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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要先天性红细胞生成障碍性贫血 (CDA) 是一组异质性遗传性疾病，其特征为无效的红细胞生成、溶血以及骨髓中的双核或多核红细胞。这 CDA 中红细胞功能障碍的机制尚不完全清楚；研究分子缺陷这些疾病可以揭示终末红细胞生成的关键途径。我们已经鉴定出 VPS4A 的突变作为三名不相关的患有红细胞生成障碍、溶血性贫血、和神经发育迟缓，指出液泡蛋白分选 4A (VPS4A) 对于终末期的重要性红细胞生成和正常红细胞存活。但其作用机制还需进一步研究调查。 VPS4A 是一种 ATP 酶，已在酵母和体外细胞培养物中显示出参与内体囊泡运输、病毒出芽、运输所需的内体分选复合物 (ESCRT)-III 和胞质分裂的脱离步骤。它是内体囊泡出芽形成多囊泡体所必需的，跨膜蛋白受体的分类、回收和去除途径中的关键步骤。我们首次在 ATP 酶显性失活突变患者中显示出一种人类疾病 VPS4A 结构域，导致红细胞生成障碍，通过细胞质桥连接的红细胞一致在脱离步骤中胞质分裂失败，并且网织红细胞有证据表明细胞运输改变转铁蛋白受体 (TfR1/CD71)。我们假设 VPS4A 对于终末红细胞生成至关重要，并且功能丧失通过胞质分裂失败和内溶酶体的结合导致红细胞生成障碍缺陷。这项工作的目标是定义这些 VPS4A 变体干扰的分子机制红细胞生成，并阐明 VPS4A 和 ESCRT 机制在人类和小鼠终末期的作用红细胞生成。我们的目标是使用转基因小鼠模型在体内模拟 VPS4 引起的红细胞缺陷已知显性失活 VPS4 突变体 (VPS4EQ) 的红系靶向表达，使得能够研究该疾病的致病机制和自然史（目标 1）。使用人类 iPSC，我们将研究胞质分裂失败和铁运输缺陷对这些患者中观察到的红细胞生成障碍的贡献使用正常、患者来源和 VPS4AEQ CRISPR 基因编辑 iPSC 的个体（目标 2）。这些研究将证明了 VPS4A 和 ESCRT-III 机制作为红细胞生成的重要分子途径的作用。