Histidine phosphorylation as a new target for cancer therapy

项目概要/摘要 蛋白质中组氨酸 (His) 的磷酸化已有 60 年的历史，最初被确定为 P 酶 中间，但随后作为细菌中信号转导所必需的调节机制 感知营养物质的表面受体。哺乳动物缺乏这种信号系统，但磷酸组氨酸 (pHis) 不仅是哺乳动物酶（例如 NME1/2、ACLY）中关键的 P 酶中间体，而且还发生 作为可逆的终态蛋白质修饰，例如组蛋白 H4 中的 pH 为 18。他的磷酸化对酸不稳定 和热量使得研究具有挑战性，为了规避这个问题，我们的小组开发了一系列单克隆抗体 以序列无关的方式识别 1-pHis 或 3-pHis 同工型的抗体 (mAb)。这些 mAb 用于通过免疫印迹和免疫荧光 (IF) 染色来检测细胞中的 pH 值，并用于 用于 MS 分析的 pHis 蛋白的亲和富集，揭示了约 700 个潜在的 pHis 蛋白，并暗示 传统方法无法检测到的大型“隐藏”磷酸化蛋白质组的存在。在一项合作研究中， 这些 mAb 用于证明 KCa3.1 K 通道的位点和亚型特异性 His 磷酸化 并推断 pHs 如何触发通道开放。在第二项合作研究中，单克隆抗体被用来 证明小鼠和人类肝细胞癌 (HCC) 中 pHis 蛋白水平升高，并且 表明这种增加是由于肿瘤中 LHPP pHis 磷酸酶表达减少所致， 表明 LHPP 作为肿瘤抑制因子，His 磷酸化升高在肿瘤抑制中发挥驱动作用 这种癌症。在此基础上，计划研究增加His磷酸化是否会发挥作用 在人类癌症中发挥更广泛的作用。最初，我们的 mAb 衍生 Fab 片段结构与 pHis 肽结合 将被用来开发更好的工具来研究癌症中的 His 磷酸化 - 具有更高亲和力的 mAb 和 scFv 用于细胞内表达，以定位和干扰 pHis 蛋白，对特异性 pHis 抗体进行测序 用于研究单个蛋白质，并改进基于 MS 的 pHis 位点识别。并行深入研究 现有的 1/3-pHis mAb 以及新上线的 pHis 试剂将在三个项目上进行 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 他的磷酸化异常可能发挥了作用。将进行免疫印迹、IF 和 IHC 染色 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 磷酸化变化。在有保证的情况下， 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，进一步的实验将以在特定情况下发现的 pHis 蛋白的身份和功能为指导。 癌症。总体而言，预计 HCC、神经母细胞瘤和 PDAC 的比较研究将有助于阐明 His 磷酸化是否在癌症中发挥普遍作用，是否存在共同机制，以及 针对他的磷酸化是否可能是一种可行的新治疗方法。

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.