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.

在反应性细胞中，I 型和 II 型干扰素诱导 15-20 不同的蛋白质被认为介导宿主细胞 调节作用，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。 然而， 迄今为止，这些蛋白质中很少有功能已被确定，尽管 这些知识对于理解和利用 干扰素作为治疗剂的潜力。 这样的一位 干扰素诱导的未知功能蛋白的分子量为 15 kDa。 这种蛋白质对于细胞的安装能力一定很重要 干扰素反应，因为它被快速诱导并积累 与抗病毒活性的出现相似。 我们之前有过 提出这种多肽，称为泛素交叉反应蛋白 (UCRP) 由于其免疫学和序列与泛素相似， 代表泛素的功能特异性同系物。 最近我们 已经表明，像泛素一样，UCRP 受到本构性和 干扰素诱导的与一系列细胞内共价结合 蛋白质。 该提案的长期目标是了解 UCRP 缀合在正常细胞调节和 对干扰素的反应。 该提案的具体目标是：（1） 通过 CD 测量来物理表征重组 UCRP 稳定性 使蛋白质结晶以进行后续结构测定； (2) 至 检查细胞内游离和结合 UCRP 池的动态 使用亲和纯化的多克隆在正常和干扰素诱导的细胞中 抗 UCRP 抗体与脉冲追踪研究相结合， 用特异性反义寡核苷酸抑制 UCRP 诱导 探针； (3)研究UCRP与细胞骨架蛋白的缀合 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 缀合和通过亲和力和 FPLC 方法分离中 负责这种独特翻译后修饰的酶； (5) 克隆、测序和表达重组 UCRP 缀合 使用当前分子生物学技术的酶。 由此产生的 UCRP 缀合系统的特性将与 平行但不同的泛素连接途径。