The role of SHROOM3 in congenital heart disease

SHROOM3在先天性心脏病中的作用

• 批准号: 10495952
• 负责人: Matthew D. Durbin
• 金额: $ 39.63万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-15 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10495952
• 关键词: Address; Affect; Amino Acid Substitution; Amino Acids; Anatomy; Animals; Binding; Binding Sites; Biological Assay; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cell Shape; Cells; Congenital Abnormality; Cytoplasmic Protein; Cytoskeleton; Data; Defect; Development; Disease; Disease model; Dsh protein; Embryo; Embryonic Heart; Etiology; Exhibits; Family; Genetic; Genomics; Growth; Heart; Heart Abnormalities; Heart Ventricle; Heterozygote; Histologic; Immune; Immunoprecipitation; Laboratories; Left; Left ventricular structure; MAPK8 gene; Maps; Mass Spectrum Analysis; Mentorship; Molecular; Molecular Biology; Monomeric GTP-Binding Proteins; Morphogenesis; Mus; Mutant Strains Mice; Neural Crest Cell; Pathogenesis; Pathway interactions; Patients; Pattern; Phosphorylation; Protein Kinase; Proteins; Research; Research Personnel; Role; Scientist; Series; Signal Pathway; Signal Transduction; Situs Inversus; Structure; TNFSF5 gene; Testing; Transfection; Ubiquitination; Variant; Ventricular; Ventricular Septal Defects; cardiogenesis; career; cell motility; cohort; congenital heart disorder; gene function; in vivo; loss of function; migration; mouse model; mutant; novel; planar cell polarity; progenitor; protein protein interaction; rho; skills; stem cells; transcriptome sequencing

ABSTRACT Congenital heart disease (CHD) is the most common birth defect, yet most etiologies remain unknown. SHROOM3 is a novel CHD candidate gene that functions through interactions with F-ACTIN, Rho Associated Coiled-Coil Containing Protein Kinase (ROCK) and Disheveled2 (DVL2) and thus participates in the noncanonical WNT/Planar Cell Polarity (PCP) signaling pathway. Disruption of PCP signaling leads to cardiac defects, including left-right patterning defects, left ventricle noncompaction (LVNC), ventricular septal defects (VSDs) and outflow tract (OFT) defects. However, SHROOM3’s precise role in the PCP signaling cascade, and in cardiac development, is only partially understood. For example, the Ware laboratory recently identified that SHROOM3 variants are associated with the left-right patterning defect heterotaxy and CHD in patients. This finding prompted me to study the impact of the loss of SHROOM3 expression on cardiac development. Utilizing a gene trap mouse model (Shroom3gt/gt), I demonstrated that SHROOM3 loss-of-function results in CHDs in embryonic hearts, including VSDs, LVNC and OFT defects in homozygous mutant animals. We also showed evidence there may be genetic interaction between Shroom3 and Dvl2 during cardiac development. Moreover, molecular studies have established a protein-protein interaction (either direct or through a common binding partner) between SHROOM3 and DVL2, though the binding site is poorly defined. Interestingly, a recent study revealed that canonical WNT/PCP signaling is controlled in part through the deubiquination of DVL2 via USPX9, a highly conserved deubiquitylase. Utilizing immunoprecipitation (IP)/mass spectrometry (MS) of SHROOM3- transfected Cos7 cells, I have shown protein-protein interaction between SHROOM3 and USPX9. These observations have collectively led to the hypothesis that signaling between SHROOM3, DVL2 and USP9X is necessary for normal cardiac development and that disruption of this signaling can contribute to CHD. There is evidence SHROOM3 interacts with USP9x and DVL2 however the interactions are at best poorly defined. Genetic interactions between Shroom3 and Dvl2 during cardiac development have not been not fully delineated and genetic interaction between Shroom3 and Usp9x are unexplored. Aim 1 will assay genetic interaction between Shroom3, Dvl2 and Usp9x, analyzing cardiac defects and disrupted PCP endpoints. Although data demonstrate SHROOM3 binds DVL2, and USP9X, the binding site for DVL2 is only roughly localized to a 490 amino acid region within SHROOM3 and SHROOM3’s USP9X binding site is completely unknown. Aim 2 will assay protein-protein interactions between SHROOM3, USP9X and DVL2. Defining SHROOM3’s interaction partners will help determine its role in PCP, cardiac development and CHD pathogenesis. The plan will also build skills, under close mentorship as an Early Career Investigator, in molecular biology, mouse disease models and genomics analysis, providing a skillset to investigate CHD pathogenesis as an independent scientist.

抽象的 先天性心脏病（CHD）是最常见的先天缺陷，但大多数病因仍然未知。 Shroom3是一种新型的CHD候选基因，它通过与F-肌动蛋白相互作用，Rho相关 含有蛋白激酶（岩石）和DINEVELED2（DVL2）的盘绕卷头卷，因此参与 非规范WNT/平面细胞极性（PCP）信号通路。 PCP信号的破坏导致心脏 缺陷，包括左右构图缺陷，左心室不合作（LVNC），心室间隔缺陷 （VSD）和出口（OFT）缺陷。但是，Shroom3在PCP信号级联中的精确作用，以及 在心脏发展中，只被部分理解。例如，商品实验室最近确定 shroom3变体与患者的左右构图缺陷异质和冠心病有关。这 发现促使我研究了shoom3表达对心脏发展的影响。利用 基因陷阱鼠标模型（Shroom3GT/GT），我证明了shroom3功能丧失导致CHD中的CHD 纯合突变动物中的胚胎心脏，包括VSD，LVNC和经常缺陷。我们也表明 在心脏发育过程中，Shoom3和DVL2之间可能存在遗传相互作用的证据。而且， 分子研究已经建立了蛋白质 - 蛋白质相互作用（直接或通过共同结合 伙伴）在shroom3和dvl2之间，尽管绑定位点的定义很差。有趣的是，最近的一项研究 揭示了Canonical Wnt/PCP信号通过DVL2通过USPX9的去泛泛泛液的部分控制 高度组成的去泛素地酶。使用免疫沉淀（IP）/质谱（MS）3- 转染的COS7细胞，我显示了shroom3和USPX9之间的蛋白质 - 蛋白质相互作用。这些 观察总共提出了以下假设：shroom3，dvl2和usp9x之间的信号是 正常心脏发育和这种信号传导的破坏可能有助于CHD。有 证据shroom3与USP9X和DVL2相互作用，但是相互作用的定义较差。 心脏发育过程中Shroom3和DVL2之间的遗传相互作用尚未完全划定 Shroom3和USP9X之间的遗传相互作用是出乎意料的。 AIM 1将分析遗传相互作用 在Shroom3，DVL2和USP9X之间，分析了心脏缺陷和PCP终点中断。虽然数据 演示shroom3绑定DVL2和USP9X，DVL2的结合位点仅大致定位于490 Shroom3和Shroom3的USP9X绑定位点内的氨基酸区域是完全未知的。 AIM 2意志 shroom3，USP9X和DVL2之间的测定蛋白质蛋白质相互作用。定义Shroom3的互动 伴侣将有助于确定其在PCP，心脏发育和CHD发病机理中的作用。该计划也将建立 在分子生物学，小鼠疾病模型和 基因组学分析，提供了一种技能，以研究作为独立科学家的CHD发病机理。