Structure and mechanism of the kidney disease-causing protein APOL1

项目总结 在美国，最近的非洲血统的人患慢性肾脏疾病的可能性是普通人的四倍 疾病(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不同和健康的社会决定因素，这种额外风险中的很大一部分可能是 归因于载脂蛋白L1(APOL1)基因的两个“高风险”变种。与APOL1相关的患者 肾脏疾病在较年轻时被诊断为慢性肾脏病，慢性肾脏病进展较快，而且病情较轻。 可能对现有的治疗方法有反应。尽管高危APOL1基因与 增加患肾脏疾病的风险是关于APOL1蛋白结构的公认的基本问题 高风险和低风险变种之间的活动差异仍然没有得到回答。经细菌提纯- 已观察到表达的APOL1蛋白具有非选择性阳离子通道活性。然而， 活性APOL1通道的齐聚状态及其选择性的结构决定因素是 未知。此外，高危变异体对APOL1通道活性的功能影响尚不清楚。 在前期工作中，我已经纯化了真核表达的重组APOL1蛋白，并表明 它们在体外可以形成活性离子通道。我假设APOL1形成低聚非选择性阳离子 通道，并且由高危变异的APOL1蛋白组成的通道允许比 由低风险变异蛋白组成的通道。为了检验这一假设，我提出了两个具体目标。在AIM 1、利用电子显微镜对APOL1蛋白的结构进行解析。在目标2中，我将重建纯净的 将APOL1蛋白导入脂质体，并用荧光分光光度法检测其活性。在……里面 从事这项工作，我将获得研究结构和生物物理学所需的关键技能和专业知识。 参与肾脏疾病发病机制的膜蛋白的性质。我会成为一名内科医生- 通过正规课程、科学规划和肾脏专家的直接指导成为科学家 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 PLAN将帮助我建立一个研究基础生化的独立调查员的职业生涯 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 一位肾病学家。

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.