MCA Pilot PUI: Use of advanced mass spectrometry techniques to study the regulation of the membrane transporter, System xc-

Hope College, Holland MI

Investigators

Abstract

This project will provide the investigator dedicated time to receive training in mass spectrometry in order to study a membrane transport system (System xc-) that plays a critical role in the acute cellular response to oxidative insult. The investigator will work collaboratively with her research partner to use advanced mass spectrometry techniques to identify molecular tags that are attached to the transporter under basal and oxidative cellular conditions. Since membrane proteins are dynamically regulated by these molecular tags, this proteomic approach will lead to the identification of novel and functionally relevant modifications and a more comprehensive understanding of the cellular response to oxidative insult. Moreover, this project will serve as a platform for the training of Hope College undergraduates who will complete this work as a part of their comprehensive education in the sciences. Students will engage in this project through independent research experiences and Course-based Undergraduate Research Experiences (CURE) in the investigator’s Neurochemistry lab. Finally, this project will facilitate a new connection between Hope College and UIC, providing new opportunities for Hope College undergraduates who are interested in post-baccalaureate training, while also expanding the Hope College and Midwest community of mass spectrometry scholars. This objective of this project is to employ proteomics to study the mechanism by which oxidants rapidly upregulate System xc-, a cystine/glutamate exchanger that is required for the synthesis of glutathione, an endogenous antioxidant. Based on the investigator’s previous work, she hypothesizes that oxidants activate cellular signaling networks that trigger changes in 1) the post-translational status of the transporter and 2) its interactions with trafficking proteins, ultimately resulting in the translocation of the transporter to the plasma membrane, an increase in cystine import and glutathione synthesis. The investigator and her research partner will use advanced bottom-up mass-spectrometry techniques to identify post-translational modifications (PTM) on the transporter in basal and oxidative cellular environments. The functional significance of each PTM will be assessed using site-directed mutagenesis coupled with a biochemical transporter activity screen. Finally, quantitative proteomics will be used to identify 1) the signaling and trafficking proteins that directly interact with the transporter and 2) the signaling networks that are acutely modulated by oxidant exposure. Thus, this project will directly address the current gap in our understanding of the molecular processes that acutely regulate System xc- and advance our understanding of cellular antioxidant defense mechanism. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.