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

 描述(申请人提供)：先天性红细胞生成性贫血II型(CDAII)是一种常染色体隐性遗传病，由红系终末成熟缺陷引起，以中度贫血和骨髓双/多核红细胞增多为特征。CDAII是由SEC23B突变引起的，SEC23B是两个密切相关的哺乳动物SEC23近亲(SEC23A和SEC23B)之一。SEC23是外壳蛋白复合体-II(COPII)囊泡的核心成分，负责将分泌蛋白从内质网(ER)运送到高尔基体。骨髓移植治愈了CDAII，提示病理缺陷仅限于造血室。尽管CDAII的遗传缺陷已被鉴定，但SEC23B缺陷导致CDAII的机制仍不清楚。我们之前的研究表明，与人类相比，SEC23B基因缺陷的小鼠会在围产期死亡，表现出大规模的胰腺变性。我们还产生了仅限于造血室的SEC23B缺陷小鼠，并表明这些小鼠缺乏贫血和其他CDAII特征。在最新的初步结果中，我们将Sec23a的cDNA基因工程到了小鼠的基因组中。在正常的时间和组织特异性SEC23B模式下表达SEC23A的小鼠看起来很健康，并在断奶后存活。这一令人惊讶的观察表明，这两个Sec23对偶基因在蛋白质功能水平上是可以互换的。更多的初步数据表明，与胰腺相比，SEC23B/SEC23A在人骨髓中的表达比率更高，而在小鼠中的表达模式则相反。综上所述，这些数据表明，在小鼠和人类之间的进化过程中，Sec23类似物的组织特异性表达模式发生了变化，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的相对较高的比例(与人类相反)。这项建议的总体目标是研究两个SEC23类似基因的功能进化，并表征CDAII的分子发病机制。首先，我将确定高度相似的SEC23同源基因在体内功能上相互补偿的程度。我将全面描述SEC23B缺陷小鼠在内源性Sec23b调控元件下表达SEC23A的特征，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，并且表现出正常的生存、生长和生育能力，这将证明当SEC23A和SEC23B在适当的组织和适当的时间和水平表达时，SEC23A和SEC23B可以在蛋白质水平上互换。其次，基于SEC23A和SEC23B的表达数据，以及我们的初步工作证明这两个SEC23同源基因在功能上是重叠的，我们假设SEC23A(或SEC23A/SEC23B)缺乏的小鼠将发生CDAII。我们将产生并鉴定这些小鼠，并期望产生一个忠实地再现人类CDAII表型的小鼠模型。第三，基于本病的内质网到高尔基体转运缺陷，我们假设CDAII是由于依赖SEC23B退出内质网，从而分泌到红细胞膜上的一个或多个关键货运蛋白的运输受损所致。我们将使用定量蛋白质组学技术来鉴定人红系细胞中依赖SEC23B退出内质网的关键货物(S)。我们将验证假定的货物(S)在CDAII病理生理学中的作用。这些研究对于理解平行的SEC23基因的进化功能，以及提高我们对货物在COPII囊泡中分选的复杂机制的基本理解具有重要的意义。该项目的结果可能导致确定新的治疗靶点以及测试这些靶点的模型系统。在红细胞发育过程中依赖SEC23B分泌的货物可能是CDAII治疗的新靶点，由于红系终末成熟缺陷，CDAII也有望转化为其他贫血。我们还希望产生一种忠实地复制人类CDAII的小鼠模型，这可能在未来用于测试新的治疗方法。确定SEC23A和SEC23B的功能重叠是至关重要的，因为这些Paralog的共同作用表明，增加红系细胞中任一Paralog表达的治疗可能对CDAII有效。

 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.