Structural Biology of Urothelial Membranes

尿路上皮膜的结构生物学

• 批准号: 6820732
• 负责人: XIANGPENG KONG
• 金额: $ 26.42万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6820732
• 关键词: SDS polyacrylamide gel electrophoresis; X ray crystallography; apical membrane; biological signal transduction; computer simulation; cryoelectron microscopy; crystallization; image processing; kidney function; membrane activity; membrane proteins; membrane structure; protein localization; protein structure function; scanning transmission electron microscopy; structural biology; urinary bladder epithelium

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.

膀胱上皮的顶部表面覆盖着刚性斑块，这些斑块由由四个主要的uroplakins（UPS）组成的16 nm蛋白质颗粒的六角形晶体阵列组成。这些尿路上皮斑块是一个有效的渗透性屏障，并且可能在排尿周期的不同阶段可逆地调整尿尿皮上顶端表面积的作用，此外，尿素病型1-纤维型大肠杆菌的附着在其尿藻蛋白Ia受体上的附着可能会导致尿路静脉内卵皮骨骼的静脉内骨骼囊泡囊肿。该项目的目的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个埃斯特罗姆分辨率冷冻EM结构，我们假设（i）两个尿布蓝蛋白对，即UPIA/II和UPIB/III，分别占据了16 nm粒子的内部和外部六个子域的内部和外部6个子域，以及（II）的传播（ii），（II II）均具有16 nm的固定式结构。通过构象变化。 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 改善3D建模，并制备ur云蛋白的3D晶体，以求解尿藻蛋白构成原子分辨率； （2）在16 nm粒子的3D结构中使用Fab片段和特定的凝集素作为探针进行几个尿布链蛋白部分； （3）研究通过比较粒子的3D冷冻EM结构在存在和不存在饱和量的细菌粘附素FIMH的情况下，通过比较粒子的3D冷冻EM结构，研究了可能的细菌结合诱导的构象变化。我们的结果应该可以更好地理解尿路上皮斑块功能的结构碱基，以及尿路上皮斑块在尿路感染中的可能作用。