The Mechanism of Manganese Transport of SLC30A10 in Neuronal and Hepatic Systems

SLC30A10 在神经元和肝脏系统中的锰转运机制

• 批准号: 9327449
• 负责人: Charles E. Zogzas
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327449
• 关键词: Active Sites; Address; Adverse effects; Affect; Affinity; Agreement; Amino Acids; Artificial Membranes; Binding; Biochemical; Biological Assay; Biology; Blood; Brain; Calorimetry; Carrier Proteins; Cell Line; Cell membrane; Cells; Chronic; Cirrhosis; Clinical; Clinical assessments; Code; Confocal Microscopy; Copper; DNA; DNA Sequence Alteration; Data; Deposition; Development; Disease; Elements; Endoplasmic Reticulum; Environment; Environmental Exposure; Excision; Female; Follow-Up Studies; Foundations; Future; Genes; Genome; Goals; Growth and Development function; Health; Hela Cells; HepG2; Hepatic; Hepatocyte; Homeostasis; Human; Idiopathic Parkinson Disease; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Ion Transport; Ions; Iron; Kinetics; Knowledge; Lead; Light; Liver; Manganese; Measurement; Measures; Metabolism; Metals; Microscopy; Midbrain structure; Modeling; Molecular; Morphology; Motivation; Motor; Mus; Mutation; Nerve Degeneration; Neurologic; Neurons; Nutritional; Occupational; Paper; Parkinson Disease; Parkinsonian Disorders; Patients; Pharmaceutical Preparations; Pharmacology Study; Physiological; Play; Process; Protein Family; Proteins; Publishing; Recording of previous events; Regulation; Reporting; Research; Role; Sequence Homology; Single Nucleotide Polymorphism; Site; Structural Models; Symptoms; System; Techniques; Testing; Thermodynamics; Titrations; Toxic Environmental Substances; Toxic effect; Training; Work; Zinc; base; biological systems; biophysical techniques; clinically significant; cognitive function; cohort; cytotoxic; cytotoxicity; deep sequencing; experimental study; improved; liver function; mutant; nervous system disorder; neurotoxicity; proteoliposomes; therapeutic development; therapy development; zinc-binding protein

Abstract: Manganese (Mn) is an essential element, but overexposure is cytotoxic and has adverse effects on neurological health. In humans, Mn-induced neurotoxicity generally occurs due to chronic exposure under occupational or environmental settings and resembles idiopathic Parkinson’s disease. In some cases, patients with compromised liver function due to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn- induced parkinsonism in the absence of high exposure. While the nutritional and clinical significance of Mn is established, cellular mechanisms of Mn homeostasis are still unknown. A breakthrough in our understanding of Mn metabolism came from the identification of a familial form of parkinsonism reported to occur due to mutations in SLC30A10. Findings in our lab have determined that SLC30A10 acts as the primary Mn efflux transporter protein to protect cells against Mn-induced toxicity. Interestingly, SLC30A10 disease-causing mutants from parkinsonian patients discussed above were unable protect against high Mn. As Mn is ubiquitous in the environment, our long term goal is to elucidate the role that SLC30A10 plays in metal-induced neurodegenerative processes, which in turn lead to Parkinson-like symptoms in patients. This gap in knowledge hinders treatment development and will persist if molecular mechanisms utilized by SLC30A10 are not understood. Our hypothesis is that SLC30A10 binds and transports Mn with higher affinity than other essential metals and that this activity is sensitive to cellular environment. We have recently identified residues of SLC30A10 that are required for Mn efflux activity. However, our studies used cell-based functional experiments and do not provide the molecular detail of their mechanistic involvement in Mn efflux activity. To shed light on this, experiments proposed here will determine the Mn transport mechanism of SLC30A10 using a combination of in vitro studies and physiologically relevant cell-based assays. In Aim 1 we will perform biochemical studies on purified SLC30A10 protein to reveal the mechanism of binding and transport of SLC30A10. First, isothermal titration calorimetry (ITC) will be used to determine the Mn binding coefficient (KD) of SLC30A10. Then a proteoliposome transport assay, with artificial membranes containing SLC30A10 will determine the Mn transport kinetics (KM and Vmax). We will perform a comparison of SLC30A10WT to SLC30A10 efflux mutants identified in our primary screens to elucidate residues directly involved in Mn binding. Aim 2 will then be performed in cell-based systems. Confocal microscopy will be employed to assess SLC30A10 function in primary neurons and a hepatic cell line. Quantitative metal measurement ICP-MS will be used to measure intracellular Mn content and corroborate microscopy findings. Taken together, the findings from this training plan will improve our understanding of cellular Mn homeostasis as it relates to neurotoxicity and provide biochemical data on SLC30A10 important for developing therapies against Mn toxicity.

抽象的： 锰（Mn）是人体必需元素，但过度接触具有细胞毒性，会对人体产生不利影响。 在人类中，锰引起的神经毒性通常是由于长期接触锰而发生的。 职业或环境环境，在某些情况下类似于特发性帕金森病。 由于肝硬化等疾病导致肝功能受损，无法排泄锰，可能会出现锰- 在没有高暴露的情况下会诱发帕金森病，而锰的营养和临床意义是。 尽管锰稳态的细胞机制尚未确定，但我们对锰稳态的理解取得了突破。 锰代谢来自于对一种家族性帕金森病的鉴定，据报道，这种帕金森病的发生是由于 我们实验室的研究结果确定 SLC30A10 是主要的 Mn 外流。 转运蛋白保护细胞免受锰诱导的毒性，提示 SLC30A10 致病。 上面讨论的帕金森病患者的突变体无法预防高锰。 由于锰在环境中无处不在，我们的长期目标是阐明 SLC30A10 在 金属引起的神经退行性过程，进而导致患者出现帕金森样症状。 知识差距阻碍了治疗的发展，并且如果利用分子机制，知识差距将持续存在 SLC30A10 尚不清楚，我们的假设是 SLC30A10 以更高的亲和力结合和转运 Mn。 比其他必需金属更重要，并且我们最近发现这种活性对细胞环境敏感。 然而，我们的研究使用了基于细胞的功能。 实验，并且没有提供它们参与锰流出活动的机制的分子细节。 为了阐明这一点，这里提出的实验将确定 SLC30A10 的 Mn 传输机制 在目标 1 中，我们将结合体外研究和生理相关的细胞测定。 对纯化的 SLC30A10 蛋白进行生化研究，揭示其结合和转运机制 首先，等温滴定量热法 (ITC) 将用于确定 Mn 结合系数 (KD)。 然后使用含有 SLC30A10 的人工膜进行蛋白脂质体转运测定。 确定 Mn 传输动力学（KM 和 Vmax） 我们将对 SLC30A10WT 与 SLC30A10 进行比较。 在我们的初步筛选中鉴定出的外排突变体可阐明直接参与 Mn 结合的残基。 然后在基于细胞的系统中进行，将采用共聚焦显微镜来评估 SLC30A10 功能。 原代神经元和肝细胞系中的金属定量测量将使用 ICP-MS 进行测量。 细胞内锰含量和证实的显微镜检查结果一起，是本次培训的结果。 计划将提高我们对细胞锰稳态的理解，因为它与神经毒性有关，并提供 SLC30A10 的生化数据对于开发针对锰毒性的疗法非常重要。