Pathobiology of VPS45 severe congenital neutropenia

内酶体-溶酶体调节蛋白VPS45突变导致一种危及生命的重症 以粒细胞缺乏、中性粒细胞功能障碍、血小板α颗粒为特征的先天性中性粒细胞减少症 缺乏症和骨髓纤维化。中性粒细胞和血小板的正常功能需要形成、动员和 胞外颗粒融合；当失控时，这些缺陷会导致VPS45的“细胞内交通堵塞” 中性粒细胞减少症和其他颗粒性疾病。VPS45调节内涵体的运输和组装- 用于SNARE介导膜融合的溶酶体SNARE复合体。 我们假设突变的VPS45蛋白错误地调节哺乳动物造血中的SNARE复合体 细胞，通过异常的细胞内蛋白运输和细胞外营养导致中性粒细胞减少和中性粒细胞功能障碍。 颗粒功能。为了验证这一假设，我们开发了体外和体内生化和 VPS45突变引起的SCN的生理学分析，包括发育和初步特征 这是首例由内切酶-溶酶体缺陷引起的中性粒细胞减少症小鼠模型。 我们的具体目标是：1.研究Vps45突变对SNARE结合的生化影响 酵母的体外和细胞器运输。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，以同源SNARE蛋白和其他伙伴，以及 测定SNARE复合体组装和膜融合率。我们正在测量酵母菌的影响 在Vps45和SNARE蛋白水平上，内吞和胞吐，内体和液泡蛋白的运输， Vps45在膜上的募集，以及Vps45与伴侣蛋白的结合。2.确定分子 体外培养的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” 通过检测致病的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、膜运输和颗粒生物发生与融合。3.确定功能 VPS45功能障碍对小鼠和患者细胞的影响。我们正在调查是否 膜转运和颗粒生物发生异常导致小鼠和小鼠功能缺陷 人类细胞..。我们将重点介绍导致细胞凋亡和吞噬功能缺陷的途径。4.测试 VPS45功能障碍在我们的VPS45中性粒细胞减少症小鼠模型中的生理后果。我们 正在调查VPS45致病突变对中性粒细胞数量、骨髓结构、 器官病理学和对感染的抵抗力，以帮助确定VPS45功能障碍如何影响 中性粒细胞在整个生物体中的功能和宿主防御。 拟议的合作研究结合了芒森博士和纽伯格博士的互补专业知识， 将阐明由VPS45突变引起的新的SCN的先前未探索的分子机制， 同时深入了解由水泡和颗粒缺陷引起的这种和其他症状。

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.