Molecular mechanisms underlying heme transport at the blood-brain barrier and its role in angiogenesis

PROJECT SUMMARY This application details career development and research plans that have been uniquely tailored to facilitate transition of the principal investigator, Dr. Rosemary J. Cater, to an independent academic position. Dr. Cater has a multidisciplinary background, and through this K99/R00 proposal, seeks to finalize her biochemical training, master the biology of a new system, and gain skills for her career as an independent investigator. The K99 phase of this award (first two years) involves a structured career development plan that will allow Dr. Cater to: advance her skills in single particle cryo-electron microscopy (cryo-EM); gain essential training in brain endothelial cell-based assays and proximity-based labeling experiments; and acquire important career skills such as grant writing, science communication, laboratory management, teaching, responsible conduct of research, and mastery of the academic job application process. The career development plan also includes clear and actionable steps for identifying and successfully obtaining an independent tenure-track faculty position by the end of the K99 phase. Dr. Cater has assembled a top-tier team of multi-disciplinary mentors, advisors, and collaborators that will oversee and guide her training, research program, and transition to independence. The research plan proposed spans both the mentored (K99) and independent (R00) phases of the award. It involves mechanistic studies of the heme transporter FLVCR2, which is expressed within blood-brain barrier endothelial cells and plays a key role in the development of brain vasculature. The research program for the K99 phase aims to characterize the structure and function of FLVCR2 at a molecular level. The R00 research program then aims to delineate how FLVCR2 regulates angiogenesis at a cellular level. Core research questions addressed include: What are the molecular determinants important for FLVCR2 to bind heme specifically? What conformational changes must FLVCR2 undertake to transport heme? What drives this transport process? And ultimately, how is FLVCR2-mediated heme transport linked to cerebral angiogenesis? To answer these questions, Dr. Cater has formed a comprehensive research plan combining structural biology, biophysics, membrane protein biochemistry, electrophysiology, and cellular biology. It involves cryo-EM structure determination of FLVCR2; functional characterization of FLVCR2 using liposome-based assays and electrophysiology; interrogation of the link between FLVCR2 and angiogenesis using brain endothelial cell-based assays; and mapping the FLVCR2 interactome using proximity-based labeling assays. This research will provide key insights into how FLVCR2 transports heme in brain endothelial cells and this in turn regulates angiogenesis. The proposed studies for the K99 phase will largely take place at Columbia University, which is home to a vibrant and collegial community of structural biologists, biophysicists, and biochemists. This environment is ideal to facilitate Dr. Cater in the successful completion of the proposed K99 research program and achieving her goal of transitioning into a successful independent researcher.