Human dysferlin and its implications in Limb-Girdle Muscular Dystrophy

描述（由申请人提供）：目前对肌营养不良症病因学的理解缺乏深入的分子描述。该提案侧重于dysferlin的结构和功能研究，其与磷脂膜的相互作用，以及致病突变对其功能的影响。Dysferlin是一种分子量为237 kDa的多C2结构域膜整合蛋白，参与骨骼肌组织的膜修复。dysferlin的突变与人类的肢带肌营养不良症（LGMD）有关。因此，我们在本申请中的主要目标是使用电子显微镜（EM）、X射线晶体学和生物化学技术组装dysferlin的完整3D结构，以开发突变如何损害dysferlin作为膜修复中的介体的功能的分子描述。该建议的中心假设是基于这样的想法，即dysferlin中的七个C2结构域中的每一个都发挥着独特的作用，并且正常的dysferlin功能取决于其C2结构域的正确空间排列。在强有力的初步数据的指导下，这一假设将通过追求三个具体目标进行检验：（一）了解 全长dysferlin蛋白的结构域组织及其与MG 53的关系，我们将使用EM技术组装dysferlin蛋白和MG 53的中等分辨率结构。我们有dysferlin和MG 53的初步图像，我们已经开始将各个方向组装成一个单一的3D结构。此外，我们将测量dysferlin的功能，使用一种新的EM为基础的脂质聚集试验;（ii）研究个别域的dysferlin在更高的分辨率，我们将追求分离的C2和FerA域的晶体结构。我们最近已经解决了dysferlin的C2 A结构域的晶体结构到2.0埃和一个新的剪接变体的C2 A到1.7埃的分辨率。剩余的C2结构域已被分离和纯化。中心FerA结构域是一个四螺旋束，可能与dysferlin的膜融合活性有关。(iii)为了了解致病突变对dysferlin结构和功能的影响，我们将40个临床分离的点突变映射到dysferlin的C2结构域。这些突变聚集成三个结构类别：Ca 2 +/磷脂结合口袋，分子的结构折叠，和C2结构域的效应物结合区。将通过EM研究每个类别的代表性突变和三个截短突变对dysferlin总结构和功能的影响。此外，将使用光谱技术研究代表性突变，以了解突变对配体结合和结构域稳定性的影响。这种方法是创新的，因为它带来了最先进的EM技术与X射线晶体学和C2结构域结构生物学的背景，以更好地了解使人衰弱的人类疾病的生物学。一旦完成，dysferlin的3D结构最终可以用于结构域靶向药物，以及设计更小的功能性dysferlin蛋白，这些蛋白更适合目前用于治疗LGMD的基因治疗技术。

DESCRIPTION (provided by applicant): The understanding of the etiology of muscular dystrophy currently lacks an in-depth, molecular description. This proposal focuses on the structural and functional studies of dysferlin, its interaction with phospholipid membranes, and the effects of disease-causing mutations on its function. Dysferlin is a 237 kDa, multi-C2 domain, integral membrane protein that is involved in membrane repair in skeletal muscle tissue. Mutations in dysferlin have been linked to Limb-Girdle Muscular Dystrophy (LGMD) in humans. Therefore, our main objective in this application is to assemble a complete 3D structure of dysferlin using electron microscopy (EM), X-ray crystallographic and biochemical techniques to develop a molecular description for how mutations impair dysferlin' s function as a mediator in membrane repair. The central hypothesis of this proposal is based on the idea that each of the seven C2 domains in dysferlin serve a unique role, and that normal dysferlin function depends on the correct spatial arrangement of its C2 domains. Guided by strong preliminary data, this hypothesis will be tested by pursuing three Specific Aims: (i) to understand the domain organization of the full- length dysferlin protein, and its relationship with MG53, we will assemble a medium-resolution structure of the dysferlin protein and MG53 using EM techniques. We have preliminary images of both dysferlin and MG53, and we have begun the process to assemble individual orientations into a single 3D structure. In addition, we will measure the function of dysferlin using a novel EM-based lipid aggregation assay; (ii) to study the individual domains of dysferlin at higher resolution, we will pursue the crystal structures of the isolated C2 and FerA domains. We have recently solved the crystal structures of the C2A domain of dysferlin to 2.0 angstroms and a novel splice variant of C2A to 1.7 angstrom resolutions. The remaining C2 domains have been isolated and purified. The central FerA domain is a four-helix bundle, and may be related to the membrane fusion activity of dysferlin. (iii) to understand the effects of disease-causing mutations on the structure and function of dysferlin, we have mapped 40 clinically isolated point mutations to the C2 domains of dysferlin. These mutations clustered into three structural categories: the Ca2+/phospholipid binding pocket, the structural ß-sheets of the molecule, and the effector binding region of the C2 domains. Representative mutations from each category, and three truncation mutations will be studied by EM for their effect on the total structure and function of dysferlin. In addition, representative mutations will be studied using spectroscopic techniques to understand the effects of mutation on ligand binding and domain stability. This approach is innovative, as it brings to bear state-of-the-art EM techniques with X-ray crystallography and a background in C2 domain structural biology to better understand the biology of a debilitating human disease. Once completed, a 3D structure of dysferlin could ultimately be used for domain-targeted drugs, as well as in the design of smaller, functional dysferlin proteins that are more amenable to current gene therapy techniques for the treatment of LGMD.