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.

项目总结/摘要 蛋白质中组氨酸的磷酸化已有60年的历史，最初被鉴定为P-酶 中间体，但随后作为细菌中信号转导所必需的调节机制， 感受营养物质的表面受体。这种信号系统在哺乳动物中是缺乏的，但磷酸组氨酸 （pHis）不仅是哺乳动物酶（例如NME 1/2、ACLY）中的关键P-酶中间体，而且还发生在哺乳动物中。 作为可逆的终态蛋白质修饰，例如组蛋白H4中的pHis 18。他的磷酸化对酸不稳定 和热量使得研究具有挑战性，为了规避这一点，我们的团队开发了一系列单克隆抗体， 以序列非依赖性方式识别1-pHis或3-pHis同种型的抗体（mAb）。这些 使用mAb通过免疫印迹和免疫荧光（IF）染色检测细胞中的pHis，并用于检测细胞中的pHis。 用于MS分析的pHis蛋白的亲和富集，揭示了约700种潜在的pHis蛋白，并暗示了 存在常规方法检测不到的大的“隐藏的”磷酸化蛋白质组。在一项合作研究中， 这些单克隆抗体用于证明KCa 3.1 K+通道的位点和亚型特异性His磷酸化 并推导出pH是如何触发通道开放的在第二项合作研究中，mAb用于 在小鼠和人肝细胞癌（HCC）中显示pHis蛋白水平增加，和 表明这种增加是由于肿瘤中LHPP pHis磷酸酶表达的减少， 这表明LHPP作为肿瘤抑制因子，并且升高的His磷酸化在肿瘤发生中起驱动作用。 这个癌症在此基础上，研究计划调查增加的His磷酸化是否起作用。 在人类癌症中发挥更广泛的作用。最初，我们的mAb衍生的Fab片段与pHis肽结合的结构 将用于开发更好的工具，用于研究癌症中His磷酸化-具有更高亲和力的mAb 和用于细胞内表达以定位和干扰pHis蛋白的scFv，序列特异性pHis抗体 用于研究单个蛋白质，并改进了基于MS的pHis位点鉴定。同时，深入研究 使用现有的1/3-pHis mAb以及新的pHis试剂，将在三个 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His磷酸化可能起作用。免疫印迹、IF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肽富集和位点 鉴定方案来定义肿瘤组织特有的His磷酸化的变化。如果有必要， 将通过肿瘤细胞系中的定点诱变来研究癌症中各个pHis位点的功能。在 在每种情况下，进一步的实验将由在特定的细胞中发现的pHis蛋白的身份和功能来指导。 癌总体而言，预计对HCC、神经母细胞瘤和PDAC的比较研究将阐明 His磷酸化是否在癌症中起普遍作用，是否有共同的机制， 靶向His磷酸化是否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