Pathogenesis of diseases caused by aberrant COPII megavesicle assembly

COPII巨泡组装异常引起的疾病的发病机制

• 批准号: 8817180
• 负责人: Jinoh Kim
• 金额: $ 36.58万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8817180
• 关键词: Accounting; Affect; Attenuated; Binding; Biochemical; Biogenesis; Biological Assay; CUL3 gene; Caliber; Cell Line; Cells; Collagen; Collagen Type II; Congenital Abnormality; Craniofacial Abnormalities; Data; Defect; Deposition; Development; Developmental Process; Dimensions; Disease; Dysplasia; Electron Microscopy; Embryo; Embryonic Development; Endoplasmic Reticulum; Exclusion; Extracellular Matrix; Fibroblasts; Fishes; Forebrain Development; Generations; Glean; Goals; Guanosine Triphosphate; Holoprosencephaly; Hydrolysis; Impairment; In Vitro; Knockout Mice; Knowledge; Lesion; Link; Membrane; Microscopy; Molecular; Monitor; Mono-S; Mus; Mutation; Pathogenesis; Patients; Phenotype; Procollagen; Protein Export Pathway; Proteins; Reporting; Research; Resolution; Retinoids; Role; Signal Pathway; Sorting - Cell Movement; Structure; Syndrome; Testing; Transport Vesicles; Vertebrates; Vesicle; base; consanguineous family; craniofacial; craniofacial development; human disease; in vivo; mutant; neurodevelopment; novel; null mutation; paralogous gene; premature; public health relevance; relating to nervous system; skeletal; skeletal abnormality; smoothened signaling pathway; ubiquitin ligase

DESCRIPTION (provided by applicant): Cranio-lenticulo-sutural-dysplasia (CLSD) is a recessive syndrome with craniofacial, skeletal and neural defects (9). CLSD was originally associated with a homozygous F382L mutation in SEC23A (8). SEC23A is an essential component of COPII vesicles, which are responsible for export of cargo molecules out of the endoplasmic reticulum (ER) (77). The SEC23A F382L mutation disrupts COPII vesicle assembly (20). Recently, we have identified a novel CLSD case with a heterozygous M702V SEC23A mutation (10, 27). Surprisingly, fibroblasts derived from this patient display a collagen-specific secretion defect. Depletion of Sec23a results in a reduction in the extracellular matrix/collagen and craniofacial/skeletal abnormalities in fish (8, 31). Combined, these findings define a critical link between COPII vesicle biogenesis, secretion of collagen and craniofacial development. In this study we investigate this link. Procollagen forms about a 300nm-long rigid fibril in the ER and requires COPII proteins for export (77). However, because of its size and rigidity, procollagen cannot be packaged into the standard COPII vesicles which have a 60nm diameter. Recently, it was shown that CUL3-KLHL12 drives the assembly of large COPII-coated structures and expedites secretion of collagen (23). However, how COPII proteins generate these "megavesicles" remains unclear. Our studies have demonstrated that the sizes of these unusually large COPII vesicles, but not the sizes of the standard COPII vesicles, are reduced in M702V SEC23A patient's fibroblasts, suggesting a pronounced lesion in the assembly of COPII megavesicles but not standard COPII vesicles. Additionally, we have recently identified a novel CLSD case in which the patient carries novel compound heterozygous mutations in SEC24D, a cargo- loading subunit of COPII. This is the first reported human disease with a SEC24D mutation. To test the in vivo function of Sec24d, we have generated Sec24d null mice. Sec24d null embryos presented with holoprosencephaly and craniofacial anomaly, features that are known to result from a defect of type II collagen (32). Therefore, the secretion block of collagens and/or other critical factors may account for the skeletal, craniofacial and neural defects of CLSD. Our long term goal is to understand how the COPII vesicle assembly is integrated into the blueprint of vertebrate development. We hypothesize that CLSD is a disease of aberrant COPII megavesicle assembly. We will test this hypothesis by pursuing following questions: 1) what are the molecular features of COPII paralogs that contribute to megavesicle assembly?; 2) what are the mechanisms that regulate the size of a COPII megavesicle?; and 3) what are the roles of COPII megavesicles during embryonic development?

描述（由申请人提供）：Cranio-Lenticulo-核定型延期（CLSD）是一种隐性综合征，具有颅面，骨骼和神经缺陷（9）。 CLSD最初与Sec23a中的纯合F382L突变有关（8）。 Sec23a是Copii囊泡的重要组成部分，这些组成部分是导致从内质网（ER）出口的货物分子（77）。 SEC23A F382L突变破坏了Copii囊泡组件（20）。最近，我们发现了一种新型的CLSD病例，其杂合M702V Sec23a突变（10，27）。令人惊讶的是，该患者得出的成纤维细胞显示胶原特异性分泌缺陷。 SEC23A的耗竭导致鱼外基质/胶原蛋白和颅面/骨骼异常的降低（8、31）。这些发现结合在一起，定义了copii囊泡生物发生，胶原蛋白和颅面发育之间的关键联系。在这项研究中，我们研究了这一链接。 Procollagen在ER中形成约300nm长的刚性原纤维，需要COPII蛋白进行出口（77）。但是，由于其尺寸和刚性，procollagen不能包装到直径为60nm的标准复合囊泡中。最近，表明CUL3-KLHL12驱动了大型copii涂层结构的组装，并加快了胶原蛋白的快速分泌（23）。但是，COPII蛋白如何生成这些“巨型”尚不清楚。我们的研究表明，在M702V SEC23A患者的成纤维细胞中，这些异常大的复合囊泡的尺寸降低了，但没有降低标准复印囊泡的尺寸，这表明在Copii Megavesicles组装但不是标准的Copii疫苗的组装中，有明显的病变。此外，我们最近确定了一种新型的CLSD病例，其中患者在Sec24d（copii的货物载荷亚基）中携带新型复合杂合突变。这是首次报道的具有SEC24D突变的人类疾病。为了测试SEC24D的体内功能，我们已经生成了SEC24D NULL小鼠。 SEC24D无效的胚胎呈现了全脑脑和颅面异常，这些特征是由于II型胶原蛋白缺陷所致（32）。因此，胶原蛋白的分泌块 和/或其他关键因素可能解释了CLSD的骨骼，颅面和神经缺陷。我们的长期目标是了解如何将Copii囊泡组件集成到脊椎动物发育的蓝图中。我们假设CLSD是一种异常的Copii Megavesicle组装疾病。我们将通过提出以下问题来检验这一假设：1）COPII旁系同源物的分子特征是什么促进巨型组装的？ 2）哪些调节Copii Megavesicle大小的机制是什么？ 3）在胚胎发育过程中，copii megavesicles的作用是什么？