Pathobiology of VPS45 severe congenital neutropenia

VPS45 严重先天性中性粒细胞减少症的病理学

• 批准号: 10331316
• 负责人: Mary Munson
• 金额: $ 66.43万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-07 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10331316
• 关键词: Affect; Age; Agranulocytosis; Alpha Granule; Apoptosis; Architecture; Bacterial Infections; Binding; Biochemical; Biochemistry; Biogenesis; Biological; Biological Assay; Biology; Blood Platelets; Bone Marrow; Cells; Cellular biology; Child; Collaborations; Complex; Congenital Abnormality; Cytoplasmic Granules; Defect; Development; Disease; Embryo; Exocytosis; Fibroblasts; Functional disorder; Genes; Genetic; Granulocyte Colony-Stimulating Factor; Hematopoietic; Host Defense; Human; In Vitro; Infection; Investigation; Lead; Leukocytes; Life; Mammalian Cell; Measures; Mediating; Membrane; Membrane Fusion; Missense Mutation; Molecular; Mus; Mutation; Myelofibrosis; Neutropenia; Organ; Organelles; Pathogenicity; Pathology; Pathway interactions; Patients; Phagocytes; Phenotype; Physiological; Protein Family; Protein Sortings; Proteins; Recombinants; Recurrence; Regulation; Resistance to infection; SNAP receptor; Secondary to; Sorting - Cell Movement; Stem cell transplant; Syndrome; System; Testing; Transgenic Mice; Vesicle; Visual; Whole Organism; Yeasts; consanguineous family; experimental study; genetic regulatory protein; granule cell; human disease; in vivo; insight; kindred; mouse model; mutant; neutrophil; novel; protein transport; recruit; success; syntaxin binding protein 1; trafficking; translational therapeutics; vesicle transport

Mutations in the endosomal-lysosomal regulatory protein VPS45 cause a life-threatening form of severe congenital neutropenia (SCN) characterized by agranulocytosis, neutrophil dysfunction, platelet alpha granule deficiency, and myelofibrosis. Normal function of neutrophils and platelets requires formation, mobilization and fusion of exocytic granules; when dysregulated, these defects lead to an “intracellular traffic jam” in VPS45 neutropenia and other granule disorders. VPS45 regulates the transport and assembly of endosomal- lysosomal SNARE complexes for SNARE-mediated membrane fusion. We hypothesize that mutant VPS45 protein misregulates SNARE complexes in mammalian hematopoietic cells, leading to neutropenia and neutrophil dysfunction through abnormal intracellular protein trafficking and granule function. To test this hypothesis, we have developed in vitro and in vivo systems for biochemical and physiological analysis of SCN caused by VPS45 mutations, including development and initial characterization of the first mouse model for neutropenia due to an endosomal-lysosomal defect. Our Specific Aims are: 1. Investigate the biochemical impact of Vps45 mutations on SNARE binding in vitro and organelle trafficking in yeast. We are quantitatively assessing binding of recombinant wild-type (wt) and mutant yeast Vps45 and human VPS45 proteins to cognate SNARE proteins and other partners, and measuring the rates of SNARE complex assembly and membrane fusion. We are measuring effects in yeast on Vps45 and SNARE protein levels, endo- and exocytosis, trafficking of endosomal and vacuolar proteins, recruitment of Vps45 to membranes, and binding of Vps45 to partner proteins. 2. Determine the molecular consequences of VPS45 mutations in neutrophils and fibroblasts in vitro. We are testing the “intracellular traffic jam” hypothesis in mammalian cells by examining the effects of pathogenic VPS45 mutations on SNARE assembly, membrane trafficking, and granule biogenesis and fusion. 3. Determine the functional consequences of VPS45 dysfunction in mouse and patient cells. We are investigating whether abnormalities in membrane trafficking and granule biogenesis lead to defects in the function of mouse and human cells.. We will focus on pathways leading to apoptosis and to defects in phagocytic function. 4. Test the physiological consequences of VPS45 dysfunction in our mouse model of VPS45 neutropenia. We are investigating the effects of VPS45 pathogenic mutations on neutrophil number, bone marrow architecture, organ pathology, and resistance to infection, in order to help determine how VPS45 dysfunction affects neutrophil function and host defense in the whole organism. The proposed collaborative studies, combining the complementary expertise of Drs. Munson and Newburger, will elucidate previously unexplored molecular mechanisms for the novel SCN caused by VPS45 mutations, while gaining insights into this and other syndromes caused by vesicle and granule defects.

内体 - 溶菌体调节蛋白VPS45中的突变导致严重的生命形式 先天性嗜中性粒细胞（SCN），其特征是粒细胞增多症，中性粒细胞功能障碍，血小板α颗粒 缺乏和骨髓纤维化。中性粒细胞和血小板的正常功能需要形成，动员和 胞外颗粒的融合；当失调时，这些缺陷导致VPS45中的“细胞内交通堵塞” 中性粒细胞减少症和其他颗粒疾病。 VPS45调节内体的运输和组装 溶酶体snare复合物，用于循环介导的膜融合。 我们假设突变体VPS45蛋白质失误会使哺乳动物造血的核心复合物 细胞，通过异常细胞内蛋白运输和 颗粒功能。为了检验这一假设，我们开发了生化和体内系统的生化和体内系统 VPS45突变引起的SCN的物理分析，包括开发和初始表征 由于内体 - 溶菌体缺陷而导致的第一个小鼠模型。 我们的具体目的是：1。研究VPS45突变对圈套结合的生化影响 体外和细胞器贩运酵母。我们正在定量评估重组野生型的结合 （WT）和突变酵母VPS45以及人类VPS45蛋白质，以同志snare蛋白和其他伴侣，以及 测量军鼓复合物组件和膜融合的速率。我们正在测量酵母的影响 在VPS45和网罗蛋白水平上，内膜和外吞作用，内体和液泡蛋白的运输， VPS45募集到膜上，并结合VPS45与伴侣蛋白的结合。 2。确定分子 体外中性粒细胞和成纤维细胞中VPS45突变的后果。我们正在测试“细胞内 通过检查致病性VPS45突变对SNARE的影响，交通拥堵的假设 组装，膜运输和颗粒生物发生和融合。 3。确定功能 小鼠和患者细胞中VPS45功能障碍的后果。我们正在调查是否 膜运输和颗粒生物发生异常导致小鼠功能缺陷 人类细胞..我们将专注于导致凋亡和吞噬功能缺陷的途径。 4。测试 VPS45功能障碍的物理后果在我们的VPS45中部减少症的小鼠模型中。我们 正在研究VPS45致病突变对中性粒细胞数量，骨髓结构的影响， 器官病理学和对感染的抵抗，以帮助确定VPS45功能障碍如何影响 中性粒细胞的功能和宿主防御在整个生物体中。 拟议的合作研究结合了DR的互补专业知识。芒森和纽伯格， 将阐明由VPS45突变引起的新型SCN的先前意外分子机制， 同时了解由囊泡和颗粒缺陷引起的该综合征和其他综合征的见解。