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，A型和B型板层在核膜的核质面处形成一个二维蛋白质网络，即板层。散布在编码A型板层蛋白的LMNA基因上的突变，以及其他核膜蛋白内的突变，都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。Emery-Dreifuss肌营养不良症(EDMD)是最突出的椎板病，是一种无法治愈的破坏性肌肉萎缩性疾病，由内核膜(INM)蛋白Emerin、板层或外核膜(ONM)Kash-Proteins 1和2的突变引起。这四种蛋白质通过LINC复合体连接，LINC复合体是INM太阳蛋白和ONM Kash-Proteins之间进化保守的蛋白质复合体，连接核膜表面并物理地将核层连接到哺乳动物细胞的细胞骨架上。综上所述，这些数据表明，EDMD是核定位异常的结果，或者是通过LINC复合体的机械信号异常的结果。此外，INM处SUN1的过度积聚是EDMD的病理效应。在R21探索性赠款的资助下，我们于2012年确定了SUN-Kash复合体的核心结构，首次提供了对LINC复合体的分子洞察。在这里，我们建立在这些数据的基础上，并建议一个研究计划，该计划应该有助于发现药物靶点，有望转化为EDMD患者的药物策略。这项提案概述了一些实验，这些实验将导致A)对人类SUN-Kash相互作用组的结构和生物化学的了解，B)LINC复合体锚定到板层的结构基础，以及C)LINC复合体的组装和拆卸的调节。我们预计，对这三个目标的追求将产生对形成EDMD基础的蛋白质网络的更好的分子描述，从而揭示可能扰乱这些过程的药物靶点。我们还预计，这项研究将促进我们对核膜的总体理解，这将对大量的病理性核膜疾病产生切实的影响。

DESCRIPTION (provided by applicant): In mammalian cells, A- and B-type lamins form a two-dimensional protein meshwork, the lamina, at the nucleoplasmic face of the nuclear envelope. Mutations scattered along the LMNA gene, 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. Emery-Dreifuss muscular dystrophy (EDMD), the most prominent laminopathy, is an incurable, devastating muscular wasting disease, caused by mutations in either, the inner nuclear membrane (INM) protein emerin, laminA, or the outer nuclear membrane (ONM) KASH-proteins nesprins 1 and 2. The four proteins are connected via LINC complexes, evolutionary-conserved protein complexes between INM SUN-proteins and ONM KASH-proteins that bridge the faces of the nuclear envelope and physically connect the nuclear lamina to the cytoskeleton of mammalian cells. Taken together, the data suggests that EDMD is the result of aberrant nuclear positioning or, alternatively, aberrant mechanical signaling through the LINC complex. Furthermore, it has been shown that the overaccumulation of Sun1 at the INM is the pathological effector of EDMD. With funding through an exploratory R21 grant we have determined the core structure of the SUN-KASH complex in 2012, providing the first molecular insight into LINC complexes. Here, we build on this data and suggest a research program that should aid in the discovery of drug targets that hopefully will translate into a medication strateg for EDMD patients. This proposal outlines experiments that will lead A) to a comprehensive structural and biochemical understanding of the human SUN-KASH interactome, B) a structural basis for LINC complex anchorage to the lamin layer, and C) insight into the regulation of LINC complex assembly and disassembly. We expect that the pursuit of these three aims will yield a much better molecular description of the protein network that forms the basis of EDMD, and consequently will unveil possible drug targets that disrupt these processes. We further anticipate that this research will advance our understanding of the nuclear envelope in general, which will have a tangible impact on the vast array of pathological nuclear envelope disorders.