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）是必不可少的元素，但过度暴露是细胞毒性的，对 神经健康。在人类中，MN诱导的神经毒性通常是由于长期暴露于 占据或环境环境，类似于特发性帕金森氏病。在某些情况下，患者 由于疾病引起的肝功能受损，例如肝硬化，无法极端MN，可能会发展出Mn- 在没有高暴露的情况下引起帕金森氏症。 MN的营养和临床意义是 建立的MN稳态的细胞机制仍然未知。我们对我们的理解的突破 MN代谢来自据报道是由于 SLC30A10中的突变。我们实验室中的发现确定SLC30A10充当主要的MN外排 转运蛋白可保护细胞免受MN诱导的毒性。有趣的是，SLC30A10引起疾病 上面讨论的帕金森氏病患者的突变体无法预防高MN。 由于MN在环境中无处不在，我们的长期目标是阐明SLC30A10在 金属诱导的神经退行性过程，进而导致患者的帕金森症状。这 知识的差距会阻碍治疗的发展，如果分子机制利用 SLC30A10尚不清楚。我们的假设是SLC30A10结合并运输具有较高亲和力的MN 比其他必需金属，这种活性对细胞环境敏感。我们最近确定了 Mn外排活动所需的SLC30A10残基。但是，我们的研究使用了基于细胞的功能 实验，不提供其机械参与Mn外排活性的分子细节。 为了阐明这一点，此处提出的实验将确定SLC30A10的MN传输机理 结合体外研究和基于物理相关的基于细胞的测定。在AIM 1中，我们将表演 纯化的SLC30A10蛋白的生化研究揭示了结合和转运的机制 SLC30A10。首先，等温滴定量热法（ITC）将用于确定MN结合系数（KD） Slc30a10。然后，具有包含SLC30A10的人工机制的蛋白质体转运测定法 确定MN转运动力学（KM和VMAX）。我们将进行SLC30A10WT与SLC30A10的比较 在我们的主要筛选中鉴定出的外排突变体，以阐明直接参与MN结合的残差。 AIM 2意志 然后在基于细胞的系统中进行。共聚焦显微镜将进行评估SLC30A10功能 在原发性神经元和肝细胞系中。定量金属测量ICP-MS将用于测量 细胞内MN含量和证实显微镜发现。综上所述，这项培训的发现 计划将提高我们对与神经毒性相关的细胞MN稳态的理解并提供 SLC30A10的生化数据对于开发针对MN毒性的疗法很重要。