Elastase and B3A Function

弹性蛋白酶和 B3A 功能

• 批准号: 7109397
• 负责人: Clinton D Lothrop
• 金额: $ 32.08万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7109397
• 关键词: CD34 molecule; biological signal transduction; bone marrow transplantation; cell adhesion; cell proliferation; confocal scanning microscopy; dogs; elastases; electron microscopy; gene expression; gene mutation; hematopoiesis; hematopoietic stem cells; immunoprecipitation; intracellular transport; ligands; myeloid stem cell; platelets; protein degradation; protein localization; protein structure function; protein transport; receptor expression; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Cyclic hematopoiesis (CH) is an unusual disorder of the hematopoietic stem cell (HSC) with on/off proliferation of HSCs that causes cycles of circulating blood cells. CH is caused by mutations in the ela 2 gene encoding neutrophil elastase in human beings and in the AP3B1 gene which encodes the B1 subunit of the adaptin AP3 in dogs. Mutations in ela 2 also cause severe congenial neutropenia, Kostmann's Syndrome. Canine CH is always associated with a diluted coat color, mild bleeding disorder due to platelet dense granule storage pool disease and cutaneous mast cell deficiency. This is consistent with the notion that AP3 is a cargo protein shuttle for lysosome like organelles such as neutrophil primary granules, platelet dense granules, melanosomes and mast cell granules. The mechanism whereby mutations in ela 2 and AP3B1 result in cyclic hematopoiesis are not well understood. We hypothesize that abnormal elastase trafficking to the plasma membrane causes increased proteolytic degradation of membrane receptors and a dampened proliferative response in myeloid progenitor cells which unmasks the inherent cyclic nature of hematopoiesis. The long range goal of this project is to determine the cellular and biochemical mechanisms causing cyclic hematopoiesis with ela 2 and AP3B1 mutations. The specific aims are 1) characterize B3A expression and function in normal and CH dogs. 2) Define the temporal regulation and intracellular trafficking of elastase during the 14 day cycle in CH dogs. 3) Determine if gene transfer of the normal B3A cDNA into CD34+ c-kit+ lin- cells corrects the cellular defects in HSCs. Cells will be transduced ex vivo with a lentiviral vector containing the normal canine B3A cDNA. Elastase trafficking to the lysosome and plasma membrane and assembly of the AP3 tetrameric complex will be determined before and after gene transfer. We will also perform autologous bone marrow transplants (n=4) with the B3A transduced cells to determine if cyclic hematopoiesis is corrected. 4) Determine if elastase trafficking to the plasma membrane causes proteolytic degradation of the c-kit membrane receptor. Western blot, receptor binding kinetics and flow cytometry will be used to determine if there is increased proteolysis or receptor recycling in CH dogs. 5) Determine if there is a platelet dense granule deficiency or abnormal storage function by the mepacrine labeling technique, determine if AP3 interacts with rab27a, a small GTPase protein necessary for dense granule storage function and screen for additional proteins interacting with AP3 by co-immunoprecipitation and Western blotting. 6) Determine if HSCs are regulated in a cell-autonomous fashion (Chalone hypothesis) or in a paracrine manner by a diffusable factor using nonmyeloablative bone marrow transplantation to establish mixed hematopoietic chimerism with DLA-identical sex-mismatched normal and CH dogs (n=6). A Y-chromosome PCR assay will be used to determine the % normal and CH cells in circulation. These studies will advance our understanding of the basic biology of HSCs, granule formation and protein trafficking using the CH dog model uniquely available in our laboratory.

描述（由申请人提供）： 周期性造血（CH）是造血干细胞（HSC）的一种不寻常的疾病，其具有导致循环血细胞周期的HSC的开/关增殖。 CH是由编码人类中性粒细胞弹性蛋白酶的ela 2基因突变和编码狗适应蛋白AP 3的B1亚基的AP 3B 1基因突变引起的。 ela 2基因突变也会引起严重的同类性中性粒细胞减少症，即Kostmann综合征。 犬CH总是与被毛颜色变淡、血小板致密颗粒储存池病和皮肤肥大细胞缺乏症引起的轻度出血性疾病相关。 这与AP 3是溶酶体样细胞器如中性粒细胞初级颗粒、血小板致密颗粒、黑素体和肥大细胞颗粒的货物蛋白穿梭物的概念一致。 ela 2和AP 3B 1突变导致周期性造血的机制尚不清楚。 我们推测，异常弹性蛋白酶运输到质膜导致膜受体的蛋白水解降解增加和骨髓祖细胞的增殖反应减弱，这揭示了造血的固有周期性。 该项目的长期目标是确定ela 2和AP 3B 1突变引起循环造血的细胞和生化机制。 具体目的是1）表征正常和CH犬中的B3 A表达和功能。 2)定义CH犬14天周期内弹性蛋白酶的时间调节和细胞内运输。 3)确定将正常B3 A cDNA基因转移到CD 34 + c-kit+ lin-细胞中是否纠正了HSC中的细胞缺陷。 将用含有正常犬B3 A cDNA的慢病毒载体离体转导细胞。 将在基因转移之前和之后确定弹性蛋白酶向溶酶体和质膜的运输以及AP 3四聚体复合物的组装。 我们还将用B3 A转导的细胞进行自体骨髓移植（n=4），以确定循环造血是否得到纠正。 4)确定弹性蛋白酶运输到质膜是否引起c-kit膜受体的蛋白水解降解。 蛋白质印迹、受体结合动力学和流式细胞术将用于确定CH犬中蛋白水解或受体再循环是否增加。 5)通过米帕林标记技术确定是否存在血小板致密颗粒缺陷或异常储存功能，确定AP 3是否与致密颗粒储存功能所必需的小GT3蛋白rab 27 a相互作用，并通过免疫共沉淀和Western印迹筛选与AP 3相互作用的其他蛋白。 6)使用非清髓性骨髓移植确定HSC是否以细胞自主方式（Chalone假说）或以旁分泌方式通过扩散因子进行调节，以建立与DLA相同的性别不匹配的正常和CH犬的混合造血嵌合体（n=6）。 将使用Y染色体PCR试验测定循环中正常细胞和CH细胞的%。 这些研究将促进我们对HSC的基础生物学，颗粒形成和蛋白质运输的理解，使用我们实验室唯一可用的CH狗模型。