细胞核膜蛋白SUN1及其复合物的结构与功能研究

Studies on the structure and function of the nuclear envelope have been a hot field of life science research. In eukaryotic cells, the nuclear envelope (NE) is a double-membrane structure that serves as a physical barrier to partition the nuclear and cytoplasmic environments. NE is composed of the inner and outer nuclear membranes (ONM and INM). The highly dynamic regulation of nuclear require the linker of nucleoskeleton and cytoskeleton (LINC) complexes. The evolutionarily conserved SUN and KASH proteins from the INM and ONM , respectively, interact with each other in the perinuclear space to form the SUN-KASH complexes that span across the nuclear envelope. The formation of the SUN-KASH complexes establishes the physical linkage between the cytoskeleton and nuclear lamina, which is instrumental for the mechanical force transmission from the cytoplasm to the nuclear interior and is essential for cellular processes such as nuclear positioning and migration, centrosome-nucleus anchorage, and chromosome dynamics. SUN1, one of the most prominent SUN proteins in mammalian, is a main component of LINC complex. However, there is no structural report of SUN1 and how it works functionally is still elusive. In this proposal, we would express and purify different boundaries, including CC, SUN domain or the nuclear region of SUN1. By using structural biology, molecular biology and biochemistry methods, we would study the molecular mechanism of formation and regulation of SUN-KASH complex. The work would provide new insights into the significant function of the LINC complex in formation and regulation of nuclear membrane.

真核细胞核膜的结构和功能研究是生命科学领域的前沿课题。核膜是由内核膜和外核膜组成的双层膜状结构，它可以分隔细胞核与细胞质，保护细胞核内的遗传物质，并维持细胞正常活动所需的机械特性。核膜高度动态调控功能的实现需要连接细胞核骨架与细胞骨架的LINC复合物的参与。LINC复合物主要由一对分别定位于细胞外核膜的KASH蛋白家族和内核膜的SUN蛋白家族组成。核膜内外SUN-KASH复合物的形成实现了跨核膜机械力的传递，为细胞核定位等重要生物学过程提供了结构基础。其重要成员SUN1的三维空间结构研究，及其行使生物学功能的分子机制处于空白的阶段。本项目拟通过重组表达、纯化SUN1含CC、SUN、核内区域的不同截短体，综合运用结构生物学和分子生物学的方法，研究SUN-KASH相互作用的分子机制及其生物活性调控的工作机制。本项目研究结果将对深入认识LINC参与组成及调控核膜的结构和功能具有重要意义。

真核细胞核膜是由内核膜和外核膜组成的双层膜状结构。核膜高度动态调控功能的实现需要连接细胞核骨架与细胞骨架的LINC复合物的参与。LINC复合物主要由一对分别定位于细胞外核膜的KASH蛋白家族和内核膜的SUN蛋白家族组成，实现了跨核膜机械力的传递，为细胞核定位等重要生物学过程提供了结构基础。其重要成员SUN1的三维空间结构研究，及其行使生物学功能的分子机制处于空白的阶段。本项目研究表达纯化了SUN1的AID-SUN结构域，并利用结构生物学晶体衍射技术对C-端区域AID-SUN片段的蛋白晶体进行了结构解析，并获了2.2 Å高分辨的蛋白晶体结构；利用分子生物学、生物化学、酵母双杂交等方法，通过突变破坏AID自身结构，以及AID和KASH-lid相互作用的界面，可以恢复SUN结合KASH的活性，验证了AID调控SUN结构域活性的机制。通过对小鼠SUN1和SUN2的AID-SUN进行结构比对，发现二者非常相似，而SUN1的结构更加紧凑。因此该研究表明AID区域对SUN蛋白自抑制的分子机制在SUN蛋白家族中高度保守。