Structure and mechanism of the kidney disease-causing protein APOL1

肾脏致病蛋白APOL1的结构和机制

• 批准号: 10525524
• 负责人: Joshua Samuel Waitzman
• 金额: $ 16.6万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10525524
• 关键词: African Trypanosomiasis; African ancestry; Age; Apolipoproteins; Biochemical Process; Biological Assay; Biophysical Process; Biophysics; Cations; Cell membrane; Characteristics; Chronic Kidney Failure; Complex; Cryoelectron Microscopy; Cytolysis; Development Plans; Disease; Disease Progression; Disputes; Electron Microscopy; Electrophysiology (science); End stage renal failure; Environment; Focal Segmental Glomerulosclerosis; Funding; Future; Genes; Genetic; Genotype; Goals; Health Services Accessibility; In Vitro; Ion Channel; Ions; Kidney; Kidney Diseases; Laboratories; Link; Lipoproteins; Liposomes; Membrane Proteins; Mentors; Mentorship; Modeling; Molecular; Negative Staining; Not Hispanic or Latino; Outcome; Pathogenesis; Pathologic; Patients; Persons; Pharmaceutical Preparations; Physicians; Physiology; Positioning Attribute; Property; Proteins; Recombinants; Renal glomerular disease; Reporting; Research; Research Personnel; Resistance; Resolution; Resource Sharing; Risk; Scientist; Serum; Structure; Techniques; Testing; Time; Toxic effect; Training; Trypanosoma; Trypanosoma brucei brucei; United States; Variant; Work; biophysical properties; career; career development; cytotoxic; disease diagnosis; drug development; high risk; improved; in vitro Assay; inhibitor; innovation; novel therapeutics; pathogen; podocyte; prevent; reconstitution; risk variant; skills; social health determinants; structural biology; targeted treatment

PROJECT SUMMARY In the United States, people of recent African ancestry are four times more likely to develop chronic kidney disease (CKD) and end stage kidney disease (ESKD) than non-Hispanic whites. While some of this risk can be attributed to differential access to care and social determinants of health, much of this excess risk can be attributed to two “high-risk” variants in the Apolipoprotein L1 (APOL1) gene. Patients with APOL1-associated kidney disease are diagnosed with CKD at younger ages, have more rapid CKD progression, and are less likely to respond to existing therapies. Although the link between the high-risk APOL1 genotype and the increased risk of kidney disease is well-established, basic questions about the structure of the APOL1 protein and the differences in activity between the high- and low-risk variants remain unanswered. Purified bacterially- expressed APOL1 proteins have been observed to have nonselective cation channel activity. However, the oligomerization state of the active APOL1 channel and the structural determinants of its selectivity are unknown. Furthermore, the functional effects of the high-risk variants on APOL1 channel activity are unknown. In preliminary work, I have purified eukaryotically-expressed, recombinant APOL1 proteins and shown that they can form active ion channels in vitro. I hypothesize that APOL1 forms oligomeric nonselective cation channels, and that channels composed of high-risk variant APOL1 proteins permit greater cation currents than channels composed of the low-risk variant protein. To test this hypothesis, I propose two specific aims. In Aim 1, I will solve the structure of the APOL1 protein using electron microscopy. In Aim 2, I will reconstitute purified APOL1 proteins into liposomes and characterize their activity using fluorometric liposome efflux assays. In pursuing this work, I will gain critical skills and expertise necessary to study the structural and biophysical properties of membrane proteins involved in the pathogenesis of kidney diseases. I will develop as a physician- scientist through formal coursework, scientific programming, and direct mentorship from experts in kidney disease, structural biology, and ion channel physiology. The proposed research aims and career development plan will help me establish a career as an independent investigator who studies the fundamental biochemical and biophysical processes that cause kidney disease, while continuing to treat patients with kidney disease as a nephrologist.

项目概要 在美国，近代非洲血统的人患慢性肾病的可能性是其他人的四倍 慢性肾病 (CKD) 和终末期肾病 (ESKD) 的风险高于非西班牙裔白人，但其中一些风险可能更高。 由于获得护理的机会不同以及健康的社会决定因素，这种过度风险的大部分可能是由于 归因于载脂蛋白 L1 (APOL1) 基因中的两个“高风险”变异。 肾脏疾病被诊断为 CKD 的年龄较轻，CKD 进展更快，且发生率更低 尽管高风险 APOL1 基因型与现有疗法之间存在联系。 肾脏疾病风险增加是关于 APOL1 蛋白结构的公认的基本问题 高风险变体和低风险变体之间的活性差异仍未得到解答。 已观察到表达的 APOL1 蛋白具有非选择性阳离子通道活性。 活性 APOL1 通道的寡聚状态及其选择性的结构决定因素是 此外，高风险变异对 APOL1 通道活性的功能影响也是未知的。 在前期工作中，我纯化了真核表达的重组 APOL1 蛋白，并表明 它们可以在体外形成活性离子通道，我敢说 APOL1 会形成寡聚非选择性阳离子。 通道，并且由高风险变体 APOL1 蛋白组成的通道允许比通道更大的阳离子电流 为了检验这一假设，我提出了两个具体目标。 1，我将使用电子显微镜解析 APOL1 蛋白的结构 在目标 2 中，我将重构纯化的蛋白。 APOL1 蛋白进入脂质体并使用荧光脂质体流出测定表征其活性。 从事这项工作，我将获得研究结构和生物物理所需的关键技能和专业知识 作为一名医生，我将研究与肾脏疾病发病机制有关的膜蛋白的特性。 科学家通过正规课程、科学编程和肾脏专家的直接指导 疾病、结构生物学和离子通道生理学提出的研究目标和职业发展。 计划将帮助我建立一个研究基础生物化学的独立研究者的职业生涯 和导致肾脏疾病的生物物理过程，同时继续治疗肾脏疾病患者 肾病专家。