Structure-Function of the Nuclear Envelope Bridge and its Role in Laminopathies

描述（由申请人提供）：在哺乳动物细胞中，细胞核的结构完整性由A型和B型核纤层蛋白赋予，A型和B型核纤层蛋白是排列在核膜的核质面并形成核纤层的蛋白质网络。沿着编码A型核纤层蛋白的LMNA散布的突变以及其他核膜蛋白内的突变与广泛的人类疾病相关，统称为核纤层蛋白病。这些疾病的分子病因学仍然未知。LINC复合物是一种进化上保守的蛋白质复合物，它连接核膜的内外膜，并将核纤层与哺乳动物细胞的细胞骨架物理连接起来，最近对LINC复合物的表征表明，细胞核直接与细胞骨架相连。这种结构连接至少有两个原因是至关重要的：（1）维持核形态本身是重要的;（2）它允许机械力施加在核上，使其在细胞内移动，例如，这是肌肉和神经细胞发育中至关重要的过程。在LINC复合物的中心是含有SUN结构域的内核膜蛋白和含有KASH肽的外核膜蛋白之间的相互作用。SUN和KASH在核周空间直接且特异地相互作用。在许多核纤层蛋白病中，包括严重肌肉疾病、Emery-Dreifuss肌营养不良、神经系统疾病如小脑共济失调和早衰症，LINC复合体内的SUN-KASH相互作用丢失，导致核形态和定位的严重后果。在人类中，至少存在五种LINC复合物，每种复合物含有一对不同的含SUN和KASH蛋白。这些组件之间的信号传递知之甚少。该提案的核心是使用生物化学和X射线晶体学工具阐明SUN-KASH相互作用的结构和功能。SUN-KASH复合物的原子结构应该为这种相互作用的调节提供有价值的见解。尽管SUN-KASH复合体对人类生理学和病理学的重要性，但其结构仍然未知。这在很大程度上可以归因于获得足够数量的蛋白质用于结构研究的问题。我们已经开发出一种方法来克服这些障碍，现在能够进行定量结构功能分析。我们预计，该项目将揭示通过LINC复合物建立的核-细胞骨架连接背后的生物物理原理。我们将通过X射线晶体学鉴定调节这种保守相互连接的特定分子细节。同时，我们将对SUN-KASH复合物进行详细的生化和突变分析。这项研究有可能使整个LINC复合物的结构和分子表征，包括所有其他组件。因此，该项目应该建立新的药物靶点，用于治疗广泛的层粘连蛋白病。 公共卫生相关性：肌营养不良对健康有很大的影响，仅在美国就有数万人受到影响。Emery-Dreifuss肌肉病的特征是某些肌肉的浪费，关节畸形和危及生命的心脏问题，可能导致过早和猝死。目前还没有确定的治疗Emery-Dreifuss肌营养不良症或相关疾病的方法;因此，该项目的工作旨在确定可导致患者治疗的分子靶点。

DESCRIPTION (provided by applicant): In mammalian cells, the structural integrity of the nucleus is conferred by A- and B-type lamins, a meshwork of proteins that lines the nucleoplasmic face of the nuclear envelope and forms the nuclear lamina. Mutations scattered along LMNA, which encodes A-type lamins, as well as mutations within other nuclear envelope proteins are associated with a broad range of human diseases, collectively called laminopathies. The molecular etiology of these diseases remains unknown. The recent characterization of the LINC complex, an evolutionary-conserved protein complex that bridges the inner and outer membranes of the nuclear envelope and physically connects the nuclear lamina to the cytoskeleton of mammalian cells, suggests that the nucleus is directly tethered to the cytoskeleton. This structural linkage is crucial for at least two reasons: (1) it is important to maintain nuclear morphology per se and (2) it allows mechanical force to be exerted upon the nucleus to move it within the cell, a process vitally important in muscle and nerve cell development, for example. At the center of the LINC complex is the interaction between SUN domain containing proteins of the inner nuclear membrane and KASH-peptide-containing proteins of the outer nuclear membrane. SUN and KASH interact directly and specifically in the perinuclear space. In many of the laminopathies, including the severe muscle disease, Emery-Dreifuss muscular dystrophy, neurological disorders such as cerebellar ataxia, and progeria the SUN-KASH interaction within the LINC complex is lost, resulting in severe consequences for nuclear morphology and positioning. In humans, at least five LINC complexes exist, each containing a different pair of SUN- and KASH-containing proteins. Signaling between these components is poorly understood. At the center of this proposal is the elucidation of the structure and function of the SUN-KASH interaction using biochemical and X-ray crystallographic tools. The atomic structure of the SUN-KASH complex should provide valuable insight into the regulation of this interaction. Despite the importance of the SUN-KASH complex for human physiology and pathology, its structure is still unknown. This can largely be attributed to the problems with obtaining sufficient quantities of the proteins for structural studies. We have developed a method to overcome these obstacles and are now able to pursue quantitative structure-function analysis. We anticipate that this project will shed light on the biophysical principles behind the nucleo-cytoskeletal connection established through LINC complexes. We will identify by X-ray crystallography the specific molecular details that regulate this conserved interconnection. In parallel, we will perform detailed biochemical and mutational analysis of the SUN-KASH complex. This study has the potential to enable the structural and molecular characterization of the entire LINC complex, including all other components. In consequence, this project should establish new drug targets for the cure of a broad array of laminopathies. PUBLIC HEALTH RELEVANCE: Muscular dystrophies collectively have a high impact on health, affecting tens of thousands of people in the United States alone. Emery-Dreifuss muscular is characterized by wasting of certain muscles, joint deformities and life-threatening heart problems that can result in premature and sudden death. There is currently no definitive therapy for Emery-Dreifuss muscular dystrophy or related diseases; therefore, the work in this project is designed to identify molecular targets that can lead to treatments for patients.