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

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。知识价值。系统xc-是一种细胞运输系统，它允许胱氨酸，一种氨基酸，在细胞外部交换谷氨酸，一种氨基酸和神经递质，在细胞内部，在各种细胞类型中。这种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，谷胱甘肽的生产中发挥重要作用，从而保护细胞免受氧化应激的损害。首席研究员（PI）和她的学生最近证明，氧化剂，如过氧化氢，通过将转运体从细胞内部重新分配到活跃的细胞膜上，似乎可以在几分钟内对xc系统进行剧烈调节。这些令人兴奋的发现提示了xc系统的一种新的调节形式，它可能作为细胞防御系统的一个重要组成部分，保护细胞免受氧化损伤，并可能作为调节其他运输系统的模型。为了证实和扩展这些发现，本项目将使用生化和细胞生物学技术来阐明系统xc-活性通过插入和从膜中移除（膜运输）来调节的机制。具体来说，本项目中概述的实验将允许在培养的人类细胞的基础条件下鉴定系统xc-蛋白组分的膜运输途径。本研究还将探讨过氧化氢对xc-系统细胞膜表达和活性的调控机制。最后，本项目将研究xc-系统在不同细胞类型中的基础和受调节的运输，以确定氧化剂对xc-系统的急性调节是否对各种细胞都是共同的。因此，一旦完成，该项目将使我们对系统xc-的基础和管制贩运的基本理解取得重大进展。由于先前对这种转运蛋白的研究只关注在遗传控制下的转运蛋白的产生，因此这项研究将为xc系统的一种新的调节形式提供见解-它允许转运蛋白活性的急性调节。氧化剂似乎对这种胱氨酸/谷氨酸交换有强烈的调节作用，这一事实很有趣，因为这种转运体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-调控的理解外，该项目还为9名本科生和6名高中生提供研究培训，其中大多数学生将来自科学领域代表性不足的群体。在这个项目中提出的大部分研究将由这些学生完成，作为他们综合科学教育的一部分。学生们将与PI一起在项目中进行团队合作，学习如何提出假设，设计和进行实验，分析数据，并报告他们的发现。在PI的经历中（在8年中指导了41名学生），参与原创性研究项目使学生对科学过程的理解、对困难和复杂问题的批判性思考能力以及科学交流能力显著提高。因此，这个项目将培养学生为未来的科学事业做更好的准备。此外，这种研究经历可以激励PI的学生追求科学事业，从而为增加科学个人的多样性做出贡献。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《神经科学学会年会》等国家科学会议上发表报告来扩大。

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.