STRUCTURE/FUNCTION ANALYSIS OF PHAGOCYTE PROTEINS

吞噬细胞特有的细胞色素b是吞噬细胞的关键成分。 产生超氧阴离子自由基的氧化物络合物。遗传性缺陷 在这一重要的宿主防御途径中导致慢性肉芽肿 疾病(CGD)。细胞色素b是91kD膜的异源二聚体。 该基因编码的糖蛋白以经典的X连锁形式突变 CGD和一种非糖基化的22kD多肽，该多肽来源于 常染色体隐性遗传性CGD的一个亚群中常染色体发生突变。这个 两个亚基的一级结构，从它们对应的 CDNA，与已知蛋白质没有明显的相似性。在建议的 研究，细胞色素-b的组装、结构和功能 调查，利用分子和生化试剂 在以前的研究中发展起来的。所选择的特定遗传损伤 将对CGD病例进行特征描述，目的是确定重要的 细胞色素异源二聚体的功能结构域。抗体升高到 每个亚基的特定区域将作为试剂进行研究 细胞色素-b的生物合成、组装及其功能研究 互动。此外，还将探索Rap1的潜在角色，a 与细胞色素-b相互作用的RAS相关蛋白，通过调节其 使用突变的衍生物和反义寡核苷酸进行表达。 最后，该提案的一个重要目标是开发适用于 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。非吞噬细胞系是否可以表达 每个亚基和组装一个稳定的杂二聚体将被研究。为 功能研究，将开发X-CGD样吞噬细胞系 利用靶向同源重组灭活KD基因 髓系原核细胞系中的细胞色素亚单位。拟议的研究 应该提供对CGD的功能基础的洞察，更广泛地说， 扩展关于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.