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

项目总结 这份申请详细说明了职业发展和研究计划，这些计划是专门为促进 首席调查员罗斯玛丽·J·卡特博士过渡到独立的学术职位。卡特尔医生 拥有多学科背景，并通过K99/R00提案，寻求最终确定她的生化 培训，掌握一个新系统的生物学，并获得作为一名独立调查员的职业技能。 该奖项的K99阶段(头两年)涉及一个结构化的职业发展计划，这将使博士。 迎合：提高她在单粒子冷冻电子显微镜(Cryo-EM)方面的技能；获得基本的大脑培训 基于内皮细胞的分析和基于邻近的标记实验；并获得重要的职业技能 如资助撰写、科学交流、实验室管理、教学、负责任的行为 研究，并掌握学术工作申请流程。职业发展计划还包括明确 确定并成功获得独立终身教职教师职位的可行步骤 K99阶段的结束。Cater博士组建了一支由多学科导师、顾问和 将监督和指导她的培训、研究计划和向独立过渡的合作者。 建议的研究计划跨越了奖项的指导(K99)和独立(R00)两个阶段。它 涉及血红素转运蛋白FLVCR2的机制研究，它在血脑屏障中表达 血管内皮细胞在脑血管发育中起着关键作用。K99的研究计划 阶段的目的是在分子水平上表征FLVCR2的结构和功能。R00研究计划 然后旨在描述FLVCR2如何在细胞水平上调节血管生成。核心研究问题 涉及的问题包括：FLVCR2与血红素特异性结合的重要分子决定因素是什么？什么 构象变化FLVCR2必须承担运输血红素？是什么推动了这一运输过程？和 最终，FLVCR2介导的血红素转运是如何与脑血管生成联系起来的？ 为了回答这些问题，卡特博士制定了一项结合结构生物学的全面研究计划， 生物物理学、膜蛋白生物化学、电生理学和细胞生物学。它涉及到低温电磁结构 FLVCR2的测定；FLVCR2的功能表征 电生理学：用脑内皮细胞研究FLVCR2和血管生成之间的联系 以及使用基于邻近的标记分析来定位FLVCR2相互作用组。这项研究将提供 对FLVCR2如何在脑内皮细胞中运输血红素，进而调节血管生成的关键见解。 K99阶段的拟议研究将主要在哥伦比亚大学进行，该大学拥有一个充满活力的 以及结构生物学家、生物物理学家和生物化学家的合伙社区。这种环境非常适合 协助Cater博士成功完成拟议的K99研究计划并实现她的目标 转变为一名成功的独立研究人员。

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.