Research Project 2

研究项目2

基本信息

批准号：
10090713
负责人：
Heather R Flanagan Steet
金额：
$ 21.42万
依托单位：
CLEMSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-10 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10090713
关键词：
Address Affect Biochemical Bioinformatics Biosensor Cartilage Cell Adhesion Molecules Centers of Research Excellence Chondrocytes Chondrogenesis Cleaved cell Congenital disorders of glycosylation Defect Development Disease Drosophila genus Embryo Environmental Risk Factor Enzymes Exhibits Fishes Foundations Future Gene Expression Genes Genetic Goals Guanosine Diphosphate Mannose Heat-Shock Response Hereditary Disease Human Genetics Impairment Individual Knowledge Laboratories Link Maps Matrix Metalloproteinases Mendelian disorder Methods Modeling Molecular Molecular Genetics Mutation N-Cadherin Pathogenesis Pathogenicity Pathology Pathway interactions Patients Penetrance Peptide Hydrolases Pharmacology Phenotype Phosphomannomutase Polysaccharides Process Production Protein Glycosylation Proteins Research Project Grants Services Severity of illness Signal Transduction Single-Gene Defect System Technology Testing Tissues Transgenic Organisms Viral Work Zebrafish cartilage development clinical phenotype confocal imaging disease-causing mutation fly genetic manipulation glycosylation in vivo mannose 1-phosphate mannose 6 phosphate mutant novel null mutation promoter stem sugar nucleotide tool transcriptome sequencing

项目摘要

PROJECT SUMMARY Congenital disorders of glycosylation (CDGs) are a heterogeneous group of rare inherited diseases caused by mutations in genes involved in protein glycosylation. The most common CDG, PMM2-CDG, results from mutations in the gene phosphomannomutase 2 (PMM2), encoding an enzyme that converts mannose 6- phosphate (M6P) to mannose 1-phosphate (M1P). Defects in PMM2 limit the production of GDP-mannose, a nucleotide sugar essential to synthesize precursors needed for N-linked glycosylation. Reduced GDP- mannose causes protein hypoglycosylation and numerous clinical phenotypes. The connection between hypoglycosylated proteins and phenotypes is unclear, creating a major gap in our knowledge of CDG disease pathogenesis. PMM2-CDG patients exhibit variable penetrance indicating that genetic and/or environmental factors modify disease. We characterized a zebrafish model of PMM2-CDG and identified two classes of enzymes, the protein proconvertases (PCs) and matrix metalloproteinases (MMPs), as candidate drivers of pathology. Analyses of cartilage defects in pmm2 mutant zebrafish revealed a block in early chondrocyte development that is associated with defective processing of the cell adhesion molecule N- cadherin. N-cadherin is sequentially cleaved by furin PCs and MMPs, and both exhibit altered activity in pmm2 mutant zebrafish. The proposed studies address the hypothesis that reduced glycosylation alters the activity of PCs like furin, initiating a cascade involving MMPs that disrupts processing of key cell adhesion molecules, including N-cadherin. We will define how hypoglycosylation of protein processing enzymes alters tissue development; determine whether there is a common mechanism among CDG subtypes; and identify genetic modifiers of CDG disease severity. Toward these goals in Aim 1 we will develop novel zebrafish lines that express wild type and glycan-deficient FLAG-tagged forms of several PCs and MMP enzymes. We will use these tools to define how hypoglycosylation of individual enzymes contributes to impaired chondrogenesis in PMM2-CDG. In Aim 2 we will perform RNA sequencing on several zebrafish models of PMM2, STT3A and STT3B-CDG to identify the pathogenic mechanisms and molecular networks that are commonly or uniquely altered in CDG. Using these analyses in combination with novel Drosophila models we will also pursue genetic modifiers of CDG disease severity. The molecular and genetic pathways identified as sensitive to hypoglycosylation will provide foundational information to develop much needed therapies. Further, the platform established within this proposal will create the road map to ultimately study how disruption of other N-glycosylation genes causes disease.

项目概要先天性糖基化障碍 (CDG) 是一组由以下原因引起的罕见遗传病：与蛋白质糖基化相关的基因突变。最常见的 CDG，PMM2-CDG，来自磷酸甘露糖变位酶 2 (PMM2) 基因发生突变，该基因编码将甘露糖 6- 转化的酶磷酸盐 (M6P) 转化为 1-磷酸甘露糖 (M1P)。 PMM2 的缺陷限制了 GDP-甘露糖的生产，GDP-甘露糖是一种合成 N-连接糖基化所需前体所必需的核苷酸糖。 GDP减少- 甘露糖会导致蛋白质低糖基化和许多临床表型。之间的联系低糖基化蛋白和表型尚不清楚，造成我们对 CDG 的了解存在重大差距疾病发病机制。 PMM2-CDG 患者表现出不同的外显率，表明遗传和/或环境因素改变疾病。我们表征了 PMM2-CDG 的斑马鱼模型，并确定了两个候选酶类：蛋白质原转化酶 (PC) 和基质金属蛋白酶 (MMP) 病理学的驱动因素。对 pmm2 突变斑马鱼软骨缺陷的分析揭示了早期的软骨缺陷软骨细胞发育与细胞粘附分子 N- 的加工缺陷有关钙粘蛋白。 N-钙粘蛋白依次被弗林蛋白酶 PC 和 MMP 裂解，并且两者都在 pmm2 中表现出改变的活性突变斑马鱼。拟议的研究提出了这样的假设：糖基化的减少会改变弗林蛋白酶等 PC 的活性，启动涉及 MMP 的级联反应，破坏关键细胞的处理粘附分子，包括 N-钙粘蛋白。我们将定义蛋白质加工的低糖基化如何酶改变组织发育；确定CDG亚型之间是否存在共同机制；并确定 CDG 疾病严重程度的遗传修饰因素。为了实现目标 1 中的这些目标，我们将开发新颖的表达野生型和聚糖缺陷型 FLAG 标记形式的多种 PC 和 MMP 的斑马鱼系酶。我们将使用这些工具来定义单个酶的低糖基化如何促进 PMM2-CDG 软骨形成受损。在目标 2 中，我们将对几条斑马鱼进行 RNA 测序 PMM2、STT3A 和 STT3B-CDG 模型，用于识别致病机制和分子网络 CDG 中常见或独特的改变。将这些分析与新型果蝇结合使用我们还将研究 CDG 疾病严重程度的基因修饰模型。分子和遗传途径被鉴定为对低糖基化敏感的药物将为开发急需的产品提供基础信息疗法。此外，该提案中建立的平台将创建最终研究的路线图其他 N-糖基化基因的破坏如何导致疾病。