ATP UBIQUITIN-DEPENDENT PROTEOLYSIS

蛋白质的稳态细胞内浓度由 合成和降解速率之间的动态平衡。 的 真核生物内降解的主要途径是由 多酶ATP，泛素依赖性蛋白水解途径， 蛋白质被26 S蛋白酶体靶向降解， 共价缀合至8.6kDa多肽泛素。 最近 有证据表明，泛素/蛋白酶体降解途径是 一些基本的监管程序，包括 异常蛋白质的蛋白水解，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、细胞器生物发生和各种癌蛋白的周转， 肿瘤抑制因子和转录因子。 长期目标是 本文旨在阐明该途径的酶学和功能 潜在的治疗干预。 这一行动的直接目标是 建议包括五个具体目标：（1）定点诱变 泛素将用于鉴定多肽上的关键残基 作为一种手段，为关键的活动场所绘制地图， 途径的酶;（2）E1内特定残基的突变， 泛素结合的第一种酶，将被用来鉴定 该酶的活性位点;（3）该酶的缺失/突变分析。 主要泛素载体蛋白E2/14 K将用于鉴定区域 与E1和E3相互作用的酶， 识别/结合酶;（4）最近发现的 泛素载体蛋白E2 EPF，被认为是发育所必需的 用于角质形成细胞的终末分化，将用于 确定网站的autoubiquitination建议，以调节自我- 该酶的靶向和细胞内浓度，然后 在培养的人细胞系中通过瞬时 （5）E2亲和性和双杂交 筛选方法将用于分离和克隆同源E3同工酶 需要E2/14 K和E2 EPF，随后是它们的表达和动力学， 分析了重组结合酶的作用机理。

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.