ATP UBIQUITIN-DEPENDENT PROTEOLYSIS

细胞内蛋白质的稳定浓度是由 合成和降解速率之间的动态平衡。这个 真核生物内降解的主要途径是由 多酶ATP，泛素依赖的蛋白分解途径，在其中 蛋白质被26S蛋白酶体通过它们的 8.6 kDa多肽泛素的共价偶联。近期 有证据表明泛素/蛋白酶体的降解途径是 一系列基本监管程序所需的 异常蛋白质的蛋白分解、有丝分裂进程、基因转录 蛋白质加工，发育程序性细胞死亡，压力 反应，细胞器的生物发生，以及各种癌蛋白的周转， 肿瘤抑制因子和转录因子。的长期目标 本建议旨在阐明该途径的酶学和功能。 进行潜在的治疗干预。这样做的直接目标是 建议包括五个具体目标：(1)定点诱变 泛素将被用来识别多肽上的关键残基 其作为映射关键字的活动站点的手段所需的 该途径的酶；(2)E1内特定残基的诱变， 将利用泛素偶联的第一种酶来鉴定 该酶的活性部位；(3)缺失/突变分析 主要泛素载体蛋白E2/14K将用于识别区域 与E1和E3相互作用的酶，底物 识别/结合酶；(4)一株新发现的突变株 泛素载体蛋白E2EPF，建议发育所需 对于角质形成细胞的终末分化，将被用于 确定自我调节的自体素化位点 该酶的靶向和胞内浓度，随后 该假说在培养的人细胞系中的直接检验 适当的E2EPF突变体的转染；(5)E2亲和性和双杂交 将采用筛选方法分离和克隆同源E3同工酶 需要E2/14K和E2EPF，然后是它们的表达和动力学 重组结合酶的作用机理分析。

The steady state intracellular concentration of proteins is regulated by the dynamic balance between rates of synthesis and degradation. The major pathway for degradation within eukaryotes is mediated by the multienzyme ATP, ubiquitin-dependent proteolytic pathway in which proteins are targeted for degradation by the 26S proteasome through their covalent conjugation to the 8.6 kDa polypeptide ubiquitin. Recent evidence indicates the ubiquitin/proteasome degradative pathway is required for a number of fundamental regulatory processes including proteolysis of abnormal proteins, mitotic progression, gene transcription and protein processing, developmentally-programmed cell death, the stress response, organelle biogenesis, and the turnover of various oncoproteins, tumor suppressors, and transcription factors. The long range goal of this proposal is to elucidate the enzymology and function of this pathway for potential therapeutic intervention. The immediate goals of this proposal constitute five specific aims: (1) Site-directed mutagenesis of ubiquitin will be utilized to identify key residues on the polypeptide required for its function as a means of mapping the active sites for key enzymes of the pathway; (2) Mutagenesis of specific residues within E1, the first enzyme of ubiquitin conjugation, will be exploited to identify the active site of this enzyme; (3) Deletion/mutation analysis of the major ubiquitin carrier protein E2/14K will be used to identify regions of the enzyme that interact with E1 and E3, the substrate recognition/conjugation enzyme; (4) Mutagenesis of a recently discovered ubiquitin carrier protein E2EPF, suggested to be required developmentally for the terminal differentiation of keratinocytes, will be utilized to identify the site of autoubiquitination proposed to regulate the self- targeting and intracellular concentration of this enzyme, followed by direct test of the hypothesis in cultured human cell lines by transient transfection of appropriate E2EPF mutants; (5) E2-affinity and two-hybrid screening methods will be used to isolate and clone cognate E3 isozymes requiring E2/14K and E2EPF, followed by their expression and kinetic analysis of the mechanisms for the recombinant conjugating enzymes.