FUNCTION OF AN INTERFERON-INDUCED UBIQUITION HOMOLOG

干扰素诱导的泛在同系物的功能

• 批准号: 3306922
• 负责人: ARTHUR L HAAS
• 金额: $ 18.42万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306922
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; autoradiography; chemical conjugate; chemical stability; circular dichroism; covalent bond; crystallization; cycloheximide; enzyme mechanism; gene induction /repression; genetic library; immunocytochemistry; interferons; isotope dilution method; laboratory rabbit; molecular cloning; posttranslational modifications; protein biosynthesis; protein degradation; protein purification; protein structure function; stable isotope double label; tissue /cell culture; ubiquitin; western blottings

In responsive cells, type I and type II interferons induce 15-20 different proteins that are thought to mediate the host of cellular regulatory effects such as changes in cell structure, activity, differentiation, proliferative capacity, presentation of major histocompatibility antigens, and anti-viral activity. However, the functions for few of these proteins have been ascribed to date, although such knowledge is fundamental to understanding and exploiting the potential of the interferons as therapeutic agents. One such interferon-induced protein of unknown function has a molecular weight of 15 kDa. This protein must be important for the ability of cells to mount an interferon response since it is rapidly induced and its accumulation parallels the appearance of antiviral activity. We have previously proposed that this polypeptide, termed Ubiquitin Cross-Reactive Protein (UCRP) because of its immunological and sequence similarity to ubiquitin, represents a function-specific homolog of ubiquitin. More recently we have shown that, like ubiquitin, UCRP is subject to both constitutive and interferon-induced covalent conjugation to a spectrum of intracellular proteins. The long-term objectives of this proposal are to understand the role of UCRP conjugation in normal cellular regulation and in response to interferon. The specific aims of the proposal are: (1) to physically characterize recombinant UCRP stability by CD measurements and to crystallize the protein for subsequent structure determination; (2) to examine the dynamics of intracellular pools of free and conjugated UCRP in normal and interferon-induced cells using affinity-purified polyclonal antibodies against UCRP in conjunction with pulse-chase studies and inhibition of UCRP induction with specific antisense oligonucleotide probes; (3) to study the conjugation of UCRP to cytoskeletal proteins using the anti-UCRP antibodies as immunohistochemical probes for localization of the adducts; (4) to characterize the enzymological steps in UCRP conjugation and isolate by affinity and FPLC methods the enzyme(s) responsible for this unique post-translational modification; and (5) to clone, sequence, and expressed recombinant UCRP conjugating enzyme(s) using current molecular biological techniques. The resulting characteristics of the UCRP conjugating system will be compared to the parallel but distinct ubiquitin ligation pathway.

在响应细胞中，I型和II型干扰素诱导15-20 被认为可以介导细胞宿主的不同蛋白质 调节效应，例如细胞结构，活性的变化， 分化，增殖能力，主要表现 组织相容性抗原和抗病毒活性。 但是， 迄今为止，这些蛋白质中很少有功能已被归因于 这样的知识是理解和利用的基础 干扰素作为治疗剂的潜力。 一个这样的 干扰素诱导的未知功能蛋白具有分子量的 15 kda。 该蛋白对于细胞安装的能力必须很重要 干扰素反应迅速诱导并积累 与抗病毒活性的出现相似。 我们以前有 提出该多肽称为泛素交叉反应蛋白 （UCRP）由于其与泛素的免疫学和序列相似性， 代表泛素特定功能的同源物。 最近我们 已经表明，像泛素一样，UCRP受本构和 干扰素诱导的共轭与细胞内的光谱 蛋白质。 该提议的长期目标是了解 UCRP共轭在正常细胞调节和 对干扰素的响应。 该提案的具体目的是：（1） 通过CD测量值和 使蛋白质结晶以进行后续结构测定； （2）至 检查自由和共轭UCRP的细胞内池的动力学 在正常和干扰素诱导的细胞中，使用亲和纯化的多克隆 针对UCRP的抗体与脉搏追踪研究和 用特异性反义寡核苷酸抑制UCRP诱导 探针； （3）研究UCRP与细胞骨架蛋白的结合 使用抗UCRP抗体作为免疫组织化学探针 加合物的定位； （4）表征酶学步骤 在UCRP结合和通过亲和力和FPLC方法分离中 酶负责这种独特的翻译后修饰； （5）克隆，序列和表达的重组UCRP结合 使用电流分子生物学技术的酶。 结果 将UCRP共轭系统的特征与 平行但独特的泛素连接途径。