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: 9298412
• 负责人: Rami Khoriaty
• 金额: $ 16.96万
• 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/9298412
• 关键词: Abnormal Red Blood Cell; Accounting; Acute Erythroblastic Leukemia; Anatomy; Anemia; Animals; Biological Models; Biological Process; Bone Marrow; Bone Marrow Transplantation; Capsid Proteins; Cell Differentiation process; Cell membrane; Cell physiology; Cells; Characteristics; Complementary DNA; Complex; Congenital dyserythropoietic anemia; 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; Impairment; K-562; Lead; Mass Spectrum Analysis; Molecular; Mus; Mutation; Organelles; Pancreas; Pathogenesis; Pathologic; Pathway interactions; Patients; Pattern; Perinatal; Phenotype; Production; Proteins; Proteome; Proteomics; Regulation; Regulatory Element; Role; Sampling; Secretory Cell; Techniques; Testing; Time; Tissues; Translating; Vesicle; Weaning; Work; base; improved; in vivo; insight; mouse model; new therapeutic target; novel; novel therapeutics; 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）：先天性红细胞生成障碍性贫血 II 型（CDAII）是一种由红系终末成熟缺陷引起的常染色体隐性遗传疾病，其特征是中度贫血和骨髓（BM）双核/多核红细胞增多。 CDAII 是由 SEC23B 突变引起的，SEC23B 是两个密切相关的哺乳动物 SEC23 旁系同源物（SEC23A 和 SEC23B）之一。 SEC23 是外壳蛋白复合物 II (COPII) 囊泡的核心成分，它将分泌蛋白货物从内质网 (ER) 转运到高尔基体。骨髓移植可治愈 CDAII，表明病理缺陷仅限于造血室。尽管已鉴定出 CDAII 背后的遗传缺陷，但 SEC23B 缺陷导致 CDAII 的机制仍不清楚。我们之前表明，与人类相比，SEC23B 缺陷小鼠在围产期死亡，表现出严重的胰腺变性。我们还培育了仅限于造血室的 SEC23B 缺陷小鼠，并表明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 Sec23a cDNA 基因工程改造到小鼠体内 Sec23b 的基因组位点中。在 SEC23B 的正常颞和组织特异性模式下表达 SEC23A 的小鼠看起来很健康，并且在断奶后存活下来。这一令人惊讶的观察结果表明，两个 Sec23 旁系同源物在蛋白质功能水平上是可以互换的。其他初步数据表明，与胰腺相比，人类骨髓中的 SEC23B/SEC23A 表达比率更高，而在小鼠中观察到的模式相反。总而言之，这些数据表明，Sec23 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 SEC23B 缺陷小鼠中 CDAII 的缺失是由于 SEC23A 的补偿，后者在这些动物的造血室中占总 SEC23 的相对较高比例（与人类相比）。该提案的总体目标是研究两个 SEC23 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 SEC23 旁系同源物在体内功能上相互补偿的程度。我将在内源性 Sec23b 调控元件下全面表征表达 SEC23A 的 SEC23B 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正常并且表现出正常的存活、生长和生育能力，则这将证明当在适当的组织中并在适当的时间和水平表达时，SEC23A和SEC23B在蛋白质水平上是可以互换的。其次，根据 SEC23A 和 SEC23B 表达数据，以及我们的初步工作证明 2 个 SEC23 旁系同源物在功能上重叠，我们假设具有造血 SEC23A（或组合 SEC23A/SEC23B）缺陷的小鼠将出现 CDAII。我们将生成并表征这些小鼠，并期望生成忠实再现人类 CDAII 表型的小鼠模型。第三，基于这种疾病中的内质网到高尔基体的运输缺陷，我们假设 CDAII 是由于一种或多种关键货物蛋白的运输受损所致，这些蛋白依赖于 SEC23B 进行内质网出口，从而分泌到红细胞质膜。我们将采用定量蛋白质组学技术来鉴定依赖于 SEC23B 从人红细胞内质网排出的关键货物。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 SEC23 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。该项目的结果可能有助于确定新的治疗靶点以及测试这些靶点的模型系统。在 RBC 发育过程中依赖 SEC23B 分泌的货物可能代表 CDAII 治疗的新靶点，预计这也将转化为由于红细胞终末成熟缺陷而导致的其他贫血。我们还期望生成忠实复制人类 CDAII 的小鼠模型，该模型将来可能用于测试新疗法。确定 SEC23A 和 SEC23B 的功能重叠至关重要，因为这些旁系同源物的共同作用表明，增加红系细胞中任一旁系同源物表达的疗法可能对 CDAII 有效。