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）：哺乳动物肾脏的中心功能是血浆通过复杂的肾小球滤过屏障的超滤清，该层肾小球滤过屏障，该屏障由肾上腺内皮，肾小球基底膜（GBM）和称为Podococytes的专门上皮细胞组成。每个足细胞在GBM顶部互连的脚步过程和狭缝隔膜相邻细胞之间的蛋白质桥。缝隙隔膜（SD）的破坏会导致肾小球泄漏，表现为蛋白尿，这是许多肾脏疾病中的典型表现。因此，保持肾小球滤过屏障的完整性对于正常肾脏功能至关重要。几种足细胞基因产物已经 发现有助于SD的结构和功能，例如肾素，CD2AP和Podocin。从搜索导致SD功能的其他因素，我们发现了一个新型的基因FAM49B，该基因在斑马鱼和小鼠肾脏中的发育和成熟足细胞中高度表达。击倒斑马鱼胚胎中的FAM49B导致了足迹脚步的效率，脚足细胞形态改变和肾衰竭。在患有蛋白尿肾小球功能障碍的小鼠的肾脏以及局灶性节段性肾小球硬化症患者（FSGS）的肾脏中，FAM49B表达的下降调节。从酵母的两杂交筛网中，我们将磷脂酰丝氨酸合酶-1（PSS1）确定为FAM49B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积极地调节PSS1活性和PS内容，并增加细胞的活性。因此，我们提出，FAM49B是一种新型蛋白质，在通过调节足细胞中的磷脂代谢来维持肾小球滤过屏障的结构和功能完整性中起着保守而关键的作用。在这项研究中，我们将利用良好的斑马鱼模型与各种体外模型系统结合使用，以探索FAM49B的功能作用。此外，我们将探索磷脂如何调节足细胞功能，从而有助于肾小球疾病的病理生理。具体而言，我们将：1）研究FAM49B在足细胞生物学中的作用，并维护缝隙膜片（SD）的结构和功能； 2）探索FAM49B通过调节磷脂代谢，脂质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了解这种新型基因FAM49B的功能，而且还探讨了磷脂在足细胞病理生理学中的作用，因此提供了治疗肾小球疾病的潜在新治疗靶标。 公共卫生相关性：肾脏是过滤血液，清除废物以及调节椎动物中水和盐平衡的关键器官。我们发现了一个新的基因FAM49B对于肾脏过滤器的形成和/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“漏水”肾脏疾病的病理生理学的贡献。