STRUCTURE AND FUNCTION OF PHAGOCYTE PROTEINS

吞噬细胞蛋白的结构和功能

• 批准号: 5200527
• 负责人: T L LETO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200527
• 关键词: NAD(P)H dehydrogenase; child (0-11); chronic granulomatous disease; enzyme activity; enzyme reconstitution; enzyme structure; gene mutation; human subject; human tissue; inflammation; leukocyte oxidative burst; molecular cloning; phagocytes; phosphorylation; protein sequence; tissue /cell culture

Structure-function relationships involved in assembly and activation of NADPH oxidase (respiratory burst) have been explored both in vitro and in transfected cell models. This enzyme is an important host defense system of phagocytes responsible for production of superoxide anion and related microbicidal oxidants; oxidase deficiencies cause chronic granulomatous disease. Recent work has also focused on the oxidase as a model for protein-protein interactions relevant in diverse intra- cellular signal transduction cascades. We have established a model in which multiple interactions of conserved Src Homology 3 (SH3) domains in two cytosolic components (p47-phox and p67-phox) with proline-rich target sequences in other oxidase components mediate NADPH oxidase assembly. Both constitutive and regulated SH3 interactions have been elucidated, based on translocation and activation phenomena observed in whole cells. A tail-tail SH3 interaction between p67-phox and p47-phox affects stability of p67-phox in transfected cells, while two other p47-phox SH3 interactions were evident during oxidase activation when the functions of truncated forms of these proteins were compared. Structural determinants for SH3 domain recognition have also been investigated by gene transfer and mutagenesis; binding epitopes were compared with other well characterized SH3-peptide ligand complexes. We also initiated screening of biased peptide libraries to broaden our understanding of binding specificities of all SH3 domains of this system. We have engineered nine phosphorylation site mutations in p47-phox to explore the role of phosphorylation in regulation of SH3 function during oxidase activation. In related work, we have cloned a cDNA encoding a third cytosolic oxidase component with an SH3 motif (p40-phox), which appears to form a high molecular weight complex with the other cytosolic components. We have expressed p40-phox cDNA in a recombinant baculovirus and have affinity purified this protein through interaction with the C- terminus of p47-phox. We have also expressed most oxidase proteins in the yeast two-hybrid system and have demonstrated interactions in these vectors between p40-phox and the N-terminus of p67-phox and between p67- phox and p21-rac, a Ras-related regulator of the oxidase. These studies may be used as a structural basis for designing drugs that can inhibit this important aspect of inflammation or provide insights on other diverse signal transduction systems involving structurally related proteins.

结构 - 功能关系涉及组装和激活 NADPH氧化酶（呼吸爆发）在体外和 在转染的细胞模型中。 这种酶是重要的宿主防御 负责产生超氧化阴离子的吞噬细胞系统和 相关的微生物氧化剂； 氧化酶缺陷引起慢性 肉芽肿性疾病。 最近的工作也集中在氧化酶 蛋白质蛋白质相互作用的模型 细胞信号转导级联。 我们已经建立了一个模型 保守的SRC同源性3（SH3）域的多次相互作用 具有富含脯氨酸靶的两个胞质成分（P47-PHOX和P67-PHOX） 其他氧化酶成分中的序列介导了NADPH氧化酶的组装。 构成和调节的SH3相互作用均已阐明， 基于在整个细胞中观察到的易位和激活现象。 P67-Phox和P47-Phox之间的尾尾SH3相互作用会影响 p67-phox在转染的细胞中的稳定性，而另外两个p47-phox SH3 当功能时，氧化酶激活期间相互作用是明显的 比较了这些蛋白质的截短形式。 结构 还研究了SH3域识别的决定因素 基因转移和诱变； 将结合表位与其他 良好的SH3肽配体配合物。 我们也发起了 筛选有偏见的肽库，以扩大我们对 该系统所有SH3域的结合特异性。 我们有 在P47-Phox中设计了9个磷酸化位点突变，以探索 磷酸化在氧化酶调节SH3功能调节中的作用 激活。 在相关工作中，我们克隆了一个编码三分之一的cDNA 具有SH3基序（p40-Phox）的胞质氧化酶成分，它出现 与其他胞质形成高分子量复合物 成分。 我们已经在重组杆状病毒中表达P40-磷cDNA 并通过与c-相互作用使该蛋白质纯化该蛋白 p47-phox的末端。 我们还表达了大多数氧化酶蛋白 酵母两杂交系统，并在这些系统中证明了相互作用 P40-Phox和P67-Phox的N端之间以及P67-之间的向量 PHOX和P21-RAC，一种与RAS相关的氧化酶调节剂。 这些研究 可以用作设计可以抑制的药物的结构基础 炎症的这一重要方面或对其他的见解 涉及结构相关的多种信号转导系统 蛋白质。