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

载脂蛋白 L1 与 SNARE 蛋白在慢性肾脏病发病机制中的相互作用

• 批准号: 10055541
• 负责人: Sethu Madhavan Madhavan
• 金额: $ 16.09万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10055541
• 关键词: APOL1 gene; Accounting; Address; Affect; African; African American; Age; American; Amino Acid Sequence; Apolipoproteins; Attenuated; Autophagosome; Binding; Biochemical; Biological; Biological Models; Cell Culture Techniques; Cell Line; Cells; Cellular biology; Chronic Kidney Failure; Co-Immunoprecipitations; Confocal Microscopy; Cytoplasm; Cytoskeleton; Data; Data Analyses; Defect; Development; Diagnosis; Dialysis procedure; Disease; Disease Progression; End stage renal failure; Event; Expenditure; Focal Segmental Glomerulosclerosis; Follow-Up Studies; Genes; Genetic Predisposition to Disease; Genetic Variation; Goals; Graft Survival; High Density Lipoprotein Cholesterol; Human; Image; Immune; Immune System Diseases; Immunofluorescence Microscopy; Immunologic Factors; Immunologic Tests; Individual; Interferon Type II; Interleukin-1 beta; Investigation; Kidney; Kidney Diseases; Kidney Failure; Knowledge; Lead; Learning; Location; Mass Spectrum Analysis; Measures; Mediating; Medicare; Membrane Fusion; Minority; Modeling; Molecular; NMR Spectroscopy; Pathogenesis; Pathologic; Pathway interactions; Patients; Pattern; Physicians; Physiological; Population; Prevalence; Prevention; Protein Conformation; Protein Family; Proteins; Proteinuria; Proteomics; Public Health; Publishing; Risk; Role; SNAP receptor; Scientist; Secondary to; Stimulus; Structural Models; Structure; TNF gene; Testing; Time; Transgenic Mice; United States; Variant; Vesicle; base; bioinformatics tool; biological adaptation to stress; clinically significant; cytotoxicity; design; disease diagnosis; experimental study; genetic variant; glomerulosclerosis; human imaging; immune activation; in silico; knock-down; live cell imaging; non-diabetic; novel strategies; novel therapeutic intervention; patient population; podocyte; public health relevance; recruit; response; risk variant; skills; stable cell line; three dimensional structure; tool; trafficking

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.

概括 慢性肾脏病 (CKD) 是影响超过 2000 万人的主要公共卫生问题 美国。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 节段性肾小球硬化症（FSGS）。 APOL1 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。这些变体发生变化 APOL1 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。这 这些 APOL1 变异导致 CKD 的发病机制尚不清楚。的羧基末端 APOL1 与 VAMP8 相互作用，VAMP8 是一种参与囊泡运输的内体/溶酶体 SNARE 蛋白， APOL1 肾病相关变异减弱了与 VAMP8 继发于蛋白质的相互作用 由变异引起的构象变化。与 SNARE 蛋白一样，APOL1 定位于囊泡结构 在健康人肾脏的足细胞中。我们假设 APOL1:SNARE 相互作用对于 减轻/减弱足细胞应激反应，APOL1 变异破坏功能性 SNARE 相互作用， 介导细胞毒性。 为了检验这一假设，我们提出以下目标： 首先，我们将使用表达 APOL1 的稳定细胞系和健康的人类肾脏来识别其他相互作用 SNARE 蛋白。我们将使用活细胞共聚焦显微镜来跟踪 APOL1:SNARE 在各种情况下的定位 在存在和不存在免疫刺激的情况下的亚细胞区室。 其次，我们将研究参考蛋白和变体 APOL1 蛋白在不存在和存在的情况下的细胞毒性 细胞培养中不存在和存在 SNARE 敲低的情况下免疫刺激和参考 APOL1 的变化 模型。 通过这些目标，我将获得解决我们长期问题所需的生化、成像和蛋白质组学数据。 确定治疗 APOL1 相关肾脏疾病的药物的目标。了解分子 APOL1 变异导致蛋白尿 CKD 的机制对于开发新药物至关重要 治疗策略。我建议使用成像和蛋白质组学工具来研究APOL1的病理作用 其功能的变体并学习生物信息学工具，使我能够开发模型来研究 CKD 机制。作为一名年轻的医师科学家，这个项目将帮助我获得新的技能和知识，同时 研究困扰我的患者的一个重要的公共卫生问题。