Structural and functional investigation of the human CLC-7/OSTM1 complex

人类 CLC-7/OSTM1 复合物的结构和功能研究

• 批准号: 443618349
• 负责人: Dr. Marina Schrecker, Ph.D.
• 金额: --
• 依托单位: Memorial Sloan Kettering Cancer Center
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/443618349?language=en
• 关键词: Structural; functional; investigation; human; CLC

Members of the chloride channel (CLC) family occur in all phyla, with nine members present in mammals. The chloride-proton antiporter CLC-7 belongs to the CLC family and is the major CLC transporter in lysosomes, late endosomes and in the ruffled border, the acid-secreting plasma membrane domain of osteoclasts. Mutations of CLC-7 in humans or mice cause osteopetrosis and lysosomal storage diseases. The Cl- transporter CLC-7 reveals several features typical for the CLC family including its dimeric topology and the architecture of the ion translocation pathway. Also, the transporter obeys the classical CLC transporter ratio of two Cl- to one H+. However, CLC-7 has some very intriguing features which distinguish it from the rest of the CLC family. For instance, the voltage-dependent activation and deactivation of CLC-7 is much slower than other CLC transporters. Furthermore, CLC-7 is the only member that requires an accessory protein, osteopetrosis-associated membrane protein 1 (OSTM1), for both stability and function. CLC-7 and OSTM1 are co-localized in lysosomes and in the ruffled border of osteoclasts. OSTM1 is a type 1 transmembrane protein containing a large, highly glycosylated N-terminus and single membrane-spanning helix and short C-terminus. CLC-7-, OSTM1- and CLC-7/OSTM1-deficient mice have short life spans and develop severe osteopetrosis, retinal degeneration, lysosomal storage diseases, and neurodegeneration. So far, the mechanism of CLC-7 regulation by its accessory protein OSTM1 remains an open question. I propose a multidisciplinary approach combining structural, electrophysiological and biochemical tools for investigation of CLC-7/OSTM1. During my first months in the host laboratory, I was able to establish the protocol for purification of CLC-7 from Gallus gallus. I then analyzed the purified sample by cryo-electron microscopy (cryo-EM), which revealed over 3,000,000 well-distributed single particles. Next, I aim to investigate the function and structure of human CLC-7 alone (Aim 1) and CLC-7 in complex with OSTM1 (Aim 2). Comparison of the cryo-EM structure of the complex with the isolated form of CLC-7 will facilitate identification of important regulatory regions, and will help unravel the unique regulatory nature of the CLC-7 protein. I will then further investigate the regulatory mechanism of CLC-7 by fluorescence-based flux assays and electrophysiological measurements, using lipid-reconstituted samples of CLC-7 and CLC-7/OSTM1. These investigations will ultimately determine the mechanism of CLC-7/OSTM1 interaction and regulation. Beyond the molecular characterization of CLC-7/OSTM1, these studies will promote our understanding of the physiological and pathological roles of the CLC-7/OSTM1 complex.

氯离子通道（CLC）家族的成员存在于所有的门，哺乳动物中有9个成员。氯质子反向转运蛋白CLC-7属于CLC家族，是溶胞体、晚期内体和破骨细胞的皱边、酸分泌质膜结构域中的主要CLC转运蛋白。人类或小鼠中CLC-7的突变导致骨硬化症和溶酶体贮积病。Cl-转运蛋白CLC-7揭示了CLC家族的几个典型特征，包括其二聚体拓扑结构和离子易位途径的结构。此外，该转运蛋白遵循经典的CLC转运蛋白比例，即两个Cl-对一个H+。然而，CLC-7有一些非常有趣的功能，使其与CLC家族的其他成员区分开来。例如，CLC-7的电压依赖性激活和失活比其他CLC转运蛋白慢得多。此外，CLC-7是唯一一个需要辅助蛋白，骨硬化相关膜蛋白1（OSTM 1），稳定性和功能的成员。CLC-7和OSTM 1共定位于溶酶体和破骨细胞的皱褶边缘。OSTM 1是一种1型跨膜蛋白，含有一个大的、高度糖基化的N-末端和单个跨膜螺旋以及短的C-末端。CLC-7、OSTM 1和CLC-7/OSTM 1缺陷型小鼠寿命短，并发生严重的骨硬化症、视网膜变性、溶酶体贮积病和神经变性。到目前为止，CLC-7通过其辅助蛋白OSTM 1调节的机制仍然是一个悬而未决的问题。我提出了一个多学科的方法相结合的结构，电生理和生物化学工具的调查CLC-7/OSTM 1。在我在宿主实验室的头几个月里，我能够建立从原鸡中纯化CLC-7的方案。然后，我通过冷冻电子显微镜（cryo-EM）分析了纯化的样品，发现了超过3，000，000个分布良好的单个颗粒。接下来，我的目标是研究单独的人CLC-7（Aim 1）和CLC-7与OSTM 1复合物（Aim 2）的功能和结构。将CLC-7的分离形式与复合物的cryo-EM结构进行比较将有助于鉴定重要的调控区域，并将有助于揭示CLC-7蛋白的独特调控性质。然后，我将进一步研究CLC-7的调节机制，通过基于荧光的通量测定和电生理测量，使用脂质重构的CLC-7和CLC-7/OSTM 1样品。这些研究将最终确定CLC-7/OSTM 1相互作用和调节的机制。除了CLC-7/OSTM 1的分子特征之外，这些研究将促进我们对CLC-7/OSTM 1复合物的生理和病理作用的理解。