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-颅-缝-骨缝发育不良（CLSD）是一种隐性综合征，伴有颅面、骨骼和神经缺陷（9）。CLSD最初与SEC 23 A中的纯合F382 L突变相关（8）。SEC 23 A是COPII囊泡的重要组分，其负责将货物分子输出内质网（ER）（77）。SEC 23 A F382 L突变破坏COPII囊泡组装（20）。最近，我们发现了一例新的CLSD病例，其具有杂合M702 V SEC 23 A突变（10，27）。令人惊讶的是，来源于该患者的成纤维细胞显示胶原特异性分泌缺陷。Sec 23 a的消耗导致鱼的细胞外基质/胶原蛋白和颅面/骨骼异常减少（8，31）。结合起来，这些发现定义了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。在这项研究中，我们调查了这种联系。前胶原在ER中形成约300 nm长的刚性原纤维，并需要COPII蛋白用于输出（77）。然而，由于其大小和刚性，前胶原不能被包装到具有60 nm直径的标准COPII囊泡中。最近，研究表明，CUL 3-KLHL 12驱动大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“巨噬细胞”仍不清楚。我们的研究已经证明，这些异常大的COPII囊泡的尺寸，但不是标准COPII囊泡的尺寸，在M702 V SEC 23 A患者的成纤维细胞中减小，这表明在COPII巨噬细胞而不是标准COPII囊泡的组装中有明显的损伤。此外，我们最近鉴定了一个新的CLSD病例，其中患者在SEC 24 D（COPII的货物装载亚基）中携带新的复合杂合突变。这是第一个报告的人类疾病与SEC 24 D突变。为了测试Sec 24 d的体内功能，我们产生了Sec 24 d缺失小鼠。Sec 24 d无效胚胎表现为前脑无裂畸形和颅面异常，这些特征已知是II型胶原蛋白缺陷所致（32）。因此，胶原蛋白的分泌阻滞 和/或其他关键因素可能导致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巨噬细胞组装异常的疾病。我们将通过以下问题来验证这一假设：1）COPII旁系同源物的分子特征是什么？2)控制COPII巨型柱大小的机制是什么？COPII巨噬细胞在胚胎发育中的作用