Pathobiology of VPS45 severe congenital neutropenia

VPS45 严重先天性中性粒细胞减少症的病理学

基本信息

批准号：
10741137
负责人：
Mary Munson
金额：
$ 6.97万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
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/10741137
关键词：
Affect Age Agranulocytosis Alpha Granule Apoptosis Architecture Bacterial Infections Binding Biochemical Biochemistry Biogenesis Biological Biological Assay Biology Blood Platelets Bone Marrow Cell Separation Cells Cellular biology Child Classification Collaborations Complex Congenital Abnormality Cytoplasmic Granules Defect Development Disease Embryo Endosomes 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 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

项目摘要

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-溶酶体调节蛋白 VPS45 的突变会导致危及生命的严重疾病先天性中性粒细胞减少症 (SCN)，特征为粒细胞缺乏、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、动员和形成。胞吐颗粒的融合；当失调时，这些缺陷会导致 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。用于 SNARE 介导的膜融合的溶酶体 SNARE 复合物。我们发现突变的 VPS45 蛋白会错误调节哺乳动物造血系统中的 SNARE 复合物细胞，通过异常的细胞内蛋白质运输导致中性粒细胞减少和中性粒细胞功能障碍，为了检验这一假设，我们开发了用于生化和体内的体外和体内系统。 VPS45 突变引起的 SCN 生理分析，包括发育和初步表征第一个因内体溶酶体缺陷而导致中性粒细胞减少症的小鼠模型。我们的具体目标是： 1. 研究 Vps45 突变对 SNARE 结合的生化影响我们正在定量评估重组野生型的结合。 (wt) 和突变酵母 Vps45 和人 VPS45 蛋白与 SNARE 蛋白和其他伙伴同源，以及测量 SNARE 复合体组装和膜融合的速率我们正在测量酵母中的效果。 Vps45 和 SNARE 蛋白水平、胞吞作用和胞吐作用、内体和液泡蛋白的运输， Vps45 募集到膜上，以及 Vps45 与伙伴蛋白的结合 2. 确定分子。我们正在体外测试中性粒细胞和成纤维细胞中 VPS45 突变的后果。通过检查致病性 VPS45 突变对 SNARE 的影响，提出哺乳动物细胞中的“交通堵塞”假说组装、膜运输以及颗粒生物合成和融合 3. 确定功能。我们正在研究 VPS45 功能障碍对小鼠和患者细胞是否造成的后果。膜运输和颗粒生物发生的异常导致小鼠和小鼠的功能缺陷人类细胞。我们将重点关注导致细胞凋亡和吞噬功能缺陷的途径。 4. 测试。在我们的 VPS45 中性粒细胞减少症小鼠模型中，VPS45 功能障碍的生理后果。正在研究 VPS45 致病性突变对中性粒细胞数量、骨髓结构的影响，器官病理学、感染抵抗力，以帮助确定 VPS45 功能障碍如何影响整个生物体中的中性粒细胞功能和宿主防御。拟议的合作研究结合了 Munson 和 Newburger 博士的互补专业知识，将阐明之前未探索过的由 VPS45 突变引起的新型 SCN 的分子机制，同时深入了解由囊泡和颗粒缺陷引起的这种综合症和其他综合症。