversible end-state protein modification, e.g. pHis18 in histone H4. His phosphorylation is labile to acid and heat making it challenging to study, and to circumvent this our group developed a series of monoclonal antibodies (mAbs) that recognize the 1-pHis or 3-pHis isoforms in a sequence-independent manner. These mAbs were used to detect pHis in cells by immunoblotting and immunofluorescence (IF) staining, and for affinity enrichment of pHis proteins for MS analysis, revealing ~700 potential pHis proteins and implying the existence of large “hidden” phosphoproteome not detectable by conventional methods. In a collaborative study, these mAbs were used to demonstrate site and isoform specific His phosphorylation of the KCa3.1 K+ channel and deduce how pHis triggers channel opening. In a second collaborative study, the mAbs were used to demonstrate increased levels of pHis proteins in mouse and human hepatocellular carcinoma (HCC), and show that the increase was due to reduced expression of the LHPP pHis phosphatase in the tumors, suggesting that LHPP acts as a tumor suppressor, and that elevated His phosphorylation plays a driver role in this cancer. On this basis, studies are planned to investigate whether increased His phosphorylation plays a broader role in human cancer. Initially, our structures of mAb-derived Fab fragments bound to pHis peptides will be exploited to develop better tools for studying His phosphorylation in cancer - mAbs with higher affinity and scFvs for intracellular expression to localize and perturb pHis proteins, sequence specific pHis antibodies for studying individual proteins, and improved MS-based pHis site identification. In parallel, in-depth studies with existing 1/3-pHis mAbs, as well as new pHis reagents as they come online, will be conducted on three selected tumor types - HCC, pediatric neuroblastoma and pancreatic cancer, where there is evidence that aberrant His phosphorylation may play a role. Immunoblotting, and IF and IHC staining will be performed on tumor tissues/cell lines and normal controls, combined with use of optimized pHis peptide enrichment and site identification protocols to define changes in His phosphorylation unique to tumor tissues. Where warranted, the function of individual pHis sites in cancer will be studied by site-directed mutagenesis in tumor cell lines. In each case, further experiments will be guided by the identity and function of pHis proteins found in a particular cancer. Overall, it is anticipated that comparative studies on HCC, neuroblastoma and PDAC will shed light on whether His phosphorylation plays a general role in cancers, whether there are common mechanisms, and whether targeting His phosphorylation could be a viable new therapeutic approach.

项目摘要/摘要 蛋白质中组氨酸（HIS）的磷酸化具有60年的历史，最初被鉴定为p-酶 中级，但随后是细菌中对信号转导的调节机制， 感知营养的表面受体。这种信号系统缺乏哺乳动物，但磷酸酶氨酸缺乏 （PHI）不仅是哺乳动物酶（例如NME1/2，acly）中的键P-酶中间体，而且还发生 作为可逆的终端蛋白质修饰，例如组蛋白H4中的Phis18。他的磷酸化不稳定 并引起热量挑战，并规避我们的小组开发了一系列单克隆 以序列无关的方式识别1-PHIS或3-PHIS同工型的抗体（mAb）。这些 使用MAB通过免疫印迹和免疫荧光（IF）染色来检测细胞中的PHI，而对于 PHIS蛋白的亲和力富集用于MS分析，揭示了约700个潜在的Phis蛋白，并暗示 常规方法无法检测到大型“隐藏”磷蛋白质组的存在。在一项协作研究中 这些mAb用于演示位点和同工型特定于KCA3.1 K+通道的磷酸化。 并推断PHIS如何触发频道的打开。在第二次合作研究中，mabs被用来 表明小鼠和人肝细胞癌（HCC）中的PHIS蛋白水平升高，并且 表明增加是由于LHPP PHIS磷酸酶在肿瘤中的表达降低所致， 表明LHPP充当肿瘤抑制剂，并且升高他的磷酸化在 这个癌症。在此基础上，计划研究是否增加其磷酸化的作用是否有 在人类癌症中更广泛的作用。最初，我们与Phis肽结合的mab衍生的Fab片段的结构 将探索以开发更好的工具来研究他在癌症中的磷酸化 - 具有较高亲和力的mAb 用于细胞内表达的SCFV，用于定位和扰动PHIS蛋白，序列特异性PHIS抗体 用于研究各个蛋白质，并改善基于MS的PHIS位点识别。并行，深入研究 使用现有的1/3 phis mabs以及在线的新PHIS试剂，将在三个上进行 选定的肿瘤类型 - HCC，小儿神经母细胞瘤和胰腺癌，有证据表明 异常的磷酸化可能起作用。免疫印迹，如果和IHC染色将在 肿瘤组织/细胞系和正常对照，结合使用优化的PHIS肽富集和部位 识别方案来定义其肿瘤组织独有的磷酸化变化。保证的地方， 癌症中各个PHI的位点的功能将通过肿瘤细胞系中定位的诱变进行研究。在 每种情况，进一步的实验将由特定的Phis蛋白的身份和功能指导 癌症。总体而言，预计HCC，神经母细胞瘤和PDAC的比较研究将揭示 他的磷酸化是否在癌症中起一般作用，是否有共同的机制和 靶向磷酸化是否可能是一种可行的新治疗方法。

项目成果

Analysis of in vitro and in vivo products of the TMV 30kDa open reading frame using antisera raised against a synthetic peptide.

使用针对合成肽产生的抗血清分析 TMV 30kDa 开放阅读框的体外和体内产物。

• DOI: 10.1016/0014-5793(83)80316-0
• 发表时间: 1983
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Kiberstis,PaulaA;Pessi,Antonello;Atherton,Eric;Jackson,Richard;Hunter,Tony;Zimmern,David
• 通讯作者: Zimmern,David