FUNCTION OF AN INTERFERON INDUCED UBIQUITIN HOMOLOG

描述(改编自申请人的摘要)：具体细胞系 干扰素的作用是通过协调诱导来实现的 在细胞因子的精确时间表达的基因的子集 回应。首席调查员已经证明了p15，一个17 kDa的早期 1型干扰素诱导的基因产物，与 一个串联的双泛素序列。随后的证据表明 泛素交叉反应蛋白(UCRP)的生物学反应是 通过共价连接传递给一小部分亚群 细胞内靶蛋白。最近的结果表明UCRP结合 靶向蛋白质的连接途径不同于 泛素的作用。这种新的干扰素作用机制将是 审查了六个具体目标。(1)UCRP的物理特性-- 重组UCRP及其前体在不同条件下的稳定性 结构扰动将通过CD和荧光猝灭进行监测 用于与泛素进行比较。(2)检查UCRP与 中间丝--UCRP-氯霉素的瞬时表达 培养的A549细胞中的转氨酶(CAT)将用于确认 早期的免疫组织化学数据表明UCRP是一种反式 靶蛋白非共价结合的作用结合决定簇 有中间细丝的。UCRP-CAT构建物的缺失分析 将被用来确定UCRP上负责 花丝协会。部分微测序将用于 三种新型低分子量(15-17 kDa)中间体的表征 发现与UCRP结合的细丝相关蛋白。(3)审查 细胞内UCRP池的动态变化--直接测定法和互补法 培养的A549细胞提取物中~(125)1-UCRP结合的研究 检测干扰素-β过程中连接反应的调节 归纳法。UCRP在干扰素反应中的潜在作用将是 通过瞬时表达UCRP抑制UCRP的合成 反义UCRP基因。(4)前UCRP处理的提纯和表征 活动。(5)UCRP结合的酶学研究--体外 (125)将使用1-UCRP结合分析来鉴定 酶(S)存在于A549提取物中，催化多肽连接。 这些酶将被提纯和鉴定，以便与 泛素结合途径。(6)UCRP结合基因的克隆 酶--UCRP结合酶的部分微测序 用于构建用于筛选干扰素诱导的A549的探针 C DNA文库。UCRP结合酶基因的克隆与表达 因为随后的机械学研究将提供功能比较 至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.