Biological Mechanism of INF2-mediated FSGS

摘要 该项目的目标是了解INF2基因突变导致局灶性病变的分子机制。 人类的节段性肾小球硬化(FSGS)。超过45种不同的FSGS相关突变 已被确认身份。一组携带FSGS导致的INF2突变的人也表现出Charcot Marie Tooth 疾病。INF2对于Forin家族成员来说是独一无二的，因为它既能加速肌动蛋白聚合，又能促进 解聚。Formins可以通过分子内相互作用自动抑制自己的活动 结构域，N-末端DID(透明抑制域)和C-末端DAD(透明 自动调节域)。INF2有两个主要的剪接变体，其中一个与内质有关 INF2-CAAX和第二种异构体INF2-NONCAAX，帮助维持高尔基体的完整性。INF2-CAAX 是足细胞的主要亚型。 在这笔赠款的最后一段时间里，我们在理解INF2生物学和 INF2功能如何因突变而改变。我们现在了解了INF2在细胞器功能中所扮演的角色，以及 对其在调控线粒体分裂中的作用有更清晰的认识。我们已经确定了一个主要的机制 抑制INF2活性，与内源蛋白复合体(环化酶相关蛋白)相互作用 与翻译后乙酰化的肌动蛋白结合)。我们还发现INF2蛋白经历了 在N-末端DID区域和C-末端之间的位置进行切割，包含FH2和DAD 区域，这可能在调节INF2功能和可能抑制两者的功能中起重要作用 蛋白质的区域。我们发现INF2经历了一个切割事件，这可能是重要的 与基本上所有其他肌动蛋白调节蛋白不同的是，INF2-DID调节INF2的功能 突变是遗传性FSGS的一种相对常见的形式，提示INF2-DID具有独特的 足细胞中的非冗余功能。我们的长期目标是了解这些功能，并最终， 利用它们来获得治疗益处。 我们有四个主要目标：(1)确定引起FSGS突变的特定生化效应。我们将测试 多个FSGS突变体对INF2与内源性抑制复合体相互作用的影响 研究INF2突变体与其结合伙伴的相互作用；(2)定义INF2功能和突变介导性 细胞功能障碍。这包括检查INF2突变如何改变其对线粒体的调节 功能；(3)定义INF2裂解的功能；(4)利用基因工程小鼠更好地理解 体内INF2功能和突变介导的功能障碍。

SUMMARY The goal of project is to understand the molecular mechanisms by which mutations in INF2 cause focal segmental glomerulosclerosis (FSGS) in humans. More than 45 different FSGS-associated mutations have been identified. A subset of people with FSGS-causing INF2 mutations also exhibit Charcot Marie Tooth disease. INF2 is unique for a formin family member in that it accelerates both actin polymerization and depolymerization. Formins can autoinhibit their own activity by an intramolecular interaction between two domains, the N-terminal DID (diaphanous inhibitory domain) and the C-terminal DAD (diaphanous autoregulatory domain). INF2 has two major splice variants, one of which is associated with the endoplasmic reticulum, INF2-CAAX, and a second isoform, INF2-nonCAAX, that helps maintain Golgi integrity. INF2-CAAX is the major podocyte isoform. During the last period of this grant, we made significant progress in our understanding of both INF2 biology and how INF2 function is altered by mutations. We now understand the role INF2 plays in organelle function, and have a clearer understand of its role in regulating mitochondrial fission. We have identified a major mechanism of inhibition of INF2 activity, an interaction with an endogenous protein complex (cyclase-associated protein bound to actin that is post-translationally acetylated). We have also found that the INF2 protein undergoes cleavage at a site between the N-terminal DID region and the C-terminus, containing the FH2 and DAD regions, which may be important in regulating INF2 function and perhaps disinhibiting the functions of both regions of the protein. We have found that INF2 undergoes a cleavage event that may be important in regulating INF2 function The fact that in contrast to essentially all other actin regulatory proteins, INF2-DID mutations are a relatively common form of inherited FSGS, suggests 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. We have four major goals: (1) Define the specific biochemical effects of FSGS-causing mutants. We will test the effects of multiple FSGS mutants on the interaction of INF2 with the endogenous inhibitory complex and examine INF2 mutant interactions with its binding partners; (2) Define INF2 function and mutation-mediated dysfunction in cells. This includes examination of how INF2 mutations alter its regulation of mitochondrial function; (3) Define the function of INF2 cleavage; (4) Use genetically engineered mice to better understand INF2 function and mutation mediated dysfunction in vivo.