Structural Biology of Urothelial Membranes

哺乳动物尿路上皮的顶端表面覆盖着刚性斑块(也称为不对称斑块 单元膜或AUM)，其是六角排列的I6 nm颗粒的晶体阵列 由四个尿板(UP)组成。这些尿路蛋白斑块有助于尿路上皮 通透性屏障功能，其中一种尿路蛋白，UPIA，可以作为该类型的受体 1-菌毛状致尿路致病性大肠杆菌(UPEC)。UPEC通过结合在尿路中获得立足点， 通过其粘附素FimH，与尿路上皮受体结合。在上一次授权期内，我们获得了6A 分辨16 nm Uroplakin颗粒的低温EM结构，表明FimH结合可以 引起胞外区和跨膜的大的构象变化 尿板的螺旋状结构。这些结果表明，细菌附着可以诱导一种新的 跨膜信号转导导致细菌侵袭的机制及建立 尿路感染(UTI)是最常见的传染病之一。在下一笔赠款中 期间，我们将继续努力研究16-nm的结构与功能的关系 颗粒和细菌结合诱导信号转导的机制。在目标一中，我们将 通过执行电磁密度辅助建模来建立i6 nm粒子的原子模型 借助低温电子显微镜的改进的分辨率和尿板蛋白的结构域结构，我们可以 对接到电子密度。我们将获得用于3D的全长重组Uroplakin分子 结晶，针对尿路蛋白的原子分辨结构。在第二个目标中，我们将携带 Out电子层析三维可视化的AUM-细胞骨架连接在正常、 机械性伸展，在感染的膀胱里。我们还将调查这些人的身份 形成连接AUM和细胞骨架的桥梁的蛋白质。我们的结果应该是 有助于更好地了解尿路上皮斑块功能的结构基础，以及 尿路上皮斑块在尿路感染中的可能作用。

The apical surface of mammalian urothelium is covered by rigid plaques (also known as asymmetric unit membrane or AUM) that are hexagonally packed crystalline arrays of i6-nm particles consisting of four uroplakins (UPs). These uroplakin plaques contribute to the urothelial permeability barrier function, and one of the uroplakins, UPIa, can serve as the receptor for the type 1-fimbriated uropathogenic E. coli (UPEC). UPEC gains a foothold in the urinary tract by binding, via its adhesin FimH, to the urothelial receptor. During the last grant period, we obtained a 6A resolution cryo-EM structure of the 16-nm uroplakin particle and showed that FimH binding can induce large conformational changes of the extracellular domains as well as the transmembrane helices of the uroplakins. These results suggest that bacterial attachment can induce a novel mechanism of transmembrane signal transduction leading to bacterial invasion and establishment of urinary tract infection (UTI), one of the most common infectious diseases. In the next grant period, we will continue our efforts in studying the structure-function relationship of the 16-nm particle and the mechanism of bacterial binding-induced signal transduction. In Aim one, we will establish an atomic model of the i6-nm particle by performing an EM density-aided model building with the help of cryo-EM of improved resolution and domain structures of uroplakins that we can dock into the electron densities. We will obtain full-length recombinant uroplakin molecules for 3D crystallization, aiming at atomic resolution structures of the uroplakins. In Aim two, we will carry out an electron tomographic 3D visualization of the AUM-cytoskeleton connections in normal, in mechanically stretched, and in infected bladder. We will also investigate the identities of the proteins that form the bridge interconnecting the AUM and the cytoskeleton. Our results should lead to a better understanding of the structural bases of urothelial plaque function, and of the possible roles of urothelial plaques in urinary tract infection.