STRUCTURE/FUNCTION ANALYSIS OF PHAGOCYTE PROTEINS

吞噬细胞特异性细胞色素b是吞噬细胞的重要组成部分 氧化酶复合物，产生超氧自由基。 遗传缺陷 在这一重要宿主防御途径中， 疾病（CGD）。 细胞色素b是一个91 kD的异源二聚体 糖蛋白编码的基因突变的经典的X-连锁形式， CGD，和衍生自CGD的非糖基化22 kD多肽， 常染色体隐性遗传CGD亚组中常染色体位点突变。 的 这两个亚基的一级结构，从它们相应的 cDNA与已知蛋白质没有明显的相似性。 拟议 研究，细胞色素b的组装，结构和功能将是 利用分子和生化试剂 在以前的研究中发展。 特定的遗传性病变， CGD病例将被描述，目的是确定重要的 细胞色素异二聚体中的功能结构域。 抗体升高至 每个亚基的特定区域将被用作研究试剂， 细胞色素-b生物合成和组装，并探讨其功能 交互. 此外，还将探讨Rap 1的潜在作用， Ras相关蛋白，通过调节其 使用突变衍生物和反义寡核苷酸表达。 最后，本提案的一个重要目标是开发适合于 通过基因转移研究细胞色素b异源二聚体， 特别是突变的cDNA 非吞噬细胞系是否能表达 将研究每个亚基并组装成稳定的异源二聚体。 为 功能研究，X-CGD样吞噬细胞系将被开发 使用靶向同源重组来扩增kD的基因 在髓样PLB细胞系中的细胞色素亚基。 拟议研究 应该提供深入了解CGD的功能基础，更广泛地说， 扩大有关吞噬细胞的超氧化物生成系统的知识。

A phagocyte-specific cytochrome-b is a critical component of the phagocyte oxidase complex which generates the superoxide radical. Inherited defects in this important host defense pathway result in chronic granulomatous disease (CGD). Cytochrome-b is a heterodimer of a 91 kD membrane glycoprotein encoded by the gene mutated in the classic X-linked form of CGD, and a non-glycosylated 22 kD polypeptide that derives from an autosomal locus mutated in a subgroup of autosomal recessive CGD. The primary structures of the two subunits, deduced from their corresponding cDNAs, have no obvious similarities to known proteins. In the proposed research, the assembly, structure and function of cytochrome-b will be investigated, taking advantage of molecular and biochemical reagents developed in previous studies. The specific genetic lesions in selected cases of CGD will be characterized, with the aim of identifying important functional domains in the cytochrome heterodimer. Antibodies raised to specific regions of each subunit will be used as reagents to study biosynthesis and assembly of cytochrome-b and to probe its functional interactions. In addition, a potential role will be explored for Rap1, a Ras-related protein that copurifies with cytochrome-b, by modulation of its expression using mutated derivatives and antisense oligonucleotides. Finally, an important goal of this proposal is to develop systems suitable for the study of the cytochrome-b heterodimer by gene transfer of specifically mutated cDNAs. Whether non-phagocytic cell lines can express each subunit and assemble a stable heterodimer will be investigated. For functional studies, an X-CGD-like phagocytic cell line will be developed using targeted homologous recombination to inactivate the gene for the kD cytochrome subunit in the myeloid PLB cell line. The proposed research should provide insight into the functional basis of CGD, and more broadly, extend knowledge about the superoxide generating system of the phagocyte.