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

总结 慢性肾脏疾病（CKD）是一个主要的公共卫生问题，影响到2000多万人， 美国的非裔美国人不成比例地受到进行性CKD的影响，尤其是局灶性CKD。 节段性肾小球硬化（FSGS）。APOL 1羧基末端结构域的遗传变异是 与非洲血统人群中的FSGS和其他非糖尿病CKD相关。这些变体会改变 APOL 1的氨基酸序列，并解释了这些人群中CKD风险增加的大部分原因。的 由这些APOL 1变异体导致CKD的发病机制尚不清楚。的羧基末端 APOL 1与VAMP 8相互作用，VAMP 8是一种参与囊泡运输的内体/溶酶体SNARE蛋白， APOL 1肾病相关变异体减弱了与VAMP 8的相互作用，继发于蛋白质 由变体引起的构象变化。与SNARE蛋白一样，APOL 1定位于囊泡结构 在健康人肾脏的足细胞中。我们假设APOL 1：SNARE相互作用是必要的， 减轻/减弱足细胞应激反应，APOL 1变体破坏功能性SNARE相互作用， 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，我们将使用表达APOL 1的稳定细胞系和健康人肾脏来鉴定额外的相互作用。 SNARE蛋白。我们将使用活细胞共聚焦显微镜跟踪APOL 1：SNARE定位在各种 在存在和不存在免疫刺激物的情况下的亚细胞区室。 其次，我们将研究在不存在和存在APOL 1蛋白的情况下， 在细胞培养物中不存在和存在SNARE敲低的情况下， 模型 通过这些目标，我将获得必要的生物化学，成像和蛋白质组学数据，以解决我们的长期 目的是鉴定用于治疗APOL 1相关肾病的药物。了解分子 APOL 1变异体引起蛋白尿性CKD的机制对于开发新的 治疗策略我建议使用成像和蛋白质组学工具来研究APOL 1的病理作用。 了解其功能的变异，并学习生物信息学工具，使我能够开发模型来研究CKD 机制等作为一个年轻的医生科学家，这个项目将帮助我获得新的技能和知识， 研究一个困扰我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.