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

摘要： 锰是一种必需元素，但过量接触对细胞有毒性，对人体健康有不良影响。 神经健康。在人类中，锰引起的神经毒性通常是由于长期接触以下物质而发生的 职业或环境环境，类似于特发性帕金森氏症。在某些情况下，患者 由于疾病，如肝硬变，肝功能受损，不能排出锰，并可能发展为锰- 在没有高暴露的情况下诱发帕金森氏症。而锰的营养和临床意义是 已经确定的，细胞机制的锰动态平衡仍不清楚。我们对……认识上的突破 锰代谢来自于一种家族性帕金森氏症的鉴定，据报道，帕金森症是由于 SLC30A10基因突变。我们实验室的发现已经确定SLC30A10作为主要的锰外流 转运蛋白，以保护细胞免受锰诱导的毒性。有趣的是，SLC30A10致病 以上讨论的帕金森病患者的突变株不能抵抗高锰。 由于锰在环境中普遍存在，我们的长期目标是阐明SLC30A10在 金属诱导的神经变性过程，进而导致患者出现帕金森样症状。这 知识上的差距阻碍了治疗的发展，如果利用分子机制治疗将持续下去 SLC30A10尚不清楚。我们的假设是SLC30A10以更高的亲和力结合和运输MN 这种活性对细胞环境很敏感。我们最近确认了 锰外排活动所需的SLC30A10残基。然而，我们的研究使用了基于细胞的功能 实验没有提供它们参与锰外排活性的分子细节。 为了阐明这一点，这里提出的实验将确定SLC30A10的锰运输机制 使用体外研究和生理相关的细胞分析相结合的方法。在目标1中，我们将表演 纯化SLC30A10蛋白的生化研究以揭示其结合和转运机制 SLC30A10。首先，将使用等温滴定量热法(ITC)来确定锰结合系数(Kd)。 SLC30A10。然后，用含有SLC30A10的人工膜进行蛋白质脂质体转运试验 测定锰的转运动力学(Km和Vmax)。我们将对SLC30A10WT和SLC30A10进行比较 在我们的初步筛选中发现了外排突变体，以阐明与锰结合直接相关的残基。目标2将 然后在基于蜂窝的系统中执行。将使用共聚焦显微镜来评估SLC30A10的功能 在原代神经元和一个肝细胞系中。定量金属测量电感耦合等离子体质谱将用于测量 细胞内的锰含量和确证的显微镜结果。综上所述，这次培训的发现 PLAN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的生化数据对开发抗锰中毒的治疗方法具有重要意义。

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.