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.

项目摘要 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、信号传导、营养感测和细胞 新陈代谢.溶酶体功能的核心是其酸性腔，其pH值可低至4.5。的 低pH值激活降解酶，这些酶分解蛋白质、受损的细胞器和其他 将大分子转化为可以回收用于细胞用途的构建块。在神经元中， 功能可导致潜在的细胞毒性大分子如Ab、α-突触核蛋白、tau和 他人因此，溶酶体失调与许多人类疾病相关，包括许多 神经退行性疾病在这个应用中，我们提出了实验，将阐明分子 两种溶酶体离子转运蛋白TMEM 175和CLC-7的突变机制可导致 溶酶体内稳态的缺陷，并与人类和小鼠的疾病有关。 TMEM 175是一种溶酶体K+通道，被鉴定为是一种高度有效的风险因子， 帕金森病的发展。在细胞中，TMEM 175在细胞之间建立膜电位 溶酶体腔和胞质溶胶，并且对于溶酶体和细胞内稳态是关键的。TMEM损失175 导致溶酶体pH调节异常，自噬和线粒体自噬缺陷， 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的重要性， TMEM 175功能的分子细节及其生理作用才刚刚开始被理解。我们 最近确定的TMEM 175在打开和关闭状态下的低温EM结构表明， TMEM 175在结构上与其他K+通道无关。此外，该结构证实了其门控， 渗透和选择性机制不同于其它K+通道的特征。通过 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 TMEM 175功能的选择性机制，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+交换剂的CLC家族的成员，其需要b-亚基， OSTM 1，在溶酶体和破骨细胞的皱褶边缘发挥作用。CLC-7是一种溶酶体Cl-/H+交换剂 其突变可导致骨硬化症、溶酶体贮积症和发育迟缓。值得注意的是， 与人类疾病相关的突变导致门控的变化。虽然研究 的原核和真核CLC蛋白已经建立了一个框架的运输周期，门控的 CLC在分子水平上了解甚少。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。这些研究将作为一个 为更好地理解CLC-7/OSTM 1在溶酶体生理学中的作用奠定了基础。