Role of VPS4A and ESCRT-III in terminal erythropoiesis

VPS4A 和 ESCRT-III 在终末红细胞生成中的作用

• 批准号: 10523545
• 负责人: Katie Giger Seu
• 金额: $ 15.29万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10523545
• 关键词: ATP phosphohydrolase; ATPase Domain; Biogenesis; Bone Marrow; Cell Culture Techniques; Cell Survival; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Congenital dyserythropoietic anemia; Critical Pathways; Cytokinesis; Defect; Development; Disease; Dominant-Negative Mutation; Erythroblasts; Erythrocytes; Erythroid; Erythropoiesis; Excision; Failure; Functional disorder; Genes; Genetic Diseases; Goals; Hematological Disease; Heme; Hemolysis; Hemolytic Anemia; Human; Impairment; In Vitro; Individual; Inherited; Integral Membrane Protein; Investigation; Iron; Iron Overload; Lysosomes; Mammalian Cell; Membrane; Mitochondria; Modeling; Molecular; Multivesicular Body; Mus; Mutation; Neurodevelopmental Disorder; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peripheral; Phenocopy; Phenotype; Play; Production; Recycling; Reporting; Reticulocytes; Role; Site; Sorting - Cell Movement; Spleen; Syndrome; TFRC gene; Time; Transferrin; Transfusion; Transgenic Mice; Vacuolar Protein Sorting; Variant; Vesicle; Viral; Work; Yeasts; base; career; de novo mutation; disease natural history; disease-causing mutation; exosome; human disease; in vivo; induced pluripotent stem cell; insight; knock-down; loss of function; mouse model; mutant; novel; peripheral blood; receptor; receptor recycling; recruit; trafficking; uptake

Project Summary Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic disorders characterized by ineffective erythropoiesis, hemolysis, and bi- or multi-nucleated erythroblasts in the bone marrow. The mechanisms of erythroid dysfunction in CDAs are incompletely understood; studying the molecular defects in these diseases can reveal critical pathways in terminal erythropoiesis. We have identified mutations in VPS4A as a novel cause of CDA in three unrelated patients with a syndrome of dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay, pointing to the importance of Vacuolar Protein Sorting 4A (VPS4A) for terminal erythropoiesis and normal red blood cell survival. However, the mechanisms of its action need further investigation. VPS4A is an ATPase that has been shown in yeast and in vitro cell cultures to participate with the Endosomal Sorting Complex Required for Transport (ESCRT)-III in endosomal vesicle trafficking, viral budding, and the abscission step of cytokinesis. It is required for budding of endosomal vesicles into multivesicular bodies, a critical step in the pathway for the sorting, recycling, and removal of transmembrane protein receptors. We have shown for the first time a human disease in patients with dominant negative mutations in the ATPase domain of VPS4A, resulting in dyserythropoiesis with erythroblasts connected by cytoplasmic bridges consistent with cytokinesis failure at the abscission step and reticulocytes with evidence of altered trafficking of the transferrin receptor (TfR1/CD71). We hypothesize that VPS4A is essential for terminal erythropoiesis and that loss of function results in dyserythropoiesis through a combination of cytokinesis failure and endolysosomal defects. The goal of this work is to define the molecular mechanisms by which these VPS4A variants disturb erythropoiesis, and to elucidate the roles of VPS4A and the ESCRT machinery in human and murine terminal erythropoiesis. We aim to model the erythroid defects due to VPS4 in vivo using a transgenic mouse model with erythroid-targeted expression of a known dominant-negative VPS4 mutant (VPS4EQ), enabling studies on the pathogenic mechanisms and natural history of the disease (Aim 1). Using human iPSCs, we will investigate the contributions of cytokinesis failure and iron trafficking defects to the dyserythropoiesis observed in these individuals using normal, patient-derived, and VPS4AEQ CRISPR gene-edited iPSCs (Aim 2). These studies will demonstrate the role of VPS4A and ESCRT-III machinery as an essential molecular pathway for erythropoiesis.

项目摘要 先天性红细胞生成性贫血(CDA)是一组不同类型的遗传性疾病，其特征是 无效的红细胞生成、溶血和骨髓中的双核或多核红细胞。这个 CDA红系功能障碍的机制尚不完全清楚；研究CDA的分子缺陷 这些疾病可以揭示终末红细胞生成的关键途径。我们已经确定了VPS4A的突变 作为三名无血缘关系的患者中CDA的新病因，这些患者患有红细胞生成障碍、溶血性贫血、 和神经发育延迟，表明空泡蛋白分类4A(VPS4A)对终末的重要性 红血球的生成和正常的红细胞存活。然而，其作用机制还需要进一步研究。 调查。VPS4A是一种ATPase，已在酵母和体外细胞培养中显示参与 运输所需的内体分选复合体(ESCRT)-III在内体囊泡运输、病毒萌发、 胞质分裂的脱落步骤。它是内体小泡萌发成多囊泡体所必需的， 跨膜蛋白受体分选、回收和移除过程中的关键一步。我们 首次发现人类疾病患者中存在ATPase的显性负突变 VPS4A的结构域，导致红细胞通过胞浆桥连接一致，导致红细胞生成障碍 在脱落阶段胞质分裂失败，网织红细胞有改变运输的证据 转铁蛋白受体(TfR1/CD71)。我们假设VPS4A是终末红细胞生成所必需的，并且 功能丧失通过胞质分裂失败和内切酶结合导致红细胞生成障碍 缺陷。这项工作的目标是确定这些VPS4A变体干扰的分子机制 红细胞生成，并阐明VPS4A和ESCRT机制在人和小鼠终末的作用 红血球生成。我们的目标是使用转基因小鼠模型在体内模拟Vps4引起的红系缺陷。 已知的显性阴性Vps4突变体(VPS4EQ)的红系靶向表达，使研究 疾病的致病机制和自然病史(目标1)。利用人类的IPSCs，我们将研究 细胞质分裂失败和铁转运缺陷对这些患者红细胞生成障碍的贡献 使用正常的、患者来源的和VPS4AEQ CRISPR基因编辑的IPSCs的个人(目标2)。这些研究将 证明VPS4A和ESCRT-III机制作为红细胞生成的基本分子途径所起的作用。