Role of the novel gene fam4b in phosphatidylserine synthesis in podocytes and mai

新基因fam4b在足细胞和麦磷脂酰丝氨酸合成中的作用

• 批准号: 8302056
• 负责人: HUA A LU
• 金额: $ 25.74万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-17 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302056
• 关键词: Acute Glomerulonephritis; Affect; Animal Model; Animals; Apoptosis; Binding; Biochemical; Biological Assay; Biological Models; Biology; Cell membrane; Cells; Co-Immunoprecipitations; Complex; Data; Diacylglycerol Kinase; Disease; Down-Regulation; Dynamin; Embryo; Endothelium; Filtration; Fluorescence; Fluorescence Recovery After Photobleaching; Focal Segmental Glomerulosclerosis; Foot Process; Functional disorder; Genes; Guanosine Triphosphate Phosphohydrolases; Human; Imaging technology; Kidney; Kidney Diseases; Kidney Failure; Lipids; Maintenance; Mediating; Membrane Microdomains; Membrane Proteins; Metabolism; Modeling; Morphology; Mus; NPHS2 protein; Organ; Patients; Phosphatidylserine Synthase; Phosphatidylserines; Phospholipid Metabolism; Phospholipids; Plasma; Play; Process; Protein Kinase; Protein Kinase C; Proteins; Proteinuria; Renal function; Renal glomerular disease; Reporter; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Molecule; Specialized Epithelial Cell; Structure; System; Testing; Ultrafiltration; Vesicle; Water; Yeasts; Zebrafish; blood filter; glomerular basement membrane; glomerular filtration; in vitro Model; in vivo; knock-down; migration; mouse model; nephrin; new therapeutic target; novel; overexpression; podocyte; protein transport; salt balance; slit diaphragm; spine bone structure; trafficking; wasting; yeast two hybrid system

DESCRIPTION (provided by applicant): The central function of mammalian kidney is ultrafiltration of plasma through the complex glomerular filtration barrier that is composed of a fenestrated endothelium, a glomerular basement membrane (GBM) and specialized epithelial cells called podocytes. The foot processes of each podocyte interconnect on the top of the GBM and protein bridges between neighboring cells from the slit diaphragm. Disruption of the slit diaphragm (SD) causes leakiness of the glomerulus, manifested as proteinuria, a typical presentation in many renal diseases. Therefore, maintaining the integrity of the glomerular filtration barrier is critical for normal kidney function. Several podocyte gene products have been found to contribute to the structure and function of the SD, such as Nephrin, CD2AP and Podocin. From a search for other factors contributing to SD function, we discovered a novel gene, fam49b, that is highly expressed in developing and mature podocytes in both zebrafish and mouse kidneys. Knocking down fam49b in zebrafish embryos led to podocyte foot process effacement, altered podocyte morphology, and renal failure. A down regulation of fam49b expression was consistently observed in kidneys from mice with proteinuric glomerular dysfunction and from patients with focal segmental glomerulosclerosis (FSGS). From a yeast two-hybrid screen we identified phosphatidylserine synthase-1 (PSS1) as a Fam49b interacting partner. Overexpression of fam49b in HEK293 cells increases the binding of a phosphatidylserine (PS)-binding reporter (Lactadherin-GFP) and enhances vesicular activity in LLCPK1 cells, suggesting that Fam49b positively regulates PSS1 activity and PS content, and increases vesicular activity in cell. Therefore, we propose that Fam49b, a novel protein, plays a conserved and critical role in maintaining the structural and functional integrity of the glomerula filtration barrier by regulating phospholipids metabolism in podocytes. For this study, we will take advantage of the well-characterized zebrafish model in combination with various in vitro model systems to explore the functional role of Fam49b. In addition, we will explore how phospholipid modulates the podocyte function therefore contribute to the pathophysiology of glomerular diseases. Specifically, we will: 1) investigate the role of Fam49b in podocyte biology and maintenance of the structure and function of slit diaphragm (SD); and 2) explore the mechanism that Fam49b regulates the structure and function of the SD through modulating phospholipids metabolism, lipid raft mediated protein trafficking and cell signaling. Our proposed study will further our understanding of not only the function of this novel gene, fam49b, but also explore the role of phospholipids in the pathophysiology of podocyte, therefore provide potentially new therapeutic targets to treat glomerular disease. PUBLIC HEALTH RELEVANCE: Kidney is a critical organ for filtering blood, removing wastes, and regulating water and salt balance in vertebral animals. We have discovered a new gene, fam49b to be critical for formation and/or function of the kidney filter, slit diaphragm. We proposed to examine the underlying mechanism of this gene and understand its contribution to the pathophysiology of "leaky" kidney diseases.

描述（由申请人提供）：哺乳动物肾脏的核心功能是通过复杂的肾小球滤过屏障对血浆进行超滤，该屏障由有孔内皮、肾小球基底膜（GBM）和称为足细胞的特殊上皮细胞组成。每个足细胞的足突在 GBM 顶部互连，并且蛋白质在狭缝隔膜的相邻细胞之间桥接。裂隙隔膜 (SD) 的破坏会导致肾小球渗漏，表现为蛋白尿，这是许多肾脏疾病的典型表现。因此，维持肾小球滤过屏障的完整性对于正常肾功能至关重要。多个足细胞基因产物已被 发现有助于 SD 的结构和功能，例如 Nephrin、CD2AP 和 Podocin。通过寻找影响 SD 功能的其他因素，我们发现了一种新基因 fam49b，该基因在斑马鱼和小鼠肾脏的发育和成熟足细胞中高度表达。在斑马鱼胚胎中敲低 fam49b 会导致足细胞足突消失、足细胞形态改变和肾衰竭。在蛋白尿性肾小球功能障碍小鼠和局灶节段性肾小球硬化症 (FSGS) 患者的肾脏中，始终观察到 fam49b 表达下调。通过酵母双杂交筛选，我们鉴定出磷脂酰丝氨酸合酶-1 (PSS1) 是 Fam49b 的相互作用伴侣。 HEK293 细胞中 fam49b 的过表达增加了磷脂酰丝氨酸 (PS) 结合报告基因 (Lactadherin-GFP) 的结合，并增强了 LLCPK1 细胞中的囊泡活性，表明 Fam49b 正向调节 PSS1 活性和 PS 含量，并增加细胞中的囊泡活性。因此，我们认为Fam49b这种新型蛋白质通过调节足细胞中的磷脂代谢，在维持肾小球滤过屏障的结构和功能完整性方面发挥着保守且关键的作用。在本研究中，我们将利用已充分表征的斑马鱼模型结合各种体外模型系统来探索 Fam49b 的功能作用。此外，我们将探讨磷脂如何调节足细胞功能，从而促进肾小球疾病的病理生理学。具体来说，我们将： 1）研究Fam49b在足细胞生物学和缝隔膜（SD）结构和功能维持中的作用； 2）探索Fam49b通过调节磷脂代谢、脂筏介导的蛋白质运输和细胞信号传导来调节SD的结构和功能的机制。我们提出的研究不仅将进一步加深我们对fam49b这一新基因功能的理解，还将探索磷脂在足细胞病理生理学中的作用，从而为治疗肾小球疾病提供潜在的新治疗靶点。 公众健康相关性：肾脏是脊椎动物过滤血液、清除废物和调节水盐平衡的重要器官。我们发现了一种新基因 fam49b，它对于肾过滤器狭缝隔膜的形成和/或功能至关重要。我们建议检查该基因的潜在机制并了解其对“渗漏”肾脏疾病的病理生理学的贡献。