A Novel Class of Enzyme Sensors to Elucidate the Biochemical Responses of Human Nasal Epithelial Cells to Heavy Metals

7.项目摘要 重金属是一类主要的化学暴露剂，对公众有重大影响 环境、职业和/或周围环境破坏后导致健康的发病率和死亡率。 吸入是重金属暴露的最常见途径之一，已知会引起呼吸道疾病。 炎症、癌症、金属烟雾热、哮喘和体能下降。2 -6重要的是，已知 Akt激酶和/或表皮生长因子受体激酶（EGFR）的异常活性发生在 呼吸道上皮组织暴露于重金属后。6 -9然而，由于固有的细胞 呼吸道上皮内细胞的异质性和单细胞对化学物质的动态反应 刺激;10重金属暴露对不同细胞间单细胞信号动力学的影响 呼吸道上皮内的细胞类型是未知的。11因此， 在原发性呼吸道上皮细胞的超小样本中获得Akt和EGFR活性的多重测量， 细胞，将提高我们对重金属暴露的生化机制的理解。 重要的是，这种技术可能使临床医生能够识别重金属的早期预警信号 诱导毒性和/或疾病诱导的个体非常小，异质的主要样品。 我的目标是改善呼吸上皮的生化研究，通过采用传感器为基础的化学 细胞仪基于传感器的化学细胞术是单细胞分析方法，其中生物分子传感器 用于测量小群体单细胞中的信号动力学。12 -16在这里，我提出了 开发一套新的荧光酶传感器，以获得Akt和EGFR的多重测量 使用采用荧光检测的毛细管电泳（CE-F）在单细胞内进行。我还打算 改进所提出的酶传感器的设计和功能;通过安装光活化部分 在磷酸化位点上，我期望提高传感器的膜渗透性，并获得对 此外，我计划控制细胞中激酶反应的停止时间 通过开发一种新的化学选择性试剂， 通过CE-F进行回收分析。 使用这些新型酶传感器的研究将加强我们对生物化学的理解。 控制疾病的诱导和/或从超小群体的恢复力的机制， 呼吸道上皮细胞此外，从这项建议中获得的知识将提高我们的 了解重金属暴露的生化机制，同时确定新的 开发细胞可渗透传感器的策略，以实现单细胞内的时间控制反应。

7. Project Summary Heavy metals comprise a major class of chemical exposure agents and have a significant impact on public health causing morbidity and mortality following environmental, occupational, and/or ambient exposures.1 Inhalation is one of the most common routes for heavy metal exposures, and is known to cause respiratory inflammation, cancers, metal fume fever, asthma, and reduced physical performance.2-6 Importantly, it is known that aberrant activity of Akt kinase and/or epidermal growth factor receptor kinase (EGFR) occurs within respiratory epithelial tissue following exposures to heavy metals.6-9 However, due to the inherent cellular heterogeneity of cells within the respiratory epithelium and the dynamic responses of single cells to chemical stimuli;10 the effects of heavy metal exposures on the signaling dynamics on single cells across different cell types within the respiratory epithelium is unknown.11 Therefore, a single cell analysis technology that can obtain multiplexed measurements of Akt & EGFR activity in ultra-small samples of primary respiratory epithelial cells, would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures. Importantly, such a technology can potentially enable clinicians to identify early warning signs of heavy metal induced toxicity and/or disease induction in individuals from very small, heterogeneous primary samples. I aim to improve biochemical investigations of the respiratory epithelium, by employing sensor based chemical cytometry. Sensor based chemical cytometry is a single cell analysis method in which biomolecular sensors are used to measure signaling dynamics in small populations of single cells.12-16 Here, I propose the development of a novel set of fluorescent enzyme sensors to obtain multiplexed measurements of Akt & EGFR within single cells using capillary electrophoresis employing fluorescence detection (CE-F). I also aim to improve the design and functionality of the proposed enzyme sensors; by installing photoactivatable moieties on the phosphorylation sites, I expect to improve membrane permeability of the sensors, and gain control over the kinase reaction start time within cells.16 Additionally, I plan to control the kinase reaction stop time in cells by developing a novel chemo-selective reagent which halts intracellular reactions, and facilitates reporter recovery for analysis via CE-F. Studies made possible using these novel enzyme sensors will bolster our understanding of the biochemical mechanisms that govern the induction of disease and/or resilience from ultra-small populations of primary respiratory epithelial cells. Additionally, the knowledge gained from this proposal would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures, while identifying novel strategies to develop cell permeable sensors to achieve temporally controlled reactions within single cells.