Structural regulation of megalin recycling in the proximal tubule

近曲小管巨蛋白循环的结构调节

• 批准号: 10802792
• 负责人: ANDREW S BEENKEN
• 金额: $ 16.65万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10802792
• 关键词: Adopted; Advisory Committees; Affinity; Albumins; Alkalinization; Amino Acids; Apical; Award; Back; Basic Science; Binding; Binding Proteins; Binding Sites; Biological Assay; Biological Markers; Bowman' s space; Cardiovascular system; Cell membrane; Cell surface; Cells; Chronic Kidney Failure; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; Dependence; Didelphidae; Disease; Dissociation; Early Endosome; Egtazic Acid; Endocytosis; Endosomes; Epithelial Cells; Event; Filtration; Future; Goals; Immunofluorescence Immunologic; Institution; Intervention; Ions; LDL-Receptor Related Protein 2; Laboratory Research; Life Cycle Stages; Ligand Binding; Ligands; Mass Spectrum Analysis; Mediating; Mentors; Molecular; Molecular Chaperones; Molecular Weight; Mutate; Mutation; Physicians; Process; Proteins; Proteinuria; Proximal Kidney Tubules; Publishing; Recycling; Regulation; Renal tubule structure; Research; Research Personnel; Research Project Grants; Scientist; Signal Transduction; Sorting; Structure; Surface; Surface Plasmon Resonance; Testing; Training; Translating; Tubular formation; Universities; Urine; Variant; Work; absorption; apical membrane; career; career development; chelation; design; experience; experimental study; glomerular filtration; kidney cell; late endosome; mutant; particle; protein complex; protein structure; receptor; receptor recycling; reconstruction; stoichiometry; structural biology; trafficking; urinary

Abstract This K08 Career Development Proposal details the candidate’s research on the structural regulation of megalin recycling in the kidney proximal tubule (PT) that will be conducted during a 5-year mentored award period. With the guidance of his co-mentors at Columbia University, Dr. Shapiro and Dr. Barasch, along with oversight from an experienced multi-institutional advisory committee, Dr. Beenken has designed a comprehensive plan of didactics and intensive laboratory research in cryo-electron microscopy (cryo-EM) and mass spectrometry. This training will enable him to differentiate himself from his mentors and develop a body of research that he can use to start an independent research laboratory as a physician-scientist. His ultimate goal is to translate the understanding of structural mechanisms of megalin recycling to new treatments for proteinuric disease. Megalin-dependent endocytosis in the PT enables protein capture that protects the kidney tubules from damage due to proteinuria. Megalin must recycle between cell surface and endosomes to fulfill its endocytic function, but the structural mechanisms that underlie this recycling are poorly understood. Dr. Beenken has published cryo-EM structures of megalin at pH 7.5 and 5.2, revealing megalin’s structure at the cell surface and in the late endosome. These structures demonstrated that megalin undergoes extensive pH-dependent structural transitions to bind ligands from the urinary filtrate at the cell surface and then shed ligands in the acidic endosomes. Dr. Beenken’s work led him to hypothesize that megalin adopts unique structures in different endosomal compartments that are regulated by specific residues in megalin and depend on changes in megalin’s coordination of Ca2+ ions. In his proposed research project, Dr. Beenken will test his hypothesis by determining structures of megalin in different endosomal compartments including early and recycling endosomes (Aim 1), characterizing pH-sensitive megalin residues during trafficking (Aim 2), and determining the dependence of megalin recycling on Ca2+-coordination (Aim 3). Pursuing these research goals will lay the groundwork for Dr. Beenken’s career in the structural biology of megalin receptor recycling in the PT and enable him to become an independent investigator. Ultimately, he plans for his discoveries in the basic science of megalin recycling in the PT to translate to clinical interventions for proteinuric disease.

抽象的 本 K08 职业发展提案详细介绍了候选人对巨蛋白结构调控的研究 肾脏近端小管 (PT) 的回收将在 5 年指导奖励期内进行。 在哥伦比亚大学的共同导师夏皮罗博士和巴拉什博士的指导下以及监督下 宾肯博士在经验丰富的多机构咨询委员会的指导下，设计了一个全面的计划 冷冻电子显微镜 (cryo-EM) 和质谱方面的教学和密集实验室研究。 这种培训将使他能够从他的导师中脱颖而出，并开展一系列研究 可以作为医生科学家启动一个独立的研究实验室。他的最终目标是翻译 了解巨蛋白循环的结构机制以用于蛋白尿疾病的新疗法。 PT 中巨蛋白依赖性内吞作用能够捕获蛋白质，从而保护肾小管免受 蛋白尿造成的损害。巨蛋白必须在细胞表面和内体之间循环才能完成其内吞作用 功能，但人们对这种回收背后的结构机制知之甚少。宾肯博士有 发表了 pH 7.5 和 5.2 下巨蛋白的冷冻电镜结构，揭示了细胞表面巨蛋白的结构 和晚期内体中。这些结构表明巨蛋白经历了广泛的 pH 依赖性 结构转变，在细胞表面结合尿滤液中的配体，然后在细胞表面脱落配体 酸性内体。 Benken 博士的工作使他推测巨蛋白在 不同的内体区室受巨蛋白中特定残基的调节并依赖于变化 巨蛋白与 Ca2+ 离子的配位。在他提出的研究项目中，Beenken 博士将通过以下方式检验他的假设： 确定不同内体区室中巨蛋白的结构，包括早期和回收 内体（目标 1），表征运输过程中 pH 敏感的巨蛋白残基（目标 2），并确定 巨蛋白回收对 Ca2+ 配位的依赖性（目标 3）。追求这些研究目标将奠定 为 Beenken 博士在 PT 和巨蛋白受体回收的结构生物学领域的职业生涯奠定了基础 使他能够成为一名独立调查员。最终，他计划在基础科学领域取得发现 PT 中巨蛋白的回收转化为蛋白尿疾病的临床干预措施。