FUNCTION OF AN INTERFERON-INDUCED UBIQUITION HOMOLOG

在应答细胞中，I型和II型干扰素诱导15-20 不同的蛋白质被认为是介导宿主细胞 调节作用，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。 但 迄今为止，这些蛋白质中很少有人被赋予功能，尽管 这些知识对于理解和利用 干扰素作为治疗剂的潜力。 一个这样 未知功能干扰素诱导蛋白的分子量为 15 kDa。 这种蛋白质对细胞的粘附能力很重要 干扰素反应，因为它是快速诱导和其积累 与抗病毒活性的出现平行。 我们先前已经 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受组成性和 干扰素诱导的共价结合到细胞内的谱 proteins. 本提案的长期目标是了解 UCRP结合在正常细胞调节和 对干扰素的反应 建议的具体目标是：（1） 通过CD测量物理表征重组UCRP稳定性， 使蛋白质结晶，用于随后的结构测定;（2） 检查游离和结合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 抗UCRP抗体结合脉冲追踪研究， 特异性反义寡核苷酸抑制UCRP诱导 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）表征酶促步骤 在UCRP缀合和通过亲和和FPLC方法分离中， 负责这种独特的翻译后修饰的酶; 和（5）克隆、测序和表达重组UCRP缀合物， 使用当前的分子生物学技术的酶。 所得 UCRP缀合系统的特性将与 平行但不同的泛素连接途径。

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.