FUNCTION OF AN INTERFERON INDUCED UBIQUITIN HOMOLOG

描述（改编自申请人摘要）：细胞系特异性 干扰素的作用是通过协调诱导 在细胞因子作用过程中， 反应 首席研究员已经表明，p15，一个17 kDa的早期 1型干扰素诱导的基因产物，与 串联二泛素序列。 随后的证据表明， 这种泛素交叉反应蛋白（UCRP）的生物学反应是 介导的共价连接到一个小的亚群 胞内靶蛋白。 最近的结果表明UCRP结合 与靶蛋白的连接途径不同于 即泛素。 这种新的干扰素作用机制将是 在六个具体目标中进行研究。 (1)UCRP的物理特性 重组UCRP及其前体对各种 通过CD和荧光猝灭监测结构扰动 用于与泛素进行比较。 (2)检查UCRP与 中间丝-UCRP-氯霉素瞬时表达 将使用培养的A549细胞中的转氨酶（CAT）来确认 早期的免疫组化数据表明UCRP作为一种反式 靶蛋白非共价结合的作用结合决定簇 具有中间细丝。 UCRP-CAT构建体的缺失分析 将被用来确定UCRP上负责 纤维团 部分微测序将用于 表征三种新的低分子量（15-17 kDa）中间体 发现与UCRP结合的免疫相关蛋白。 (3)检查 细胞内UCRP库的动力学--直接测定和互补 在培养的A549细胞提取物中的（125）1-UCRP结合研究将 检查在干扰素-β作用期间连接反应的调节， 诱导 UCRP在干扰素应答中的潜在作用将是 通过瞬时表达UCRP来阻断UCRP的合成， 反义UCRP mRNA。 (4)纯化和表征preUCRP处理 活动 (5)检查UCRP结合的酶学-体外 （125）1-UCRP结合试验将用于鉴定 A549提取物中存在的催化多肽连接的酶。 这些酶将被纯化和表征以与本发明的酶进行比较。 泛素结合途径 (6)UCRP缀合物的克隆 酶-UCRP结合酶的部分微测序将 用于构建筛选干扰素诱导的A549的探针， cDNA文库 UCRP结合酶的克隆和表达 为后续机制研究提供功能比较 平行的泛素连接途径。

DESCRIPTION (adapted from the applicant's abstract): Cell line specific effects of the interferons are mediated through the coordinated induction of a subset of genes expressed at precise times during the cytokine response. The principal investigator has shown that p15, a 17 kDa early gene product of type 1 interferon induction, bears marked homology to a tandem di-ubiquitin sequence. Subsequent evidence suggests the biological response of this Ubiquitin Cross Reactive Protein (UCRP) is mediated through covalent ligation to a small subpopulation of intracellular target proteins. Recent results indicate UCRP conjugation to target proteins proceeds through a ligation pathway distinct from that of ubiquitin. This novel mechanism for interferon action will be examined in six specific aims. (1) Physical characterization of UCRP-- the stability of recombinant UCRP and its precursor to various structural perturbants will be monitored by CD and fluorescence quench for comparison to ubiquitin. (2) Examine the binding of UCRP to intermediate filaments--transient expression of UCRP-chloramphenicol aminotransferase (CAT) in cultured A549 cells will be used to confirm earlier immunohistochemical data suggesting UCRP serves as a trans acting binding determinant for noncovalent association of target proteins with intermediate filaments. Deletion analysis of UCRP-CAT constructs will be utilized to identify the binding motif on UCRP responsible for filament association. Partial microsequencing will be used to characterize three novel low molecular weight (15-17 kDa) intermediate filament-associated proteins found to bind UCRP. (3) Examine the dynamics of intracellular UCRP pools--direct assays and complementation studies of (125)1-UCRP conjugation in cultured A549 cell extracts will examine the regulation of the ligation reaction during interferon-beta induction. Potential roles for UCRP in the interferon response will be tested by blocking UCRP synthesis through transient expression of antisense UCRP MRNA. (4) Purify and characterize the preUCRP processing activity. (5) Examine the enzymology of UCRP conjugation--in vitro (125)1-UCRP conjugation assays will be employed to identify the enzymes(s) present in A549 extracts catalyzing polypeptide ligation. These enzymes will be purified and characterized for comparison to the ubiquitin conjugation pathway. (6) Cloning of the UCRP conjugating enzymes--partial microsequencing of the UCRP conjugating enzymes will be used to construct probes for screening an interferon-induced A549 cDNA library. Cloning and expression of the UCRP conjugating enzymes for subsequent mechanistic studies will provide functional comparisons to the parallel ubiquitin ligation pathway.