Mechanisms of lysosomal ion transport proteins involved in pH homeostasis

溶酶体离子转运蛋白参与 pH 稳态的机制

• 批准号: 10365563
• 负责人: Richard Kevin Hite
• 金额: $ 44.25万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10365563
• 关键词: Albers-Schonberg disease; Autophagocytosis; Biochemical; Biological; Biological Assay; Biophysics; CLC Gene; Carrier Proteins; Cells; Cellular Immunity; Chloride Channels; Chlorides; Complex; Cryoelectron Microscopy; Cytosol; Data; Defect; Development; Developmental Delay Disorders; Disease; Electrophysiology (science); Enzymes; Family; Foundations; Functional disorder; Goals; Health; Homeostasis; Human; Hydrophobicity; In Vitro; Ion Transport; Ions; Lead; Ligands; Lipids; Lysosomal Storage Diseases; Lysosomes; Membrane; Membrane Potentials; Metabolism; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nutrient; Organelles; Osteoclasts; Parkinson Disease; Pathology; Phosphatidylinositols; Phosphotransferases; Physiological; Physiology; Point Mutation; Potassium Channel; Predisposition; Process; Proteins; Protons; RAC-Alpha Serine/Threonine Kinase; Recycling; Regulation; Reporting; Research; Risk Factors; Role; Signal Transduction; Stimulus; Stress; Structure; alpha synuclein; antiporter; constriction; cytotoxic; detection of nutrient; experimental study; human disease; improved; in vitro Assay; insight; macromolecule; member; molecular dynamics; pH Homeostasis; potassium ion; repaired; tau Proteins; vacuolar H+-ATPase; voltage

PROJECT SUMMARY Lysosomes are small, membrane-bound organelles that participate in numerous critical processes including macromolecular degradation, secretion, membrane repair, signaling, nutrient sensing and cellular metabolism. Central to lysosomal function is its acidic lumen, which can reach pH values as low as 4.5. The low pH activates degradative enzymes that break down proteins, damaged organelles and other macromolecules into building blocks that can be recycled for cellular use. In neurons, defects in lysosomal function can lead to accumulation of potentially cytotoxic macromolecules such as ab, a-synuclein, tau and others. Consequently, lysosomal dysregulation is associated with numerous human diseases, including many neurodegenerative diseases. In this application, we propose experiments that will elucidate the molecular mechanisms of two lysosomal ion transport proteins, TMEM175 and CLC-7, whose mutation can lead to defects in lysosomal homeostasis and are associated with disease in humans and mice. TMEM175 is a lysosomal K+ channel that was identified in as a highly potent risk-factor for the development of Parkinson’s Disease. In cells, TMEM175 establishes a membrane potential between the lysosomal lumen and the cytosol and is critical for lysosomal and cellular homeostasis. Loss of TMEM175 leads to dysregulation of lysosomal pH, deficiencies in autophagy and mitophagy and an increased susceptibility to cytotoxic stress. Despite its importance in Parkinson’s Disease and cellular homeostasis, both the molecular details of TMEM175 function and its physiological roles are only starting to be understood. We recently determined cryo-EM structures of TMEM175 in open and closed states that demonstrated that TMEM175 is structurally unrelated to other K+ channels. Moreover, the structure confirmed that its gating, permeation and selectivity mechanisms are distinct from those characterized for other K+ channels. Through a combination of biophysical, biochemical and structural analyses, we will determine the permeation, gating and selectivity mechanisms underlying TMEM175 function and gain insights into how its mutation can lead to lysosome dysfunction and disease. CLC-7 is a member of the CLC family of Cl- channels and Cl-/H+ exchangers that requires a b-subunit, OSTM1, to function in lysosomes and the ruffled border of osteoclasts. CLC-7 is a lysosomal Cl-/H+ exchanger whose mutation can lead osteopetrosis, lysosomal storage disease and developmental delay. Notably, several of the mutations associated with associated with disease in humans result in changes in gating. While studies of prokaryotic and eukaryotic CLC proteins have established a framework for the transport cycles, the gating of CLCs is poorly understood at the molecular level. Using structural and electrophysiological approaches, we will elucidate mechanisms by which pH and ligands regulate the gating of CLC-7. These studies will serve as a foundation for better understanding the role of CLC-7/OSTM1 in lysosomal physiology.

项目概要 溶酶体是小型的膜结合细胞器，参与许多关键过程 包括大分子降解、分泌、膜修复、信号传导、营养传感和细胞 代谢。溶酶体功能的核心是其酸性腔，pH 值可低至 4.5。这 低 pH 值会激活降解酶，分解蛋白质、受损的细胞器和其他物质 将大分子转化为可回收用于细胞使用的构件。在神经元中，溶酶体缺陷 功能可能导致潜在细胞毒性大分子的积累，例如 ab、a-突触核蛋白、tau 和 其他的。因此，溶酶体失调与许多人类疾病有关，包括许多 神经退行性疾病。在此应用中，我们提出了实验来阐明分子 两种溶酶体离子转运蛋白 TMEM175 和 CLC-7 的机制，其突变可导致 溶酶体稳态缺陷与人类和小鼠的疾病有关。 TMEM175 是一种溶酶体 K+ 通道，已被确定为 帕金森病的发展。在细胞中，TMEM175 在 溶酶体腔和细胞质，对于溶酶体和细胞稳态至关重要。 TMEM175 丢失 导致溶酶体 pH 值失调、自噬和线粒体自噬缺陷以及增加 对细胞毒性应激的敏感性。尽管它在帕金森病和细胞稳态中很重要，但 TMEM175 功能的分子细节及其生理作用才刚刚开始被了解。我们 最近确定了 TMEM175 在打开和关闭状态下的冷冻电镜结构，证明 TMEM175 在结构上与其他 K+ 通道无关。此外，该结构证实了它的门控， 渗透和选择性机制与其他 K+ 通道的特征不同。通过一个 结合生物物理、生物化学和结构分析，我们将确定渗透、门控和 TMEM175 功能背后的选择性机制，并深入了解其突变如何导致 溶酶体功能障碍和疾病。 CLC-7 是 Cl- 通道和 Cl-/H+ 交换器 CLC 家族的成员，需要 b 亚基， OSTM1，在溶酶体和破骨细胞的褶皱边界中发挥作用。 CLC-7 是一种溶酶体 Cl-/H+ 交换剂 其突变可导致骨石症、溶酶体贮积病和发育迟缓。值得注意的是，几个 与人类疾病相关的突变会导致门控的变化。在学习的同时 原核和真核 CLC 蛋白的研究已经建立了运输循环、门控的框架 CLC 在分子水平上的了解还很有限。使用结构和电生理学方法，我们将 阐明 pH 值和配体调节 CLC-7 门控的机制。这些研究将作为 为更好地了解 CLC-7/OSTM1 在溶酶体生理学中的作用奠定了基础。