Elastase and B3A Function

描述(由申请人提供)： 循环性造血是一种罕见的造血干细胞(HSC)功能紊乱，可引起循环血细胞的循环。CH是由人类编码中性粒细胞弹性蛋白酶的ELA 2基因和狗的AP3B1基因突变引起的，AP3B1基因编码Adaptin AP3的B1亚基。ELA2基因突变还会导致严重的同源性中性粒细胞减少症，即柯斯特曼综合征。犬CH总是与毛色变淡、由于血小板致密颗粒储存池疾病和皮肤肥大细胞缺乏而导致的轻度出血障碍有关。这与AP3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、黑素小体和肥大细胞颗粒的货运蛋白穿梭相一致。ELA2和AP3B1基因突变导致循环造血的机制尚不清楚。我们假设，弹性蛋白酶异常转运到质膜会导致膜受体蛋白降解增加，并抑制髓系祖细胞的增殖反应，从而揭示了造血的内在周期性。该项目的长期目标是确定导致ELA 2和AP3B1突变的周期性造血的细胞和生化机制。其具体目的是：1)研究B3A在正常犬和CH犬中的表达和功能。2)明确弹性蛋白酶在CH犬14天周期中的时间调节和细胞内转运。3)检测CD34+c-kit+Lin-细胞中正常B3A基因转移是否能纠正HSCs的细胞缺陷。细胞将通过含有正常犬B3A基因的慢病毒载体进行体外转导。在基因转移前后，弹性酶向溶酶体和质膜的转运以及AP3四聚体复合体的组装将被确定。我们还将对B3A转导的细胞进行自体骨髓移植(n=4)，以确定周期性造血是否得到纠正。4)确定弹性酶转运到质膜是否导致c-kit膜受体的蛋白降解。免疫印迹、受体结合动力学和流式细胞术将被用来确定CH犬是否存在蛋白分解或受体循环增加。5)用MEPACRINE标记技术确定是否存在血小板致密颗粒缺乏或储存功能异常，确定AP3是否与致密颗粒储存功能所必需的小GTP酶蛋白Rab27a相互作用，并通过免疫共沉淀和Western blotting筛选与AP3相互作用的额外蛋白。6)通过非清髓性骨髓移植建立DLA相合、性别不合的正常犬和CH犬(n=6)的混合造血嵌合体，确定HSCs是以细胞自主的方式(Chone假说)调节还是以旁分泌的方式由可扩散因子调节。Y染色体聚合酶链式反应将被用来确定循环中的正常细胞和CH细胞的百分比。这些研究将促进我们对HSCs的基本生物学、颗粒形成和蛋白质运输的理解，使用我们实验室独一无二的CH狗模型。

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.