Biological Mechanism of INF2-mediated FSGS

描述（由申请人提供）：该项目的目标是了解倒置双胍蛋白2基因INF2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，表明INF2突变是最常见的常染色体显性形式的FSGS。INF2对于双胍家族成员来说是独特的，因为它可以加速肌动蛋白的聚合和解聚。Formins通过两个结构域的相互作用自动抑制其活性，n端DID（透明抑制结构域）和c端DAD（透明自调节结构域），并且通常通过将小GTPase结合到DID附近的n端来激活。INF2有两个主要的剪接变体，一个通过c端戊烯基与内质网（ER）相关，另一个缺乏戊烯基。在这项资助的第一阶段，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。我们已经定义了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效应物的透明双胍（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INF2及其相关突变。与所有其他肌动蛋白调控蛋白相比，INF2-DID突变是人类FSGS的一种相对常见的形式，这表明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，并最终将其用于治疗。现在，我们的目标是：(1)确定具有代表性的致病突变体的特定生化效应。我们将检验这一假设，即已知的30多个INF2突变每个都可以通过四种可能的缺陷导致疾病：1。INF2蛋白结构不稳定，导致不稳定/降解；2. 通过DID/DAD相互作用破坏INF2的自我抑制；3. 通过INF2-DID与Dia- dad相互作用改变Dia家族形成的调控4. 破坏其他分子间相互作用。(2)明确FSGS突变对INF2细胞功能的影响。我们将：1；确定足细胞中INF2和Dia蛋白的定位和异构体表达；2. 确定FSGS突变对细胞内INF2和Dia功能的影响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敲低模型来比较一系列不同的干扰对INF2（敲低，转基因）的直接表型影响，以响应RhoA/Rac/Cdc42信号的变化；2. 利用这些研究结果指导小鼠点突变（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.