Loeb K R, Haas A L
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1992 Apr 15;267(11):7806-13.
We have previously identified a 15-kDa interferon-induced protein that is recognized by affinity-purified rabbit polyclonal antibodies against ubiquitin (Haas, A. L., Ahrens, P., Bright, P. M., and Ankel, H. (1987) J. Biol. Chem. 262, 11315-11323). This ubiquitin cross-reactive protein (UCRP) possesses significant homology to a tandem diubiquitin sequence. Since the biological effects of ubiquitin arise from its covalent ligation to intracellular target proteins, we hypothesized that the multiple cellular responses to inteferon are mediated in part by an analogous conjugation pathway for UCRP. Rabbit polyclonal antibodies specific for UCRP were prepared against homogeneous recombinant protein. Affinity-purified anti-UCRP antibodies detected the induction of UCRP in interferon-beta-treated A549 cells and recognized a group of high molecular weight UCRP conjugates on immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved cell extracts. Both free and conjugated UCRP are constitutively present at low levels in untreated cells, suggesting a role for UCRP ligation in normal cellular regulation, and significantly accumulate following interferon treatment. The temporal induction of free UCRP following interferon treatment preceded a delayed increase in UCRP conjugates. Treatment of A549 cells with type I interferons (alpha and beta) strongly induced the expression of free and conjugated UCRP, whereas the response to type II interferon (gamma) was significantly less. A survey of selected cultured cell lines showed differential induction of free versus conjugated UCRP pools in response to interferon. Interferon-beta treatment of A549, MG63, and U937 cells induced high levels of both free and conjugated UCRP, whereas only free UCRP levels increased in Daudi, Namalwa, and K562 cells. These results confirm that UCRP represents a functional ubiquitin homolog participating in a parallel pathway of post-translational ligation and provides a novel mechanism for the response of susceptible cells to the effects of interferon exposure.
我们之前鉴定出一种15 kDa的干扰素诱导蛋白，它能被针对泛素的亲和纯化兔多克隆抗体识别（哈斯，A. L.，阿伦斯，P.，布莱特，P. M.，以及安克尔，H.（1987年）《生物化学杂志》262卷，第11315 - 11323页）。这种泛素交叉反应蛋白（UCRP）与串联双泛素序列具有显著同源性。由于泛素的生物学效应源于其与细胞内靶蛋白的共价连接，我们推测细胞对干扰素的多种反应部分是由UCRP的类似共轭途径介导的。针对同源重组蛋白制备了对UCRP特异的兔多克隆抗体。亲和纯化的抗UCRP抗体在经干扰素-β处理的A549细胞中检测到UCRP的诱导，并在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离的细胞提取物免疫印迹上识别出一组高分子量的UCRP共轭物。游离和共轭的UCRP在未处理细胞中均以低水平组成性存在，这表明UCRP连接在正常细胞调节中起作用，并且在干扰素处理后显著积累。干扰素处理后游离UCRP的时间诱导先于UCRP共轭物的延迟增加。用I型干扰素（α和β）处理A549细胞强烈诱导游离和共轭UCRP的表达，而对II型干扰素（γ）的反应明显较弱。对选定培养细胞系的调查显示，响应干扰素时游离与共轭UCRP池的诱导存在差异。用干扰素-β处理A549、MG63和U937细胞诱导游离和共轭UCRP的高水平表达，而在Daudi、Namalwa和K562细胞中只有游离UCRP水平增加。这些结果证实UCRP代表一种参与翻译后连接平行途径的功能性泛素同源物，并为易感细胞对干扰素暴露效应的反应提供了一种新机制。
