P56C2, A NOVEL C2 DOMAIN PROTEIN OF LEUKOCYTES

P56C2，一种新型白细胞 C2 结构域蛋白

基本信息

批准号：
6624548
负责人：
Bernard M Babior
金额：
$ 37.23万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-12-01 至 2004-11-30
项目状态：
已结题

项目摘要

The leukocyte NADPH oxidase catalyzes the production of O2 from oxygen and NADPH. The yeast 2-hybrid system was used to look for proteins that interact with p67PHOX, one of the cytosolic subunits of the oxidase. This system identified a B lymphocyte cDNA that encoded a 60 kDa protein containing two Ca++- and lipid-binding C2 domains in its C-terminal half. Rim, a Rab3-binding protein involved in synaptic vesicle trafficking, has a homologous N-terminal half and a similar C-terminal C2 domain structure, suggesting that the new protein, formerly named p56C2 but now renamed p62MCSP, might be involved in the trafficking of secretory vesicles in leukocytes. Experiments with recombinant p62MCSP fragments expressed in PC12 cells support this idea. We propose to express p62MCSP and characterize it in terms of its location in neutrophils and EBV- transformed B lymphocytes, its interactions with other leukocyte proteins and with the cortical cytoskeleton, and its association with lipids. The function of p62MCSP will be studied by 1) examining the effects of recombinant p62MCSP and a dominant negative p62MCSP mutant on the cell-free phosphorylation-dependent and SDS-dependent oxidase activating systems; 2) examining oxidase activation in EBV-transformed B lymphocytes expressing a dominant negative mutant of p62MCSP; and 3) examining the effects of a dominant negative mutant on degranulation and oxidase activation in streptolysin O-permeabilized neutrophils. In addition, we will investigate the possibility that p62MCSP interacts with Rab, a family of small guanine nucleotide-binding proteins (GNBP) involved in membrane trafficking, and with Rap1A, a GNBP that copurifies with the membrane-associated subunits of the oxidase. Through these experiments we hope to achieve a deeper understanding of the relationship between membrane vesicle fusion and the activation of the O2-forming oxidase of leukocytes, an enzyme important in host defense and in the harmful side effects of inflammation.

白细胞 NADPH 氧化酶催化氧气和 NADPH 产生 O2。酵母 2-杂交系统用于寻找与 p67PHOX（氧化酶的胞质亚基之一）相互作用的蛋白质。该系统鉴定出 B 淋巴细胞 cDNA，其编码 60 kDa 的蛋白质，在其 C 末端一半含有两个 Ca++ 和脂质结合 C2 结构域。 Rim 是一种参与突触小泡运输的 Rab3 结合蛋白，具有同源的 N 端半部和相似的 C 端 C2 结构域结构，表明这种新蛋白（以前称为 p56C2，但现在更名为 p62MCSP）可能参与运输白细胞中的分泌囊泡。在 PC12 细胞中表达的重组 p62MCSP 片段的实验支持了这一想法。我们建议表达 p62MCSP 并根据其在中性粒细胞和 EBV 转化的 B 淋巴细胞中的位置、其与其他白细胞蛋白和皮质细胞骨架的相互作用以及其与脂质的关联来表征它。 p62MCSP的功能将通过以下方式研究：1)检查重组p62MCSP和显性失活p62MCSP突变体对无细胞磷酸化依赖性和SDS依赖性氧化酶激活系统的影响； 2)检查表达p62MCSP显性失活突变体的EBV转化的B淋巴细胞中氧化酶的激活； 3) 检查显性失活突变体对链球菌溶血素 O 通透性中性粒细胞脱粒和氧化酶激活的影响。此外，我们将研究 p62MCSP 与 Rab（参与膜运输的小鸟嘌呤核苷酸结合蛋白 (GNBP) 家族）以及与 Rap1A（一种与氧化酶膜相关亚基共纯化的 GNBP）相互作用的可能性。通过这些实验，我们希望更深入地了解膜囊泡融合与白细胞 O2 形成氧化酶激活之间的关系，这种酶在宿主防御和炎症的有害副作用中发挥着重要作用。