The Evolution of the Mammalian SEC23 Paralogs and the Molecular Pathogenesis of Congenital Dyserythropoietic Anemia type II

哺乳动物 SEC23 旁系同源物的进化和先天性红细胞生成不良性贫血 II 型的分子发病机制

• 批准号: 8947436
• 负责人: Rami Khoriaty
• 金额: $ 13.39万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8947436
• 关键词: Abnormal Red Blood Cell; Accounting; Acute Erythroblastic Leukemia; Anemia; Animals; Biological Models; Biological Process; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Capsid Proteins; Cell membrane; Cell physiology; Cells; Characteristics; Complementary DNA; Complex; Cytolysis; Data; Defect; Development; Disease; Endoplasmic Reticulum; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Evolution; Exhibits; Fertility; Financial compensation; Foundations; Functional disorder; Future; Genes; Genetic Engineering; Genetic Models; Genomics; Golgi Apparatus; Growth; Hematopoietic; Hematopoietic stem cells; Human; K-562; Lead; Mass Spectrum Analysis; Modeling; Molecular; Mus; Mutation; Organelles; Pancreas; Pathogenesis; Pathologic; Pathway interactions; Patients; Pattern; Phenotype; Production; Proteins; Proteome; Proteomics; Regulation; Regulatory Element; Role; Sampling; Serum; Sorting - Cell Movement; Techniques; Testing; Time; Tissues; Translating; Vesicle; Weaning; Work; base; improved; in vivo; insight; mouse model; new therapeutic target; novel; paralogous gene; protein complex; protein function; public health relevance; secretory protein; trafficking; type II Congenital dyserythropoietic anemia

 DESCRIPTION (provided by applicant): Congenital dyserythropoietic anemia type-II (CDAII) is an autosomal recessive disease resulting from a defect in terminal erythroid maturation, characterized by moderate anemia and increased bone marrow (BM) bi/multi- nucleated erythroblasts. CDAII results from mutations in SEC23B, one of two closely related mammalian SEC23 paralogs (SEC23A and SEC23B). SEC23 is a core component of coat protein complex-II (COPII) vesicles, which transport secretory protein cargos from the Endoplasmic Reticulum (ER) to the Golgi apparatus. BM transplantation cures CDAII, suggesting that the pathologic defect is restricted to the hematopoietic compartment. Despite the identification of the genetic defect underlying CDAII, the mechanism by which SEC23B deficiency results in CDAII remains unknown. We previously showed that, in contrast to humans, SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration. We also generated mice with SEC23B deficiency restricted to the hematopoietic compartment and showed that these mice lack anemia and other CDAII characteristics. In recent preliminary results, we genetically engineered the Sec23a cDNA into the genomic locus of Sec23b in mice. Mice expressing SEC23A under the normal temporal and tissue specific patterns of SEC23B appear healthy and survive past weaning. This surprising observation suggests that the two Sec23 paralogs are interchangeable at the level of protein function. Additional preliminary data demonstrate that the SEC23B/SEC23A expression ratio is higher in human BM compared to pancreas, with the reverse pattern observed in mice. Taken together, these data suggest that the tissue-specific expression patterns of the Sec23 paralogs have shifted during evolution between mouse and human and that the absence of CDAII in SEC23B-deficient mice is due to compensation from SEC23A, the latter accounting for a relatively high fraction of total SEC23 in the hematopoietic compartment of these animals (in contrast to humans). The overall objective of this proposal is to study the functional evolution of the two SEC23 paralogs and to characterize the molecular pathogenesis of CDAII. First, I will determine the extent to which the highly similar SEC23 paralogs functionally compensate for one another in-vivo. I will fully characterize SEC23B deficient mice expressing SEC23A under the endogenous Sec23b regulatory elements and assess for subtle abnormalities in pancreas function. If these mice are anatomically normal and exhibit normal survival, growth, and fertility, this will demonstrate that SEC23A and SEC23B are interchangeable at the protein level when expressed in the appropriate tissues and at the appropriate times and levels. Second, based on SEC23A and SEC23B expression data, and on our preliminary work demonstrating that the 2 SEC23 paralogs overlap in function, we hypothesize that mice with hematopoietic SEC23A (or combined SEC23A/SEC23B) deficiency will develop CDAII. We will generate and characterize these mice, and expect to generate a murine model faithfully reproducing the human CDAII phenotype. Third, based on the ER-to-Golgi transport defect in this disease, we hypothesize that CDAII results from the impaired trafficking of one or more key cargo proteins that depend on SEC23B for ER-exit, and therefore for secretion to the RBC plasma membrane. We will perform quantitative proteomic techniques to identify the critical cargo(s) that depend on SEC23B for exit from the ER in human erythroid cells. We will validate the putative cargo(s) for their roles in CDAII pathophysiology. These studies have important implications for understanding the evolutionary functions of the paralogous SEC23 genes, and for improving our fundamental understanding of the complex mechanisms by which cargos are sorted in COPII vesicles. The results from this project may lead to the identification of new therapeutic targets as well as model systems in which to test these targets. Cargos that depend on SEC23B for secretion during RBC development may represent novel targets for CDAII therapy, which are also expected to translate to other anemias due to defects of terminal erythroid maturation. We also expect to generate a mouse model faithfully reproducing human CDAII, which may be used in the future to test novel therapies. Determining the functional overlap of SEC23A and SEC23B is critical because a shared role of these paralogs would suggest that therapies that increase the expression of either paralog in erythroid cells might be effective in CDAII.

 描述（由申请人提供）：先天性红细胞生成不良性贫血II型（CDAII）是一种常染色体隐性遗传疾病，由终末红细胞成熟缺陷引起，特征为中度贫血和骨髓（BM）双核/多核成红细胞增加。CDAII由SEC 23 B中的突变引起，SEC 23 B是两种密切相关的哺乳动物SEC 23旁系同源物（SEC 23 A和SEC 23 B）之一。SEC 23是外壳蛋白复合物-II（COPII）囊泡的核心组分，其将分泌蛋白质货物从内质网（ER）运输到高尔基体。骨髓移植治愈CDAII，表明病理缺陷仅限于造血区室。尽管鉴定了CDAII的遗传缺陷，但SEC 23 B缺陷导致CDAII的机制仍然未知。我们以前的研究表明，与人类相反，SEC 23 B缺陷小鼠围产期死亡，表现出大规模的胰腺变性。我们还产生了具有限于造血区室的SEC 23 B缺陷的小鼠，并显示这些小鼠缺乏贫血和其他CDAII特征。在最近的初步结果中，我们将Sec 23 a cDNA基因工程改造到小鼠Sec 23 b的基因组位点中。在SEC 23 B的正常时间和组织特异性模式下表达SEC 23 A的小鼠看起来健康并存活过断奶。这一令人惊讶的观察结果表明，两种Sec 23旁系同源物在蛋白质功能水平上是可互换的。额外的初步数据表明，与胰腺相比，SEC 23 B/SEC 23 A表达比率在人BM中更高，在小鼠中观察到相反的模式。总而言之，这些数据表明，Sec 23旁系同源物的组织特异性表达模式在小鼠和人类之间的进化过程中发生了变化，并且SEC 23 B缺陷小鼠中CDAII的缺失是由于SEC 23 A的补偿，后者占总SEC的相对较高比例这些动物的造血区室中的SEC 23（与人类相反）。该提案的总体目标是研究两个SEC 23旁系同源物的功能进化并表征CDAII的分子发病机制。首先，我将确定高度相似的SEC 23旁系同源物在体内功能上相互补偿的程度。我将充分表征在内源性Sec 23 b调控元件下表达SEC 23 A的SEC 23 B缺陷小鼠，并评估胰腺功能的细微异常。如果这些小鼠在解剖学上是正常的并且表现出正常的存活、生长和生育力，则这将证明当在适当的组织中以适当的时间和水平表达时，SEC 23 A和SEC 23 B在蛋白质水平上是可互换的。其次，基于SEC 23 A和SEC 23 B表达数据，以及我们的初步工作表明2种SEC 23旁系同源物在功能上重叠，我们假设具有造血SEC 23 A（或组合的SEC 23 A/SEC 23 B）缺陷的小鼠将发展CDAII。我们将产生并表征这些小鼠，并期望产生忠实地再现人CDAII表型的鼠模型。第三，基于这种疾病中ER-高尔基体转运缺陷，我们假设CDAII是由一种或多种关键货物蛋白的运输受损引起的，这些货物蛋白依赖SEC 23 B进行ER-出口，因此分泌到RBC质膜。我们将进行定量蛋白质组学技术，以确定关键货物（S），依赖于SEC 23 B退出人类红系细胞的ER。我们将验证假定的货物在CDAII病理生理学中的作用。这些研究对于理解旁系同源SEC 23基因的进化功能，以及提高我们对COPII囊泡中货物分类的复杂机制的基本理解具有重要意义。该项目的结果可能会导致新的治疗靶点的识别以及测试这些靶点的模型系统。在RBC发育过程中依赖于SEC 23 B分泌的货物可能代表CDAII治疗的新靶点，由于终末红细胞成熟的缺陷，其也被预期转化为其他贫血。我们还期望产生忠实地再现人CDAII的小鼠模型，其可在未来用于测试新疗法。确定SEC 23 A和SEC 23 B的功能重叠是至关重要的，因为这些旁系同源物的共同作用将表明增加红系细胞中任一旁系同源物的表达的疗法可能在CDAII中有效。