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）：颅骨豆状缝线发育不良 (CLSD) 是一种伴有颅面、骨骼和神经缺陷的隐性综合征 (9)。 CLSD 最初与 SEC23A 中的纯合 F382L 突变相关 (8)。 SEC23A 是 COPII 囊泡的重要组成部分，负责将货物分子从内质网 (ER) 中输出 (77)。 SEC23A F382L 突变会破坏 COPII 囊泡组装 (20)。最近，我们发现了一个新的 CLSD 病例，其具有杂合 M702V SEC23A 突变 (10, 27)。令人惊讶的是，来自该患者的成纤维细胞表现出胶原蛋白特异性分泌缺陷。 Sec23a 的消耗会导致鱼类细胞外基质/胶原蛋白减少和颅面/骨骼异常 (8, 31)。综合起来，这些发现明确了 COPII 囊泡生物发生、胶原蛋白分泌和颅面发育之间的关键联系。在本研究中，我们研究了这一联系。原胶原在 ER 中形成约 300 nm 长的刚性原纤维，并需要 COPII 蛋白进行输出 (77)。然而，由于其尺寸和刚性，原胶原无法包装到直径为 60 nm 的标准 COPII 囊泡中。最近，研究表明 CUL3-KLHL12 驱动大型 COPII 包被结构的组装并加速胶原蛋白的分泌 (23)。然而，COPII 蛋白如何产生这些“大泡”仍不清楚。我们的研究表明，M702V SEC23A 患者的成纤维细胞中这些异常大的 COPII 囊泡的尺寸减小，但标准 COPII 囊泡的尺寸没有减小，这表明 COPII 巨囊泡的组装存在明显的病变，但标准 COPII 囊泡的组装不存在明显的病变。此外，我们最近发现了一个新的 CLSD 病例，其中患者在 SEC24D（COPII 的一个货物装载亚基）中携带新的复合杂合突变。这是第一个报道的具有 SEC24D 突变的人类疾病。为了测试 Sec24d 的体内功能，我们生成了 Sec24d 无效小鼠。 Sec24d 无效胚胎出现前脑无裂畸形和颅面异常，这些特征已知是由 II 型胶原蛋白缺陷引起的 (32)。因此，胶原蛋白的分泌受阻 和/或其他关键因素可能会导致 CLSD 的骨骼、颅面和神经缺陷。我们的长期目标是了解 COPII 囊泡组装如何整合到脊椎动物发育的蓝图中。我们假设 CLSD 是一种 COPII 大泡组装异常的疾病。我们将通过提出以下问题来检验这一假设：1）有助于巨囊泡组装的 COPII 旁系同源物的分子特征是什么？ 2) 调节 COPII 大泡大小的机制是什么？ 3) COPII 大泡在胚胎发育过程中的作用是什么？