Development and Application of Organelle Chemotype Fingerprinting for the Functional Investigation of Organellar Chloride

摘要/摘要 氯离子作为人体内含量最丰富的阴离子之一，对人体健康起着至关重要的作用。氯化物 动态平衡在细胞内维持，而氯离子水平则根据细胞器的功能而变化。 氯通道突变引起的氯稳态失调导致不同的人类 囊性纤维化(全球70,000人)、蛋白尿和肾结石(CLC-5，39)等疾病 骨质疏松症(CLC-7，美国1000万人，风险组4300万人)。虽然 已经报道了五种FDA批准的氯通道调节剂，它们只针对质膜氯 渠道由于技术障碍。没有FDA批准的或针对细胞器的临床试验药物 氯离子通道。氯通道靶向治疗领域仍处于研究阶段(5种FDA批准的药物，2种 临床试验)与钙、钾和钠等其他渠道进行比较。 对有机氯的生理作用缺乏了解，并对其进行了很好的表征 氯通道是氯通道靶向治疗发展的最大障碍。 因此，合适的研究工具具有较高的分辨能力来检测活体中的细胞器氯 细胞是非常迫切的需要，这是阐明有机氯的生理作用所必需的。 并对氯离子通道进行了表征。然而，目前的氯离子测量采用一维方法 分析只显示平均离子水平。它不能观察到氯离子水平的变化 由细胞途径触发的细胞器，如叮咬和自噬。此外，典型的 荧光测量只能告诉平均氯水平的变化(增加、减少和NO 重大变化)在某些条件下。目前的方法严重阻碍了对失活的识别 基于氯离子水平测量的细胞途径或蛋白质。 建议的研究将细胞器选择性双报告、单细胞器测量、亚细胞器测量 细胞成像和三维分析，以指纹细胞器的化学类型以及刺 途径、自噬和线粒体呼吸。建议的研究完成后，将会发现 有机氯的生理作用为氯通道靶向治疗提供了线索。这个 细胞器化学类型指纹技术的发展也将提供表征氯化物的工具 评估氯通道调节剂，并识别失活的细胞通路或蛋白质。

SUMMARY/ABSTRACT As one of the most abundant anions in the human body, chloride plays a crucial role in human health. Chloride homeostasis is maintained inside the cell while the chloride level is varied based on the function of organelles. Dysregulation of chloride homeostasis caused by the mutation of chloride channels results in various human diseases such as cystic fibrosis (CFTR, >70,000 people worldwide), proteinuria and kidney stones (ClC-5, 39 million people in US), Osteoporosis (ClC-7, 10 million people in US, 43 million people in the risk group). Although five FDA-approved chloride channel modulators have been reported, they only target plasma membrane chloride channels due to the technical barrier. There is no FDA-approved or clinical trial drug that targets organellar chloride channel. The field of chloride channel-targeted therapy is still under-studied (5 FDA-approved drugs, 2 clinical trial) compared to other channels such as calcium, potassium, and sodium. The lack of understanding of the physiological role of organellar chloride and the well-characterized chloride channel are the biggest roadblocks for the development of chloride channel-targeted therapy. Therefore, suitable research tools with a high resolving ability to examine the organelle chloride in live cells is a highly urgent need, which is essential to elucidate the physiological role of organellar chloride and characterize the chloride channel. However, the current chloride measurement with one-dimensional analysis only shows the average ion level. It cannot observe the chloride level change in a minor subset of organelles triggered by the cellular pathway such as STING and autophagy. Furthermore, the typical fluorescence measurement can only tell the variation of the average chloride level (increase, decrease, and no significant change) in certain conditions. The current methods significantly hinder the identification of deactivated cell pathways or protein based on the chloride level measurement. The proposed research integrate organelle selective dual reporters, single organelle measurement, sub- cellular imaging, and the three-dimensional analysis, to fingerprint the chemotype of organelles along with STING pathway, autophagy, and mitochondrial respiration. Completion of the proposed study will find out the physiological role of organellar chloride which shed light on the chloride channel-targeted therapy. The development of the organelle chemotype fingerprinting technique will also provide tools to characterize chloride channels, evaluate chloride channel modulators and identify the deactivated cell pathways or proteins.