Role of Uroplakins in Urinary Tract Development and CAKUT

尿斑蛋白在尿路发育和 CAKUT 中的作用

• 批准号: 8821265
• 负责人: Gerard L Apodaca
• 金额: $ 45.54万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8821265
• 关键词: Adult; Affect; Apical; Binding; Biochemical; Biochemistry; Biological Models; C-terminal; Cell Line; Cells; Complex; Cytoplasmic Tail; Defect; Development; Ectopic Expression; Ensure; Epithelial; Epithelial Cells; Extravasation; Fetus; Fostering; Functional disorder; Gene Targeting; Genetic; Human; Injection of therapeutic agent; Integral Membrane Protein; Internet; Kidney; Knockout Mice; Larva; Lead; Live Birth; Lower urinary tract; MDCK cell; Membrane; Metanephric Diverticulum; Molecular; Morphogenesis; Mutate; Mutation; NMR Spectroscopy; Organogenesis; Orthologous Gene; Phenocopy; Phenotype; Phosphorylation; Play; Process; Pronephric structure; Protein Isoforms; Proteins; Recruitment Activity; Role; Structure; Surface; Tail; Testing; Tissues; Tracer; Urinary tract; Urogenital Sinus; Work; Zebrafish; apical membrane; atypical protein kinase C; base; cellular microvillus; congenital anomaly; developmental disease; ezrin; insight; kidney cell; kidney epithelial cell; malformation; moesin; mutant; protein complex; protein kinase C zeta; public health relevance; radixin protein; solute

DESCRIPTION (provided by applicant): Congenital anomalies of the kidney and urinary tract (CAKUT) are developmental disorders that occur in 1 out of every 500 live births, yet the cellular and molecular basis of these malformations has not been revealed. UPK3a, which encodes the type I transmembrane protein uroplakin 3a (UPK3a), is one gene targeted in CAKUT. Substitution of a Pro residue for a Leu (P273L) in the cytoplasmic domain of UPK3a leads to renal adysplasia and other urinary tract defects. However, the function(s) of UPK3a during urinary tract development, and the reason why mutations in this protein lead to CAKUT are not known. Using zebrafish larvae, we find that loss of expression of the UPK3a ortholog Upk3l leads to urinary tract (i.e., pronephros) dysfunction as a result of altered expression of Par polarity complex proteins (Par3, Par6, aPKC?) and defects in ezrin activation and microvilli formation. Moreover, our recent genetic and biochemical studies demonstrate that UPK3a/Upk3l may exert its influence by binding to aPKC? and then by fostering interactions between aPKC? and ezrin at the apical pole of pronephric tubule epithelial cells. Based on these observations, we propose that urinary tract development is dependent on the ability of UPK3a to promote UEEC differentiation, and mutations in UPK3a (e.g., P273L) perturb this function, leading to CAKUT. In our first aim we will use biochemistry to further define the cytoplasmic motifs in UPK3a and Upk3l that bind to the Par complex proteins and vice-versa. We will also define whether recruitment of the Par complex to the apical membrane of pronephric tubule cells depends on interactions with UPK3a/Upk3l. Furthermore, we will ectopically express UPK3a/Upk3l in MDCK cells, which do not normally express these proteins, and assess what impact this has on Par complex recruitment. In the second aim we will define how ezrin interacts with UPK3a/Upk3l and whether this interaction is critical for microvilli formation. We will further define whether aPKC? is responsible for phosphorylating ezrin-T567, a critical step in ezrin activation. In the third aim we will use MDCK cells as a model system to explore whether the P273L mutation alters ER exit and apical surface delivery of UPK3a. We will also use biochemistry and NMR spectroscopy to define whether the C-terminal tail of UPK3a contains structure, whether this structure is perturbed by the P273L mutation, and whether this mutation affects interactions with its binding partners. The proposed work is important because it will provide fundamental new insights into the basic mechanisms of lower urinary tract development and epithelial differentiation, into the role of UPK3a in these processes, and into the molecular and cellular basis of CAKUT.

描述（由申请人提供）：先天性肾脏和泌尿道异常（CAKUT）是一种发育障碍，每500例活产中有1例发生，但这些畸形的细胞和分子基础尚未揭示。UPK 3a编码I型跨膜蛋白uroplakin 3a（UPK 3a），是CAKUT中靶向的一个基因。在UPK 3a的胞质结构域中，Pro残基取代Leu（P273 L）导致肾发育不良和其他泌尿道缺陷。然而，UPK 3a在泌尿道发育过程中的功能以及该蛋白质突变导致CAKUT的原因尚不清楚。使用斑马鱼幼虫，我们发现UPK 3a直系同源物Upk 3l表达的缺失导致泌尿道（即，Par极性复合蛋白（Par 3、Par 6、aPKC？）以及埃兹蛋白活化和微绒毛形成的缺陷。此外，我们最近的遗传和生化研究表明，UPK 3a/Upk 3l可能发挥其影响，结合aPKC？然后通过促进aPKC？Ezrin位于原肾小管上皮细胞的顶极。基于这些观察，我们提出尿路发育依赖于UPK 3a促进UEEC分化的能力，并且UPK 3a中的突变（例如，P273 L）扰乱该功能，导致CAKUT。在我们的第一个目标中，我们将使用生物化学来进一步定义与Par复合物蛋白结合的UPK 3a和Upk 3l中的细胞质基序，反之亦然。我们还将确定是否招募Par复合物的前肾小管细胞的顶膜依赖于与UPK 3a/Upk 3l的相互作用。此外，我们将在MDCK细胞中异位表达UPK 3a/Upk 3l，这些细胞通常不表达这些蛋白质，并评估这对Par复合物募集的影响。在第二个目标中，我们将定义ezrin如何与UPK 3a/Upk 3l相互作用，以及这种相互作用是否对微绒毛形成至关重要。我们将进一步 定义是否aPKC？负责磷酸化ezrin-T567，这是ezrin激活的关键步骤。在第三个目标中，我们将使用MDCK细胞作为模型系统来探索P273 L突变是否改变ER出口和UPK 3a的顶端表面递送。我们还将使用生物化学和NMR光谱来确定UPK 3a的C末端尾部是否包含结构，该结构是否受到P273 L突变的干扰，以及该突变是否影响与其结合伴侣的相互作用。这项工作很重要，因为它将为下尿路发育和上皮分化的基本机制，UPK 3a在这些过程中的作用以及CAKUT的分子和细胞基础提供新的见解。