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

描述(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型板层赋予，A型和B型板层是排列在核膜核质表面并形成核板层的蛋白质网络。散布在编码A型板层蛋白的LMNA上的突变，以及其他核膜蛋白内的突变与广泛的人类疾病有关，统称为椎板病。这些疾病的分子病因尚不清楚。LINC复合体是一种进化保守的蛋白质复合体，连接核膜的内膜和外膜，并将核层与哺乳动物细胞的细胞骨架物理连接。最近对LINC复合体的表征表明，细胞核直接与细胞骨架相连。这种结构连接是至关重要的，至少有两个原因：(1)保持细胞核本身的形态很重要，(2)它允许对细胞核施加机械力，使其在细胞内移动，例如，这是一个在肌肉和神经细胞发育中至关重要的过程。LINC复合体的中心是内核膜含SUN结构域的蛋白质与外核膜含Kash多肽的蛋白质之间的相互作用。太阳和卡什在核周空间直接和具体地相互作用。在许多椎板病变中，包括严重的肌肉疾病、Emery-Dreifuss肌营养不良症、神经疾病如小脑性共济失调和早衰症，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，至少存在五个LINC复合体，每个复合体包含一对不同的含有SUN和KASH的蛋白质。这些组件之间的信号传递知之甚少。这一提议的核心是利用生化和X射线结晶学工具阐明SUN-KASH相互作用的结构和功能。太阳-卡什复合体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复合体对人体生理学和病理学具有重要意义，但其结构仍不清楚。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克服这些障碍的方法，现在能够进行定量的结构-功能分析。我们预计这个项目将阐明通过LINC复合体建立的核-细胞骨架连接背后的生物物理原理。我们将通过X射线结晶学确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复合体进行详细的生化和突变分析。这项研究有可能使整个LINC复合体的结构和分子表征成为可能，包括所有其他成分。因此，该项目应该为治疗广泛的椎板病建立新的药物靶点。 公共卫生相关性：肌肉营养不良总体上对健康有很大影响，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萎缩、关节畸形和危及生命的心脏问题，这些问题可能导致过早和猝死。目前还没有针对Emery-Dreifuss肌营养不良症或相关疾病的确切治疗方法；因此，该项目的工作旨在确定可以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.