Apolipoprotein L1 Interaction with SNARE Proteins in the Pathogenesis of Chronic Kidney Disease

摘要 慢性肾脏疾病(CKD)是一个主要的公共卫生问题，影响着全球2000多万人 美国。非裔美国人不成比例地受到进行性慢性KD的影响，特别是局灶性 节段性肾小球硬化(FSGS)。APOL1的羧基末端结构域的遗传变异是 在具有非洲血统的人群中，与FSGS和其他非糖尿病CKD相关。这些变种会发生变化 APOL1的氨基酸序列，并解释了这些人群中CKDS风险增加的主要原因。这个 这些APOL1变异导致CKD的致病机制尚不清楚。的羧基末端 APOL1与VAMP8相互作用，VAMP8是一种内体/溶酶体圈套蛋白，参与囊泡运输和 APOL1肾病相关变异体减弱VAMP8与蛋白质的相互作用 变异体引起的构象变化。与SNARE蛋白一样，APOL1定位于泡状结构 在健康的人类肾脏的足细胞中。我们假设APOL1：SNARE相互作用是必要的 减轻/减弱足细胞应激反应，而APOL1变异体破坏功能性SNAR相互作用和 介导细胞毒性。 为了验证这一假设，我们提出了以下目标： 首先，我们将使用稳定表达APOL1的细胞系和健康的人肾脏来鉴定其他相互作用 圈套蛋白。我们将使用活细胞共聚焦显微镜追踪APOL1：SNARE在不同组织中的定位 在免疫刺激存在和不存在的情况下的亚细胞间隔。 其次，我们将研究参考和变异的APOL1蛋白在没有和存在的情况下的细胞毒性 在细胞培养中没有和存在SNARE击倒的情况下免疫刺激和参考APOL1的研究 模特们。 通过这些目标，我将获得必要的生化、成像和蛋白质组学数据，以解决我们的长期问题 确定治疗APOL1相关肾脏疾病的药物的目标。理解分子 APOL1变异导致蛋白尿性CKD的机制对新的 治疗策略。我建议使用成像和蛋白质组学工具来研究APOL1的病理效应 并学习生物信息学工具，使我能够开发模型来研究CKD 机械装置。作为一名年轻的内科科学家，这个项目将帮助我获得新的技能和知识，同时 研究一个困扰我的病人的重要公共卫生问题。

Summary Chronic kidney diseases (CKDs) are a major public health problem affecting more than 20 million people in the United States. African Americans are disproportionately affected by progressive CKDs, especially focal segmental glomerulosclerosis (FSGS). Genetic variants in the carboxy-terminal domain of APOL1 are associated with FSGS and other non-diabetic CKDs in populations with African ancestry. These variants change the amino acid sequence of APOL1 and explain much of this increased risk for CKDs in these populations. The pathogenetic mechanisms that lead from these APOL1 variants to CKD are not known. The carboxy-terminus of APOL1 interacted with VAMP8, an endosomal/lysosomal SNARE protein involved in vesicular trafficking and APOL1 kidney disease-associated variants attenuated the interaction with VAMP8 secondary to protein conformational changes induced by the variants. Like SNARE proteins, APOL1 localized to vesicular structures in the podocytes of the healthy human kidney. We hypothesize that APOL1:SNARE interaction is necessary to mitigate/attenuate podocyte stress response, and APOL1 variants disrupt functional SNARE interaction and mediate cytotoxicity. To test this hypothesis, we propose the following aims: First, we will use stable cell lines expressing APOL1 and healthy human kidneys to identify additional interacting SNARE proteins. We will use live-cell confocal microscopy to track APOL1:SNARE localization in various subcellular compartments in the presence and absence of immune stimuli. Second, we will investigate the cytotoxicity of reference and variant APOL1 proteins in the absence and presence of immune stimuli and reference APOL1 in the absence and presence of SNARE knockdown in cell culture models. Through these aims, I will obtain biochemical, imaging and proteomics data necessary to address our long term goal of identifying agents for the treatment of APOL1-associated kidney diseases. Understanding the molecular mechanisms by which APOL1 variants cause proteinuric CKD is essential for the development of new therapeutic strategies. I propose to use imaging and proteomics tools to study the pathological effects of APOL1 variants on its function and to learn bioinformatics tools to allow me to develop models to investigate CKD mechanisms. As a young physician-scientist, this project will help me to acquire new skills and knowledge while studying an important public health problem that afflicts my patients.