Structure and mechanism of the kidney disease-causing protein APOL1

项目摘要 在美国，最近的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）的发病率高于非西班牙裔白人。虽然其中一些风险可能是 由于获得保健的机会和健康的社会决定因素不同，这种过度风险大部分可能是 这归因于载脂蛋白L1（APOL 1）基因中的两种“高危”变体。APOL 1相关患者 肾脏疾病在年轻时被诊断为CKD，CKD进展更快， 可能对现有疗法有反应。尽管高危APOL 1基因型与 肾脏疾病的风险增加是一个关于APOL 1蛋白结构的基本问题 并且高风险和低风险变体之间的活性差异仍然没有答案。经过细菌净化- 已观察到表达的APOL 1蛋白具有非选择性阳离子通道活性。但 活性APOL 1通道的寡聚化状态及其选择性的结构决定因素是 未知此外，高风险变体对APOL 1通道活性的功能影响尚不清楚。 在初步工作中，我已经纯化了真核表达的重组APOL 1蛋白，并表明， 它们在体外可形成活性离子通道。我假设APOL 1形成寡聚的非选择性阳离子 通道，并且由高风险变体APOL 1蛋白组成的通道允许比 由低风险变体蛋白组成的通道。为了验证这一假设，我提出了两个具体目标。在Aim中 1，我将使用电子显微镜解决APOL 1蛋白的结构。在目标2中，我将重组纯化的 将APOL 1蛋白导入脂质体中，并使用荧光脂质体流出试验表征其活性。在 从事这项工作，我将获得必要的关键技能和专业知识，研究结构和生物物理 肾脏疾病发病机制中涉及的膜蛋白的性质。我要成为一名医生- 科学家通过正式的课程，科学的编程，并从肾脏专家的直接指导 疾病、结构生物学和离子通道生理学。建议的研究目标和职业发展 计划将帮助我建立一个职业生涯，作为一个独立的调查员谁研究的基本生物化学 以及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， 肾病学家

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.