Structure and Function of Lens Membrane Proteins

描述(申请人提供)：人工晶状体是一种独特的结构，设计精巧，可以将光线聚焦到视网膜上，这一过程需要根据眼睛与所聚焦对象的距离来改变晶状体的形状。为了实现它的功能，透镜也必须是透明的，并且它必须提供高折射率。我们感兴趣的是晶状体中膜蛋白的结构和功能，它们在维持晶状体的动态平衡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体是微循环系统的基础，该系统为深层纤维细胞提供营养，并清除它们的废物。此外，膜蛋白调节纤维细胞的紧密堆积，从而将细胞之间的间距缩小到比光波长更小的距离，这是避免晶体组织光散射的重要前提。这项建议侧重于在晶状体纤维细胞中发现的两种主要的膜蛋白，即Tetraspanin MP20和Aquaglycerporin MIP(也称为Aquaporin-0)。这两种膜蛋白中任何一种的突变都会导致白内障的形成，表明它们对晶状体正常功能的重要性。 关于MP20和其他Tetraspanins的结构信息仍然很稀少。因此，具体目标1主要是通过电子显微镜来确定MP20的结构，但也要寻求X射线结晶学方法。获得的MP20的结构信息将有助于对Tetraspanin家族其他成员的结构进行建模，这些成员在许多细胞过程中都是重要的，如细胞黏附、增殖、激活、迁移和凋亡。具体目标2旨在通过进一步研究MP20与显著的黏附调节剂Galectin-3的相互作用来表征其作为细胞黏附分子的功能。我们还将确定MP20是否能与晶状体特异性整合素结合，因为许多Tetraspanins已知与整合素相互作用，特别是当这些整合素包含b1亚单位时。 MIP作为水和非电荷小分子的孔的功能得到了很好的表征，但与其他水通道蛋白不同的是，MIP还具有粘附性，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的目标是使用X射线和电子晶体学相结合的方法为MIP介导的膜连接创建一个伪原子模型。膜连接的结构将揭示相互作用的MIP四聚体中水孔的排列，这对晶状体微循环系统的功能具有重要的意义。

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.