Histidine phosphorylation as a new target for cancer therapy

项目摘要/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，但随后在细菌中作为一种调节机制，对信号转导至关重要，通过 感知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(如NME1/2，ACLY)中的关键P酶中间体，而且还存在于 作为可逆的终态蛋白修饰，例如组蛋白H4中的pH为18。他的磷酸化对酸不稳定。 和高温使研究变得具有挑战性，为了绕过这一点，我们小组开发了一系列单克隆 以序列无关的方式识别1-PHI或3-PHI异构体的抗体(MAb)。这些 用单抗通过免疫印迹和免疫荧光(IF)染色检测细胞内的PHI，并用于 用于MS分析的PHIS蛋白的亲和富集，揭示了~700个潜在的PHIS蛋白，并暗示 存在用常规方法检测不到的大的“隐藏的”磷酸蛋白质组。在一项合作研究中， 这些单抗用于证明KCa3.1K通道的位点和异构体特异性的His磷酸化 并推断PHI如何触发通道开放。在第二项合作研究中，mAbs被用来 证明在小鼠和人类肝细胞癌中PHIS蛋白水平增加，以及 表明增加是由于LHPP PHIS磷酸酶在肿瘤中的表达减少， 提示LHPP作为一种肿瘤抑制因子，并且使其磷酸化水平升高在 这种癌症。在此基础上，计划进行研究，以调查增加的磷酸化是否对 在人类癌症中发挥更广泛的作用。最初，我们的单抗衍生的Fab片段与PHIS多肽结合的结构 将被利用来开发更好的工具来研究他在癌症中的磷酸化-具有更高亲和力的单抗 和单链抗体用于细胞内表达以定位和干扰PHIS蛋白、序列特异性PHI抗体 用于研究单个蛋白质，并改进了基于MS的PHIs位点识别。同时，深入研究 对于现有的1/3-PHIs单抗，以及新的PHI试剂上线后，将在三个 选定的肿瘤类型--肝癌、儿童神经母细胞瘤和胰腺癌，有证据表明 异常的His磷酸化可能起到一定作用。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多肽浓缩和位点 确定肿瘤组织特有的His磷酸化变化的鉴定方案。在有必要的情况下， 单个PHI位点在癌症中的功能将通过肿瘤细胞系中的定点突变来研究。在……里面 在每一种情况下，进一步的实验都将以在特定的 癌症。总体而言，预计对肝细胞癌、神经母细胞瘤和PDAC的比较研究将有助于 他的磷酸化是否在癌症中起着普遍作用，是否有共同的机制，以及 靶向他的磷酸化是否可能是一种可行的新的治疗方法。

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.