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.

项目总结 溶酶体是一种小的、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、信号传递、营养感知和细胞 新陈代谢。溶酶体功能的中心是它的酸性管腔，它可以达到低至4.5的pH值。这个 低pH值会激活降解酶，从而分解蛋白质、损坏细胞器和其他 将大分子制成积木，可回收用于细胞使用。在神经元中，溶酶体的缺陷 功能可导致潜在的细胞毒性大分子的积聚，如ab，a-突触核蛋白，tau和 其他。因此，溶酶体调节失调与许多人类疾病有关，包括许多 神经退行性疾病。在这一应用中，我们建议进行实验，以阐明分子 两种溶酶体离子转运蛋白TMEM175和ClC-7的突变可导致 溶酶体动态平衡的缺陷，与人类和小鼠的疾病有关。 TMEM175是一种溶酶体K+通道，在文献中被认为是一种高度有效的风险因子。 帕金森氏症的发展。在细胞中，TMEM175在细胞和细胞之间建立了膜电位 溶酶体腔和胞浆，对溶酶体和细胞动态平衡至关重要。TMEM175丢失 导致溶酶体pH的失调，自噬和有丝分裂的缺陷，以及 对细胞毒性应激的敏感性。尽管它在帕金森氏病和细胞内稳态方面很重要，但两者都 TMEM175的功能及其生理作用的分子细节才刚刚开始被了解。我们 最近测定的TMEM175在开放和关闭状态下的低温电子显微镜结构表明 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， 渗透和选择性机制不同于其他K+通道的特征。通过一个 结合生物物理、生化和结构分析，我们将确定渗透、浇注和 TMEM175功能的选择性机制并深入了解其突变是如何导致 溶酶体功能障碍和疾病。 ClC-7是需要b-亚基的ClC-通道和Cl-/H+交换器家族的成员， OSTM1，在溶酶体和破骨细胞的褶皱边缘发挥作用。ClC-7是一种溶酶体Cl-/H+交换体 其突变可导致骨质疏松症、溶酶体储存症和发育迟缓。值得注意的是，有几个 与人类疾病相关的突变会导致门控的变化。一边学习一边学习 原核生物和真核生物的CLC蛋白已经建立了运输循环的框架，门控 在分子水平上，人们对CLCS知之甚少。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。这些研究将作为一项 为更好地理解ClC-7/OSTM1在溶酶体生理学中的作用奠定了基础。