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

细胞器化学型指纹图谱在氯化细胞器功能研究中的开发和应用

• 批准号: 10664018
• 负责人: Ka Ho Leung
• 金额: $ 36.26万
• 依托单位: CLARKSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664018
• 关键词: Anions; Autophagocytosis; Calcium; Cell membrane; Cells; Chloride Channels; Chlorides; Clinical Trials; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Development; FDA approved; Fingerprint; Fluorescence; Health; Healthcare; Homeostasis; Human; Human body; Image; Investigation; Ions; Kidney Calculi; Measurement; Methods; Minor; Mitochondria; Mutation; Organelles; Osteoporosis; Pathway interactions; Persons; Pharmaceutical Preparations; Physiological; Play; Potassium; Proteins; Proteinuria; Reporter; Reporting; Research; Respiration; Risk; Role; Sodium; Techniques; Three-dimensional analysis; Variant; care burden; effective therapy; human disease; targeted therapy trials; targeted treatment; tool

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.

总结/摘要 作为人体内最丰富的阴离子之一，氯离子对人体健康起着至关重要的作用。氯化 在细胞内维持体内平衡，而氯水平基于细胞器的功能而变化。 氯离子通道突变引起的氯离子稳态失调导致各种人类 囊性纤维化（CFTR，全世界> 70，000人）、蛋白尿和肾结石（ClC-5，39 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批准的氯离子通道调节剂，它们仅针对质膜氯离子 由于技术壁垒的原因。没有FDA批准或临床试验药物靶向细胞器 氯离子通道氯离子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）与其他通道（如钙、钾和钠）相比。 缺乏对细胞器氯化物的生理作用的理解， 氯离子通道是氯离子通道靶向治疗发展的最大障碍。 因此，合适的研究工具，具有高分辨率的能力，以检查细胞器中的氯在生活 细胞是一个非常迫切的需要，这是必不可少的阐明生理作用的细胞器氯 并表征氯离子通道。然而，目前的一维氯化物测量 分析仅显示平均离子水平。它不能观察到氯化物水平的变化，在一个小的子集， 细胞器由细胞途径如STING和自噬触发。此外，典型的 荧光测量只能告诉平均氯化物水平的变化（增加、减少和不增加）， 在某些条件下发生重大变化。目前的方法严重阻碍了对失活的 细胞途径或蛋白质的基础上的氯化物水平的测量。 该研究整合了细胞器选择性双报告基因、单细胞器测量、亚细胞测量和细胞内信号转导等技术， 细胞成像和三维分析，以利用STING对细胞器的化学型进行沿着指纹分析 途径，自噬和线粒体呼吸。完成拟议的研究后， 细胞器氯的生理作用，揭示了氯通道靶向治疗。的 细胞器化学型指纹技术的发展也将提供表征氯化物的工具 通道，评估氯离子通道调节剂，并确定失活的细胞通路或蛋白质。