Structure and Function of Lens Membrane Proteins

描述（申请人提供）：目镜透镜是一种独特的结构，其被精细地设计成将光聚焦到视网膜上，该过程需要根据眼睛与其聚焦的物体的距离而使透镜的形状发生实质性变化。为了实现其功能，透镜还必须是透明的，并且它必须提供高折射率。我们对透镜中的膜蛋白的结构和功能感兴趣，这些膜蛋白在维持透镜内环境稳定和透明度方面起着至关重要的作用。一系列高度专业化的膜通道和转运蛋白是微循环系统的基础，为深层纤维细胞提供营养，并清除它们的废物。此外，膜蛋白介导纤维细胞的紧密堆积，从而将细胞之间的空间减小到小于光的波长的距离，这是避免光被透镜组织散射的重要先决条件。该提议集中于在透镜纤维细胞中发现的两种主要膜蛋白，即四跨膜蛋白MP 20和水甘油孔蛋白MIP（也称为水通道蛋白-0）。这两种膜蛋白中的任何一种突变都会导致白内障的形成，证明了它们对正常透镜功能的重要性。 关于MP20和其他四跨膜蛋白的结构信息仍然很少。因此，具体目标1是主要通过电子显微镜确定MP20的结构，但也采用X射线晶体学方法。获得的MP20的结构信息将是有用的模型四跨膜蛋白家族，这是重要的许多细胞过程，如细胞粘附，增殖，活化，迁移和凋亡的其他成员的结构。具体目标2是通过进一步研究MP20与半乳糖凝集素-3（一种重要的粘附调节剂）的相互作用来表征MP20作为细胞粘附分子的功能。我们还将确定MP20是否可以结合晶状体特异性整合素，因为已知许多四跨膜蛋白与整合素相互作用，特别是如果这些包含b1亚基。 MIP作为水和小的不带电分子的孔的功能被很好地表征，但与其他水通道蛋白不同，MIP还具有粘附性质，并且可以形成细胞间连接，可能在纤维细胞之间产生连续的水孔。具体目标3的目的是使用X射线和电子晶体学的组合创建MIP介导的膜连接的伪原子模型。膜连接的结构将揭示相互作用的MIP四聚体中的水孔的排列，这对透镜中微循环系统的功能具有重要意义。

DESCRIPTION (provided by applicant): The ocular lens is a unique structure exquisitely designed to focus light onto the retina, a process that requires substantial shape changes of the lens according to the distance of the eye from the object it is focusing on. To accomplish its function, the lens also has to be transparent, and it has to provide a high index of refraction. We are interested in the structure and function of membrane proteins in the lens, which play crucial roles in maintaining lens homeostasis and transparency. A highly specialized array of membrane channels and transporters is the basis for a microcirculation system that supplies deeper-lying fiber cells with nutrients and clears them of waste products. Moreover, membrane proteins mediate the tight packing of the fiber cells, thus reducing spaces between cells to a distance smaller than the wavelength of light, an important prerequisite to avoid light scattering by the lens tissue. This proposal focuses on the two major membrane proteins found in lens fiber cells, the tetraspanin MP20 and the aquaglyceroporin MIP (also referred to as aquaporin-0). Mutations in either one of these two membrane proteins lead to the formation of cataracts, demonstrating their importance for proper lens function. Structural information on MP20 as well as other tetraspanins is still sparse. Specific Aim 1 is thus to determine the structure of MP20 primarily by electron microscopy but also pursuing an X-ray crystallographic approach. The structural information obtained for MP20 will be useful to model the structure of other members of the tetraspanin family, which are important in many cellular processes, such as cell adhesion, proliferation, activation, migration, and apoptosis. Specific Aim 2 is directed towards characterizing the function of MP20 as a cell adhesion molecule by further studies of its interaction with galectin-3, a prominent adhesion modulator. We will also determine whether MP20 can bind to lens-specific integrins, as many tetraspanins are known to interact with integrins, especially if these contain a b1 subunit. The function of MIP as a pore for water and small uncharged molecules is well characterized, but unlike other aquaporins MIP also has adhesive properties and can form intercellular junctions, possibly creating continuous water pores between fiber cells. The objective of Specific Aim 3 is to create a pseudo-atomic model for the MIP-mediated membrane junction using a combination of X-ray and electron crystallography. The structure of the membrane junction will reveal the arrangement of the water pores in interacting MIP tetramers, which has important implications for the functioning of the microcirculation system in the lens.