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.

在有反应的细胞中，I型和II型干扰素诱导15-20 不同的蛋白质被认为是细胞宿主的中介 调节效应，如细胞结构、活性、 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， 到目前为止，很少有这些蛋白质的功能被归因于，尽管 这些知识是理解和利用 干扰素作为治疗剂的潜力。这样的一个 功能未知的干扰素诱导蛋白的分子量为 15 kDa。这种蛋白质对细胞的装配能力肯定很重要。 一种干扰素反应，因为它是快速诱导和积累的 与抗病毒活性的外观相似。我们之前已经 提出了这种名为泛素交叉反应蛋白的多肽 (UCRP)由于其免疫学和序列上与泛素相似， 代表泛素的特定功能同系物。最近，我们 已经表明，像泛素一样，UCRP既受结构性的影响，也受 干扰素诱导的细胞内谱的共价结合 蛋白质。这项建议的长期目标是理解 UCRP结合在正常细胞调节中的作用 对干扰素的反应。该建议的具体目标是：(1) 通过CD测量和CD来物理表征重组UCRP的稳定性 使蛋白质结晶，以供后续结构测定；(2) 检测细胞内游离和结合的UCRP池的动力学 亲和纯化多克隆在正常细胞和干扰素诱导的细胞中的应用 抗UCRP抗体与脉搏追踪研究和 特异性反义寡核苷酸抑制UCRP的诱导 (3)UCRP与细胞骨架蛋白的偶联研究 用抗UCRP抗体作为免疫组织化学探针 加合物的定位；(4)酶学步骤的表征 在UCRP接合和亲和和FPLC法分离中 酶(S)负责这种独特的翻译后修饰； (5)克隆、测序和表达重组UCRP结合物 酶(S)使用当前的分子生物学技术。由此产生的 UCRP结合系统的特性将与 平行但不同的泛素连接途径。