Biological Mechanism of INF2-mediated FSGS

描述(申请人提供)：本项目的目标是了解倒置形式2基因INF2突变导致人类局灶性节段性肾小球硬化(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FSGS最常见的常染色体显性形式。INF2对于Forin家族成员来说是独一无二的，因为它同时加速了肌动蛋白的聚合和解聚。Formins通过N端DID(透明抑制域)和C端DAD(透明自动调节域)之间的相互作用来自动抑制它们的活性，通常通过将一个小的GTP酶与DID附近的N端结合来激活。INF2有两个主要的剪接变异体，一个通过C末端的戊烯基团与内质网(ER)相连，另一个缺失戊烯基基。在这笔赠款的第一阶段，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效应器的透明形成蛋白(Dia)家族的活性。我们已经建立了小鼠和斑马鱼模型，用于体内研究INF2及其相关突变。与几乎所有其他肌动蛋白调节蛋白相反，INF2-DID突变是人类FSGS的一种相对常见的形式，表明INF2-DID在足细胞中具有独特的和非冗余的功能。我们的长期目标是了解这些功能，并最终利用它们来治疗。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+INF2突变都可以通过四种可能的缺陷导致疾病：1.INF2蛋白结构的不稳定，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.通过INF2-DID与DIA-DAD的相互作用改变Dia家族福尔马林的调节；4.破坏其他分子间的相互作用。(2)明确FSGS突变对INF2细胞功能的影响。我们将：1.确定INF2和Dia蛋白在足细胞中的定位和异构体表达；2.确定FSGS突变对INF2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)体外和基于细胞的研究与体内模型(斑马鱼、小鼠)的相关性。我们将：1.使用斑马鱼INF2基因敲除模型来比较INF2(基因敲除、转基因)中一系列不同扰动对RhoA/RAC/CDC42信号变化的直接表型影响；2.使用这些研究结果来指导小鼠点突变(基因敲除)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。

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.