Myoferlin in muscle membrane fusion and repair

描述(申请人提供)：deferlin基因突变会引起肌营养不良症，而deferlin基因缺失导致肌营养不良症的机制与其他形式的肌营养不良症有根本的不同。脱铁蛋白是一种膜相关蛋白，参与囊泡运输活动。在肌肉中，这包括对质膜修复很重要的囊泡运动。我们的研究还表明，与费林家族的其他成员一样，迪弗林也调节着囊泡的循环。我们鉴定了myoferlin和Fer1L5这两个与deferlin高度相关的蛋白质，以了解这些蛋白质是否具有重叠的功能来替代deferlin。这些Ferlin蛋白每个都有至少六个C2结构域，这些结构域对蛋白质-膜和蛋白质-蛋白质相互作用很重要。我们发现脱铁蛋白的第一个C2结构域以一种钙敏感的方式与带负电荷的磷脂结合。我们随后发现Ferlin C2结构域直接与羧基末端EPS 15同源结构域(EHD)蛋白结合，并且EHD蛋白是Ferlin细胞内转运所必需的。EHD蛋白在体外和细胞内均能诱导小管形成。我们将研究Ferlins、EHD和BIN1在肌肉横管发生中的相互作用。然后，我们将研究费林蛋白丢失使肌肉容易脱管的机制，以及恢复小管形成和功能的分子要求。最后，我们将研究由Ferlin蛋白质介导的循环事件。在每一步中，我们将确定deferlin的结构域，或相关的myoferlin和Fer1L5，它们介导了这些事件，并评估这些功能的恢复是否纠正了deferlin相关性肌营养不良的潜在病理。

DESCRIPTION (provided by applicant): Mutations in dysferlin cause muscular dystrophy, and the mechanism by which loss of dysferlin leads to muscular dystrophy is a fundamentally different than other forms of muscular dystrophy. Dysferlin is a membrane-associated protein that participates in vesicle trafficking events. In muscle this includes vesicle movement important for plasma membrane repair. Our work has also shown that dysferlin, like other members of the ferlin family, regulates vesicle recycling. We characterized myoferlin and Fer1L5, two highly related proteins to dysferlin to understand whether these proteins have overlapping function to substitute for dysferlin. These ferlin proteins each have at least six C2 domains, domains important for protein-membrane and protein-protein interactions. We identified that the first C2 domain of dysferlin binds negatively charged phospholipids in a calcium sensitive manner. We subsequently showed that ferlin C2 domains bind directly to carboxy-terminal Eps 15 Homology Domain (EHD) proteins, and that EHD proteins are required for ferlin intracellular trafficking. EHD proteins can induce tubule formation in vitro and in cell. We will study the interaction between ferlins, EHD, and Bin1 in the genesis of transverse tubule in muscle. We will then examine the mechanisms by which loss of ferlin proteins renders the muscle susceptible to detubulation and the molecular requirements to restore tubule formation and function. Finally, we will examine recycling events mediated by ferlin proteins. In each step, we will determine the domains of dysferlin, or the related myoferlin and Fer1L5 that mediate these events and assess whether restoration of these functions corrects the underlying pathology in dysferlin-associated muscular dystrophy.