Structural Biology of Urothelial Membranes

膀胱上皮的顶面覆盖着坚硬的斑块，这些斑块由四种主要的尿斑蛋白（UPs）组成的16 nm蛋白颗粒的六边形排列的晶体阵列组成。这些尿路上皮斑块作为一种有效的通透性屏障，在排尿周期的不同阶段可能在尿路上皮顶面面积的可逆性调节中发挥作用。大肠杆菌对尿斑蛋白Ia受体的依赖性可引起尿路上皮细胞骨架重排、细胞凋亡和细菌侵袭。本项目的目标是了解尿路上皮斑块功能的结构基础。基于我们最近获得的16 nm小鼠尿路上皮颗粒的10埃分辨率冷冻-EM结构，我们假设（i）两个尿斑蛋白对，即，UPIa/II和UPIb/III，分别占据16 nm颗粒的内部和外部六个子域，并且（ii）16 nm uroplakin颗粒的相对灵活的结构可以通过构象变化介导跨膜信号转导。为了验证这些假设，并进一步了解尿路上皮斑块的结构-功能关系，我们将进行三个系列的研究：（1）通过获得分辨率为7 Angiol的16 nm颗粒的冷冻-EM结构来可视化尿斑蛋白的单个跨膜螺旋，将UPIa和Ib的原子模型对接到冷冻-EM密度图中， （2）以Fab片段和特异性凝集素为探针，在16 nm颗粒的三维结构中定位几个尿斑蛋白部分;和（3）通过比较3D冷冻，研究细菌结合诱导的16 nm尿斑蛋白颗粒可能的构象变化。存在和不存在饱和量细菌粘附素FimH时颗粒的EM结构。我们的研究结果将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 bladder epithelium is covered by rigid-looking plaques consisting of hexagonally packed crystalline arrays of 16 nm protein particles made up of four major uroplakins (UPs). These urothelial plaques serve as an effective permeability barrier, and may play a role in the reversible adjustment of the urothelial apical surface area during different phases of the micturition cycle, In addition, the attachment of uropathogenic type 1-piliated E. coli to their uroplakin Ia receptor can cause urothelial cytoskeletal rearrangement, apoptosis and bacterial invasion. The goal of this project is to understand the structural basis of urothelial plaque functions. Based on our recently obtained 10 Angstrom resolution cryo-EM structure of the 16 nm mouse urothelial particles, we hypothesize that (i) the two uroplakin pairs, i.e., UPIa/II and UPIb/III, occupy the inner and outer six subdomains of the 16 nm particle, respectively, and that (ii) the relatively flexible structure of the 16 nm uroplakin particle can mediate transmembrane signal transduction through conformational changes. To test these hypotheses and to further understand the structure-function relationship of the urothelial plaques, we will perform three series of studies that will: (1) visualize the individual transmembrane helices of the uroplakins by obtaining a cryo-EM structure of the 16nm particle at the resolution range of 7 Angstroms dock the atomic models of UPIa and Ib into the cryo-EM density maps thus improving the 3D modeling, and prepare 3D crystals of uroplakins aiming at solving uroplakin strucure to atomic resolution; (2) localize in the 3D architecture of the 16 nm particle several uroplakin moieties using Fab fragments and specific lectins as the probes; and (3) study the possible bacterial binding-induced conformational changes of the 16 nm uroplakin particle by comparing the 3D cryo-EM structures of the particle in the presence and absence of saturating amounts of the bacterial adhesin FimH. 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.