Biological Mechanism of INF2-mediated FSGS

INF2介导的FSGS的生物学机制

• 批准号: 8970699
• 负责人: MARTIN R. POLLAK
• 金额: $ 43.65万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970699
• 关键词: Actins; Binding; Biochemical; Biological; C-terminal; Cell physiology; Cells; Defect; Disease; Endoplasmic Reticulum; Family; Family member; Focal Segmental Glomerulosclerosis; Gene Transfer Techniques; Genes; Goals; Grant; Health; Human; In Vitro; Injury; Kidney; Kidney Diseases; Kidney Glomerulus; Knock-in; Knock-out; Lead; Mediating; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mus; Mutation; N-terminal; Organelles; Protein Isoforms; Proteins; RNA Splicing; Regulation; Role; Signal Transduction; Testing; Therapeutic; Time; Variant; Zebrafish; base; depolymerization; formin-2; genetic regulatory protein; human disease; in vivo Model; knock-down; mutant; podocyte; polymerization; prenyl; protein function; protein structure; research study; response

DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms by which mutations in the Inverted Formin 2 gene INF2 cause focal segmental glomerulosclerosis (FSGS) in humans. We and others have identified numerous FSGS-segregating mutations, demonstrating that INF2 mutations are the most common autosomal dominant form of FSGS. INF2 is unique for a formin family member in that it accelerates both actin polymerization and depolymerization. Formins autoinhibit their activity by an interaction between two domains, the N-terminal DID (diaphanous inhibitory domain) and the C-terminal DAD (diaphanous autoregulatory domain), and are generally activated by binding of a small GTPase to the N-terminus near the DID. INF2 has two major splice variants, one associated with the endoplasmic reticulum (ER) via a C-terminal prenyl group, and another lacking the prenyl group. In the first period of this grant, we have made significant progress towards understanding how mutations in INF2 cause human kidney disease. We have defined roles for INF2 in cells and organelles. We have shown that INF2 binds to and modulates the activity of the diaphanous formin (Dia) family of RhoA effectors. We have developed mouse and zebrafish models for the in vivo study of INF2 and its associated mutations. In contrast to essentially all other actin regulatory proteins, INF2-DID mutations are a relatively common form of human FSGS, suggesting that INF2-DID possesses unique and non-redundant functions in the podocyte. Our long-term goal is to understand these functions and, ultimately, exploit them for therapeutic benefit. Now, we aim to: (1) Define the specific biochemical effects of representative disease-causing mutants. We will test the hypothesis that the 30+ known INF2 mutations can each lead to disease through four possible defects: 1. Destabilization of INF2 protein structure, leading to instability/degradation; 2. Disruption of auto- inhibition of INF2 through the DID/DAD interaction; 3. Altered regulation of Dia family formins through interaction of INF2-DID with Dia-DAD; 4. Disruption of other inter-molecular interactions. (2) Define the effects of FSGS mutations on INF2 cellular function. We will: 1. Define INF2 and Dia protein localization and isoform expression in podocytes; 2. Determine cellular effects of FSGS mutations on INF2 and Dia function; 3. Assess the influence of Dia proteins on FSGS mutant effects (3) Correlate in vitro and cell-based studies with in vivo models (zebrafish, mouse). We will: 1. Use a zebrafish INF2 knockdown model to compare the direct phenotypic effects of a range of different perturbations in INF2 (knockdown, transgenesis) in response to changes in RhoA/Rac/Cdc42 signaling; 2. Use the results from these studies to guide experiments in mouse point mutant (knockin) and knockout models, investigating the molecular mechanisms of injury in a mammalian kidney.

描述（由申请人提供）：该项目的目标是了解倒置 Formin 2 基因 INF2 的突变导致人类局灶节段性肾小球硬化症 (FSGS) 的机制。我们和其他人已经鉴定了许多 FSGS 分离突变，证明 INF2 突变是 FSGS 最常见的常染色体显性形式。 INF2对于formin家族成员来说是独特的，因为它加速肌动蛋白聚合和解聚。 Formins 通过两个结构域（N 端 DID（透明抑制结构域）和 C 端 DAD（透明自动调节结构域））之间的相互作用来自动抑制其活性，并且通常通过小 GTP 酶与 DID 附近的 N 端结合来激活。 INF2 有两种主要剪接变体，一种通过 C 端异戊二烯基与内质网 (ER) 相关，另一种缺乏异戊二烯基。在这笔资助的第一期，我们在了解 INF2 突变如何导致人类肾脏疾病方面取得了重大进展。我们已经确定了 INF2 在细胞和细胞器中的作用。我们已经证明 INF2 结合并调节 RhoA 效应子的透明福明 (Dia) 家族的活性。我们开发了小鼠和斑马鱼模型，用于 INF2 及其相关突变的体内研究。与基本上所有其他肌动蛋白调节蛋白相比，INF2-DID 突变是人类 FSGS 的相对常见形式，表明 INF2-DID 在足细胞中具有独特且非冗余的功能。我们的长期目标是了解这些功能，并最终利用它们获得治疗益处。现在，我们的目标是：（1）定义代表性致病突变体的特定生化效应。我们将测试以下假设：30 多种已知的 INF2 突变均可以通过四种可能的缺陷导致疾病： 1. INF2 蛋白质结构不稳定，导致不稳定/降解； 2.通过DID/DAD相互作用破坏INF2的自身抑制； 3. INF2-DID与Dia-DAD相互作用改变Dia家族福尔明的调节； 4.其他分子间相互作用的破坏。 (2)定义FSGS突变对INF2细胞功能的影响。我们将： 1. 定义 INF2 和 Dia 蛋白在足细胞中的定位和亚型表达； 2.确定FSGS突变对INF2和Dia功能的细胞影响； 3. 评估 Dia 蛋白对 FSGS 突变效应的影响 (3) 将体外和基于细胞的研究与体内模型（斑马鱼、小鼠）相关联。我们将： 1. 使用斑马鱼 INF2 敲低模型来比较 INF2 中一系列不同扰动（敲低、转基因）响应 RhoA/Rac/Cdc42 信号传导变化的直接表型效应； 2. 利用这些研究的结果指导小鼠点突变（敲入）和基因敲除模型的实验，研究哺乳动物肾脏损伤的分子机制。