RUI: Constitutive and Regulated Trafficking of the Cystine/Glutamate Exchanger, System xc-

该奖项由2009年《美国复苏和再投资法案》（公法111-5）资助。智力价值。 系统xc-是一种细胞转运系统，允许细胞外的胱氨酸（一种氨基酸）与细胞内的谷氨酸（一种氨基酸和神经递质）进行交换。 转运蛋白存在于大脑、视网膜、肾脏和小肠中。 系统xc-已被证明在调节抗氧化剂谷胱甘肽的产生中起重要作用，从而保护细胞免受氧化应激的损害。 首席研究员（PI）和她的学生最近表明，过氧化氢等氧化剂似乎可以在几分钟内通过将转运蛋白从细胞内部重新分配到细胞膜上来调节系统xc。 这些令人兴奋的发现表明系统xc的一种新的调节形式-可能作为细胞防御系统的重要组成部分，保护细胞免受氧化损伤，并可能作为其他运输系统调节的模型。 为了证实和扩展这些发现，本项目将使用生物化学和细胞生物学技术来阐明系统xc-活性通过插入和从膜上移除（膜运输）来调节的机制。 具体而言，本项目中概述的实验将允许在基础条件下在培养的人类细胞中鉴定System xc蛋白组分的膜运输途径。 该研究还将研究过氧化氢急性调节细胞膜表达和系统xc-活性的机制。 最后，本项目将研究系统xc-在不同细胞类型中的基础和调节运输，以确定氧化剂对系统xc-的急性调节是否对各种细胞都有影响。 因此，完成后，该项目将导致我们对系统xc-的基础和受管制贩运的基本理解取得重大进展。 由于以前对这种转运蛋白的研究只集中在转运蛋白的产生上，而这种转运蛋白是在遗传控制下产生的，因此这项研究将深入了解系统xc的一种新的调节形式-它允许对转运蛋白活性进行急性调节。氧化剂似乎能剧烈调节这种胱氨酸/谷氨酸交换剂，这一事实令人感兴趣，因为转运蛋白为细胞提供合成抗氧化剂谷胱甘肽所需的胱氨酸。 因此，这些研究可能揭示了细胞在暴露于氧化剂后立即利用的重要早期防御机制。更广泛的影响。 除了提高我们对系统xc-监管的理解外，该项目还为9名本科生和6名高中生提供研究培训，其中大多数人将来自科学代表性不足的群体。 该项目中提出的大部分研究将由这些学生完成，作为其科学综合教育的一部分。 学生将在团队中与PI并肩合作，学习开发假设，设计和进行实验，分析数据，并报告他们的发现。 在PI的经验（41名学生在8年的指导），在原始研究项目的参与导致学生的科学过程的理解，批判性思考困难和复杂的问题的能力，以及沟通科学的能力显着收益。 因此，该项目将培养学生谁将更好地为未来的科学职业生涯做好准备。 此外，这种研究经验可以激励PI的学生追求科学事业，从而有助于增加科学中个人的多样性。 该项目的影响将通过在《生物化学杂志》等期刊上发表科学发现以及在实验生物学或神经科学年会等国家科学会议上发表演讲来扩大。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit. System xc- is a cell transport system that allows for the exchange of cystine, an amino acid, on the outside of the cell for glutamate, an amino acid and neurotransmitter, on the inside in of the cell in a variety of cell types. The transporter is found in the brain, retina, kidney and small intestine. System xc- has been shown to play an important role in regulating the production of the antioxidant, glutathione, and thus protecting cells from damage as a result of oxidative stress. The principal investigator (PI) and her students have recently shown that oxidants such as hydrogen peroxide appear to acutely (within minutes) regulate System xc- by redistributing the transporter from the inside of the cell to the cell membrane where it is active. These exciting findings suggest a novel form of regulation of System xc- that may serve as an important component of the cellular defense system in protecting cells from oxidative damage and may serve as a model for the regulation of other transport systems. To confirm and extend these findings, this project will use biochemical and cell biological techniques to elucidate the mechanism by which System xc- activity is regulated by insertion and removal from the membrane (membrane trafficking). Specifically, the experiments outlined in this project will allow for the identification of the membrane trafficking pathways of the protein components of System xc- under basal conditions in cultured human cells. The research will also investigate the mechanism by which hydrogen peroxide acutely regulates the cell membrane expression and activity of System xc-. Finally, this project will examine the basal and regulated trafficking of System xc- in distinctly different cell types in order to determine if the acute regulation of System xc- by oxidants is common to a variety of cells. Thus, when completed, this project will lead to significant advancements in our fundamental understanding of the basal and regulated trafficking of System xc-. Since previous studies of this transporter have focused only on the production of the transporter, which is under genetic control, this study will provide insight into a novel form of regulation of System xc- which allows for acute modulation of transporter activity. The fact that oxidants appear to acutely regulate this cystine/glutamate exchanger is intriguing since the transporter provides cells with the cystine required to synthesize the antioxidant glutathione. Therefore, these studies may reveal an important early defense mechanism utilized by cells immediately after exposure to an oxidative agent. Broader impacts. In addition to improving our understanding of the regulation of System xc-, this project provides research training for nine undergraduate students and six high school students, most of whom will be from groups underrepresented in science. The majority of the research proposed in this project will be completed by these students as a part of their comprehensive education in the sciences. Students will work collaboratively in teams on the proposed project side-by-side with the PI, learning to develop hypotheses, to design and conduct experiments, to analyze data, and to report their findings. In the PI's experience (41 students mentored in 8 years), engagement in original research projects leads to significant gains in students' understanding of the scientific process, ability to think critically about difficult and complex problems, and ability to communicate about science. Thus, this project will train students who will be better prepared for future careers in science. Additionally, this research experience could inspire the PI's students to pursue scientific careers, and thus, to contribute to increasing the diversity of individuals in science. The impact of this project will be extended by publishing the scientific findings in journals such as the Journal of Biological Chemistry and by presentations made at national scientific meetings such as Experimental Biology or the Annual Society for Neuroscience Meeting.