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.

摘要 这份K08职业发展建议书详细介绍了候选人对巨蛋白结构调节的研究 肾脏近端小管(PT)循环，将在5年的指导获奖期内进行。 在他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 来自经验丰富的多机构咨询委员会，Beenken博士设计了一个全面的计划 在教学方面和在低温电子显微镜(CRYO-EM)和质谱学方面的密集实验室研究。 这种培训将使他能够将自己与他的导师区分开来，并发展出一套他认为 可以用来创办一个独立的研究实验室，成为一名内科科学家。他的最终目标是翻译 了解巨蛋白循环的结构机制，为蛋白尿疾病的新治疗提供依据。 PT中依赖巨蛋白的内吞作用使蛋白捕获能够保护肾小管免受 蛋白尿造成的损害。巨蛋白必须在细胞表面和内小体之间循环，以完成其内吞 功能，但这种循环背后的结构性机制却知之甚少。宾肯博士已经 发表了pH为7.5和5.2的巨蛋白的冷冻-EM结构，揭示了细胞表面的巨蛋白结构 在晚期的内吞体内。这些结构表明，巨蛋白经历了广泛的pH依赖性 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的结构转变 酸性内噬菌体。Beenken博士的工作使他提出假设，巨型蛋白在 不同的内体隔室，受巨蛋白中特定残基的调节并依赖于变化 在巨蛋白对钙离子的协调作用中。在他提出的研究项目中，宾肯博士将通过以下方式检验他的假设 测定巨蛋白在不同内体隔室中的结构，包括早期和再循环 内体(目标1)，运输过程中对pH敏感的巨蛋白残留物的特征(目标2)，并确定 巨蛋白循环对钙离子配位的依赖(目标3)。追求这些研究目标将为 Beenken博士在甲状旁腺和丘脑中巨蛋白受体循环的结构生物学方面的职业生涯的基础 使他成为一名独立的调查员。最终，他计划在基础科学方面的发现 将巨蛋白在PT中的再循环转化为对蛋白尿病的临床干预。