Biological Mechanism of INF2-mediated FSGS

INF2介导的FSGS的生物学机制

• 批准号: 8517110
• 负责人: MARTIN R. POLLAK
• 金额: $ 39.66万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517110
• 关键词: Actins; Alleles; Amino Acids; Binding; Biochemical; Biological; C-terminal; Cell physiology; Cells; Defect; Disease; Endoplasmic Reticulum; Engineering; Exons; Family; Family member; Focal Segmental Glomerulosclerosis; Genes; Glomerular Filtration Rate; Histologic; Human; Inherited; Injury; Kidney; Kidney Diseases; Kidney Failure; Lead; Mediating; Microfilaments; Microtubules; Modeling; Monomeric GTP-Binding Proteins; Mus; Mutate; Mutation; N-terminal; Pattern; Phenotype; Physiology; Point Mutation; Proteins; Proteinuria; RNA Splicing; Renal function; Role; Structure; Subgroup; Testing; Variant; base; depolymerization; formin-2; gain of function; glomerular function; human disease; in vivo; in vivo Model; loss of function; member; mouse model; mutant mouse model; podocyte; polymerization; public health relevance; research study; rho

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.

描述（由申请人提供）：我们已经鉴定了一种新的基因，该基因突变时会导致一种以蛋白尿、肾小球滤过率降低和以局灶性和节段性肾小球硬化（FSGS）为特征的损伤组织学模式为特征的进行性肾病。这个基因，INF 2（反向形成蛋白2），是一个成员的透明亚组的基因家族。INF 2，像其他成员的透明的肌动蛋白亚家族，功能是加速肌动蛋白丝聚合。与其他成员不同，INF 2也可以加速肌动蛋白的解聚。该formin亚家族的成员能够通过两个结构域（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））之间的分子内相互作用来自动抑制其活性。我们在11个常染色体显性遗传FSGS家系中发现了INF 2基因DID的点突变。这些突变与疾病分离，不存在于对照中，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均定位于DID，表明存在功能获得效应。在本申请中，我们提出了实验来帮助定义INF 2突变导致人类疾病的机制，并了解INF 2在肾功能中的作用。在目的1中，我们建议探索INF 2突变对其生化功能的影响。我们将检验这一假设，即FSGS相关的突变在INF 2破坏分子内的相互作用，导致功能的变化INF 2。我们将测试FSGS引起的突变对INF 2分子内相互作用（DID与DAD）的影响，对INF 2介导肌动蛋白丝聚合和解聚的能力，以及INF 2与肌动蛋白丝和微管的相互作用。在目标2中，我们将定义INF 2突变对细胞功能的影响。我们将检验INF-2突变通过抑制INF-2活性而改变肌动蛋白基细胞功能的假说.我们将研究INF 2在细胞中的作用，以及INF 2突变对肌动蛋白结构、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种新的INF 2突变小鼠模型。我们将使用这些模型来测试INF 2在足细胞中的作用，并观察INF 2点突变在体内模型中的作用。我们将设计一种具有疾病相关INF 2点突变的小鼠模型，并开发一种足细胞特异性INF 2缺陷小鼠。我们将在体内研究INF 2突变和缺陷对肾小球功能的影响。 公共卫生相关性：我们已经确定了一个新的局灶节段性肾小球硬化症（FSGS）基因。当突变时，这种基因INF 2会导致人类肾脏疾病。更好地了解这种基因缺陷如何导致人类疾病将对理解并最终治疗常见形式的肾衰竭和肾衰竭进展具有重要和直接的意义。