Human dysferlin and its implications in Limb-Girdle Muscular Dystrophy

描述(由申请人提供)：目前对肌营养不良的病因缺乏深入的、分子的描述。这项建议侧重于对脱铁蛋白的结构和功能的研究，它与磷脂膜的相互作用，以及致病突变对其功能的影响。去铁蛋白是一个237 kDa、多个C2结构域的完整膜蛋白，参与骨骼肌组织的膜修复。功能障碍蛋白的突变与人类的肢体吉氏肌营养不良症(LGMD)有关。因此，我们在这项应用中的主要目标是利用电子显微镜(EM)、X射线结晶学和生物化学技术组装一个完整的脱铁蛋白的三维结构，以开发关于突变如何损害脱铁蛋白S在膜修复中的中介作用的分子描述。这一建议的中心假设是基于这样的想法，即deferlin中的七个C2结构域中的每一个都具有独特的作用，并且正常的deferlin功能依赖于其C2结构域的正确空间排列。在强劲的初步数据的指导下，将通过追求三个具体目标来检验这一假设：(I)了解 为了进一步研究异铁蛋白全长的结构域组织及其与MG53的关系，我们将利用EM技术组装出异铁蛋白和MG53的中等分辨率结构。我们已经有了迪弗林和MG53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，我们将使用一种新的基于EM的脂质聚集实验来测量deferlin的功能；(Ii)为了以更高的分辨率研究disferlin的单个结构域，我们将追踪分离的C2和ferA结构域的晶体结构。最近，我们已经解决了disferlin的C2a结构域的晶体结构到2.0埃，以及一个新的C2a剪接变体的晶体结构到1.7埃的分辨率。其余的C2结构域已经被分离和纯化。中央FERA结构域是一个四螺旋结构束，可能与deferlin的膜融合活性有关。(Iii)为了了解致病突变对deferlin结构和功能的影响，我们将40个临床分离的点突变定位到dyferlin的C2结构域。这些突变聚为三种结构类型：钙/磷脂结合口袋、分子的结构伯片和C2结构域的效应器结合区。来自每个类别的代表性突变和三个截断突变将被EM研究它们对deferlin总结构和功能的影响。此外，还将使用光谱技术研究具有代表性的突变，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，因为它将最先进的EM技术与X射线结晶学和C2领域结构生物学的背景结合在一起，以更好地了解一种使人衰弱的人类疾病的生物学。一旦完成，deferlin的3D结构最终可以用于针对结构域的药物，以及设计更小的、功能更好的dyferlin蛋白，这些蛋白更适合于目前治疗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.