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: 10532170
• 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/10532170
• 关键词: Albers-Schonberg disease; Autophagocytosis; Binding; 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; Albers-Schonberg disease; Autophagocytosis; Binding; 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.

项目摘要 溶酶体是小的，膜结合的细胞器，参与许多关键过程 包括大分子降解，分泌，膜修复，信号传导，营养灵敏度和细胞 代谢。溶酶体功能的中心是其酸性管腔，可以达到低至4.5的pH值。这 低pH激活降解酶，这些酶破坏了蛋白质，受损细胞器和其他 大分子进入可以回收用于细胞使用的构件。在神经元中，溶酶体缺陷 功能可能导致潜在的细胞毒性大分子的积累，例如AB，A-核蛋白，TAU和 其他的。因此，溶酶体失调与许多人类疾病有关，包括许多人 神经退行性疾病。在此应用中，我们提出了将阐明分子的实验 两种溶酶体离子转运蛋白TMEM175和CLC-7的机制，它们的突变可能导致 溶酶体体内平衡的缺陷，与人类和小鼠的疾病有关。 TMEM175是一种溶酶体K+通道，被确定为高度潜在的风险因素 帕金森氏病的发展。在细胞中，TMEM175在 溶酶体管腔和细胞质，对于溶酶体和细胞稳态至关重要。 TMEM175的损失 导致溶酶体pH的失调，自噬和线粒体的缺陷以及增加 对细胞毒性应激的敏感性。尽管它在帕金森氏病和细胞稳态中的重要性，但都 TMEM175功能及其物理作用的分子细节才开始被理解。我们 最近确定了在开放和封闭状态下TMEM175的冷冻EM结构，这表明 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 生物物理，生化和结构分析的结合，我们将确定渗透，门控和 TMEM175功能的选择性机制，并获得有关其突变如何导致其突变的见解 溶酶体功能障碍和疾病。 CLC-7是Clc nanclels和Cl-/h+交换器的CLC家族的成员，需要B套件， OSTM1，在溶酶体和破骨细胞的褶皱边界中起作用。 CLC-7是溶酶体CL-/H+交换器 其突变会导致骨质疏松症，溶酶体储存疾病和发育延迟。值得注意的是，有几个 与人类疾病相关的突变导致门控变化。研究 原核生物和真核CLC蛋白已经建立了传输周期的框架 在分子水平上，CLC的理解很少。使用结构和电生理方法，我们将 阐明pH和配体调节Clc-7门控的机制。这些研究将作为 更好地理解CLC-7/OSTM1在溶酶体生理学中的作用的基础。