Biological Mechanism of INF2-mediated FSGS

描述(申请人提供)：我们已经确定了一种新的基因，当突变时会导致一种以蛋白尿、肾小球滤过率降低为特征的进行性肾脏疾病，以及以局灶性和节段性肾小球硬化(FSGS)为特征的损害的组织学模式。这个基因，INF2(代表倒福明2)，是福明家族透明亚群的成员。INF2和透明的Forin亚家族的其他成员一样，具有促进肌动蛋白细丝聚合的功能。与其他成员不同，INF2还可以加速肌动蛋白解聚。这个亚家族的成员能够通过N端DID(透明抑制域)和C端DAD(透明自动调节域)两个结构域之间的分子内相互作用来自动抑制它们的活性。我们在11个常染色体显性遗传性FSGS无关家系中发现了INF2基因DID的点突变。这些突变与疾病分离，在对照组中不存在，并改变高度保守的氨基酸。遗传方式、缺乏明确的功能丧失等位基因，以及所有突变定位于DID，都表明了功能获得效应。在这一应用中，我们建议进行实验，以帮助确定INF2突变导致人类疾病的机制，并了解INF2在肾功能中的作用。在目标1中，我们建议探索INF2突变对其生化功能的影响。我们将检验INF2中FSGS相关突变破坏分子内相互作用导致INF2功能变化的假设。我们将测试导致FSGS的突变对INF2分子内相互作用(DID与DAD)的影响，对INF2介导肌动蛋白细丝聚合和解聚的能力的影响，以及INF2与肌动蛋白细丝和微管的相互作用。在目标2中，我们将定义INF2突变对细胞功能的影响。我们将检验INF2突变通过抑制INF2活性改变基于肌动蛋白的细胞功能的假设。我们将研究INF2在细胞中的作用，以及INF2突变对基于肌动蛋白的结构、肌动蛋白动力学、内质网动力学和相互作用蛋白的影响。在目标3中，我们将开发和分析两个新的INF2突变小鼠模型。我们将使用这些模型来测试INF2在足细胞中的作用，并在体内模型中观察INF2点突变的影响。我们将设计一种带有疾病相关INF2点突变的小鼠模型，并开发一种足细胞特异性INF2缺陷小鼠。我们将在体内检测INF2突变和缺陷对肾小球功能的影响。 公共卫生相关性：我们发现了一种新的局灶性节段性肾小球硬化(FSGS)基因。当发生突变时，这种名为INF2的基因会导致人类的肾脏疾病。更好地了解该基因缺陷是如何导致人类疾病的，将对理解并最终治疗常见形式的肾功能衰竭和肾功能衰竭进展具有重要和直接的意义。

DESCRIPTION (provided by applicant): We have identified a new gene which when mutated leads to a form of progressive kidney disease characterized by proteinuria, reduced glomerular filtration rate, and a histologic pattern of injury characterized by focal and segmental glomerulosclerosis (FSGS). This gene, INF2 (for Inverted Formin 2), is a member of the diaphanous subgroup of the formin family. INF2, like other members of the diaphanous formin subfamily, functions to accelerate actin filament polymerization. Unlike other members, INF2 can also accelerate actin depolymerization. Members of this subfamily of formins are able to autoinhibit their activity by an intramolecular interaction between two domains, the N-terminal DID (diaphanous inhibitory domain) and the C-terminal DAD (diaphanous autoregulatory domain). We have found point mutations in the DID of INF2 in eleven unrelated families with autosomal dominant FSGS. These mutations segregate with disease, are absent from controls, and alter highly conserved amino acids. The mode of inheritance, the absence of clear loss-of-function alleles, and the localization of all of the mutations to the DID, suggest a gain-of-function effect. In this application, we propose experiments to help define the mechanism by which INF2 mutations lead to human disease and to understand the role of INF2 in kidney function. In Aim 1, we propose to explore the effects of INF2 mutations on its biochemical functions. We will examine the hypothesis that FSGS- associated mutations in INF2 disrupt intramolecular interactions leading to functional changes in INF2. We will test the effects of FSGS-causing mutations on INF2 intramolecular interactions (DID with DAD), on the ability of INF2 to mediate actin filament polymerization and depolymerization, and the interactions of INF2 with actin filaments and microtubules. In Aim 2, we will define the effects of INF2 mutations on cellular functions. We will examine the hypothesis that INF2 mutations, by disinhibiting INF2 activity, alter actin- based cell function. We will study the role of INF2 in cells and the effect of INF2 mutations on actin-based structures, on actin dynamics, on the dynamics of the endoplasmic reticulum, and on interacting proteins. In Aim 3, we will develop and analyze two new INF2 mutant mouse models. We will use these models to test the role of INF2 in the podocyte and observe the effects of an INF2 point mutation in an in vivo model. We will engineer a mouse model with a disease-associated INF2 point mutation and also develop a podocyte-specific INF2 deficient mouse. We will examine the effect of INF2 mutation and deficiency on glomerular function in vivo. PUBLIC HEALTH RELEVANCE: We have identified a new focal segmental glomerulosclerosis (FSGS) gene. When mutated, this gene, INF2, causes kidney disease in humans. Better understanding how defects in this gene cause human disease will have significant and direct implications for understanding, and ultimately, treating, common forms of renal failure and renal failure progression.