Heavy metal-stimulated signal transduction: new metal-regulatory and -responsive mechanisms

项目总结/摘要 从人体生理学的角度来看，重金属（HM）分为两类：必需重金属（eHM）和 毒性（tHM）。eHM和tHM过度表达都与许多病理生理学状况有关;然而， eHM稳态失调和tHM暴露刺激的信号转导途径和基因组活性 还有待充分确立。例如，哺乳动物的铜传感器蛋白负责传递 细胞外Cu水平的变化转化为信号应答尚未被鉴定。此应用程序的目标 定义（i）eHM和tHM刺激和调节信号传导网络（包括产生的基因组 活动），和（ii）潜在的分子生物学，生物化学和生物物理机制。通过 在这项工作中，我已经表征了铜暴露诱导的信号传导和基因组反应， 确定了铜刺激表皮生长因子受体（EGFR，一种细胞表面酪氨酸受体）的机制 激酶（RTK））信号转导，从而将EGFR鉴定为候选的Cu- 传感器蛋白。生物化学实验正在研究EGFR-Cu结合位点和几何形状。 相关地，通过应用新开发的下一代测序（NGS）、分子生物学和基因测序技术， 转录组学（体外和体内）技术，我发现了一种新的机制，细胞内铜水平 是有规律的。升高的细胞外Cu水平刺激EGFR/MAPK/ERK活化，其诱导表达 转录因子（TF）EGR 1和EGR 1-辅阻遏物NAB 1/2; EGR 1-NAB 1/2复合物随后 抑制哺乳动物铜输入者CTR 1的转录。EGR 1 ChIP-seq实验正在进行中， 进一步证实。这项工作将与评估铜分子伴侣抑制剂协同作用的实验配对 和MAPK/ERK信号传导（以增加CTR 1水平）与铂类抗癌药物（顺铂，通过 CTR 1），以及EGFR驱动的肺癌中EGFR 1的TF活性的基因组见解（这一作用是 目前未知）。我的研究建立铜调控网络奠定了基础，我的计划 独立的职业（i）评估eHM和tHM的调节和响应机制，以及（ii）表征 通过tHM破坏天然信号传导。在我独立的职业生涯中，我将应用磷酸化阵列和NGS 技术与非铜eHM和高优先级环境污染物tHM刺激细胞，重点是 tHM暴露在破坏eHM调节和响应信号转导网络中的作用。在 总之，我鉴定EGFR作为将升高细胞外Cu水平传递到基因组中的管道， 通过MAPK/ERK途径的活性连接了一个主要的信号转导网络（与癌症有关 病理生理学，例如）的eHM调节，从而打开了新的途径，了解HM- 一般的监管网络。更广泛地说，该提案中建立的综合研究计划 站在确定生物标志物的伤害之前的发展和进展的癌症和其他人类 疾病，建立新的HM调节机制，并丰富tHM毒理机制的理解。

PROJECT SUMMARY/ABSTRACT From a human physiological perspective, heavy metals (HM) are divided into two groups: essential (eHM) and toxic (tHM). eHM and tHM overexposure are both linked to numerous pathophysiological conditions; however, the signal transduction pathways and genomic activities stimulated by eHM dyshomeostasis and tHM exposure have yet to be adequately established. For example, the mammalian Cu-sensor protein responsible for relaying changes in extracellular Cu levels into signaling responses has not been identified. The goals of this application are to define (i) eHM- and tHM-stimulated and -regulatory signaling networks (including resultant genomic activities), and (ii) the underlying molecular biological, biochemical, and biophysical mechanisms. Through the work in this application, I have characterized the signaling and genomic responses induced by Cu exposure and identified a mechanism of Cu-stimulated epidermal growth factor receptor (EGFR, a cell surface receptor tyrosine kinase (RTK)) signal transduction along the MAPK/ERK pathway, thereby identifying EGFR as a candidate Cu- sensor protein. Biochemical experiments are underway investigating the EGFR-Cu binding site and geometry. Relatedly, through application of newly-developed next generation sequencing (NGS), molecular biological, and transcriptomic (in vitro and in vivo) techniques, I discovered a novel mechanism by which intracellular Cu levels are regulated. Elevated extracellular Cu levels stimulate EGFR/MAPK/ERK activation, which induces expression of the transcription factor (TF) EGR1 and EGR1-corepressor NAB1/2; the EGR1-NAB1/2 complex subsequently represses transcription of the mammalian Cu-importer, CTR1. EGR1 ChIP-seq experiments are underway for further corroboration. This work will pair with experiments assessing synergism of an inhibitor of Cu-chaperones and MAPK/ERK signaling (to increase CTR1 levels) with a platinum anticancer drug (cisplatin, imported via CTR1), as well as genomic insights into the TF activities of EGR1 in EGFR-driven lung cancer (a role that is currently unknown). My research establishing the Cu-regulatory network lays the foundation for my planned independent career (i) evaluating eHM- and tHM-regulatory and -responsive mechanisms and (ii) characterizing disruption of native signaling by tHM. In my independent career, I will apply phosphorylation arrays and NGS techniques to cells stimulated with non-Cu eHM and high-priority environmental pollutant tHM, with emphasis on the effects of tHM exposure in disrupting the eHM-regulatory and -responsive signal transduction networks. In summary, my identification of EGFR as a conduit for relaying elevated extracellular Cu levels into genomic activities via the MAPK/ERK pathway has linked a major signal transduction network (implicated in cancer pathophysiology, for example) to eHM-regulation, and thereby opens new avenues of understanding HM- regulatory networks in general. More broadly, the comprehensive research program established in this proposal stands to identify biomarkers of harm preceding development and progression of cancer and other human diseases, establish novel HM-regulatory mechanisms, and enrich tHM-toxicological mechanistic understanding.