Biological Mechanism of INF2-mediated FSGS

描述（由申请人提供）：本项目的目的是了解倒位形成蛋白2基因INF 2突变导致人类局灶节段性肾小球硬化（FSGS）的机制。我们和其他人已经确定了许多FSGS分离突变，证明INF 2突变是FSGS最常见的常染色体显性形式。INF 2对于肌动蛋白家族成员是独特的，因为它加速肌动蛋白聚合和解聚。形成蛋白通过两个结构域（N-末端DID（透明抑制结构域）和C-末端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，并且通常通过小的GTdR与DID附近的N-末端结合来激活。INF 2有两个主要的剪接变体，一个通过C-末端异戊烯基与内质网（ER）相关，另一个缺乏异戊烯基。在该资助的第一阶段，我们在了解INF 2突变如何导致人类肾脏疾病方面取得了重大进展。我们已经定义了INF 2在细胞和细胞器中的作用。我们已经表明，INF 2结合并调节RhoA效应子的透明质酸（Dia）家族的活性。我们已经开发了小鼠和斑马鱼模型，用于体内研究INF 2及其相关突变。与基本上所有其他肌动蛋白调节蛋白相比，INF 2-DID突变是人类FSGS的相对常见形式，表明INF 2-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，并最终将其用于治疗。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。我们将测试这一假设，即30多个已知的INF 2突变可以通过四种可能的缺陷导致疾病：1. INF 2蛋白质结构失稳，导致不稳定/降解; 2.通过DID/DAD相互作用破坏INF 2的自抑制; 3.通过INF 2-DID与Dia-DAD的相互作用改变Dia家族形成蛋白的调节; 4.破坏其他分子间相互作用。(2)定义FSGS突变对INF 2细胞功能的影响。我们将：1.确定足细胞中INF 2和Dia蛋白的定位和亚型表达; 2.确定FSGS突变对INF 2和Dia功能的细胞效应; 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INF 2敲低模型来比较响应于RhoA/Rac/Cdc 42信号传导的变化的INF 2中的一系列不同扰动（敲低、转基因）的直接表型效应; 2.使用这些研究的结果来指导小鼠点突变（敲入）和敲除模型的实验，研究哺乳动物肾脏损伤的分子机制。

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.