E2-25K--MULTIUBIQUITINATION AND PROTEIN DEGRADATION

多泛素链与细胞蛋白质的共价连接 代表了一种有效的降解信号机制。 多泛素化 蛋白质被特定的蛋白酶识别;靶蛋白是 降解，而泛素再生。 连续泛素在 多泛素链通过涉及侧链的异肽键连接， Lys-48链和泛素C-末端。 哺乳动物的泛素- 结合酶E2- 25 K在合成 多泛素链从分离的泛素，并具有高（氨基 酸）序列相似性的一个亚家族的酵母E2的发挥作用， 在蛋白质降解中的重要作用。 全面的研究是 提出了解决E2- 25 K起主导作用的假设 在哺乳动物细胞中的多泛素化和蛋白质降解。 一 反向遗传学方法（反义RNA）将用于干扰 E2- 25 K的表达;对蛋白质周转率的影响，以及对 将测定细胞内游离多泛素链的水平。 E2- 25 K的缺失分析表明，其C-末端是一个与E2 - 25 K同源的氨基酸残基。 结合特异性的决定因素;我们将进一步测试 通过评估嵌合体的接合特异性， 一种蛋白质，其中E2- 25 K的C-末端融合到一个 不同的E2 这些结果将根据详细的 通过X射线晶体学分析获得的结构信息， E2，以及催化E2-泛素硫醇酯的类似物 中间体 建议的研究结果可以确定E2- 25 K 作为一种泛素结合酶， 在哺乳动物细胞中的周转，而且有潜力提供 E2结构之间的第一个直接连接（在任何系统中），共轭的 特异性和生物学功能。 其他实验将解决 结构-功能关系的后共轭方面， ubiquitin本身 带有反应基团的多泛素链. 链表面上的限定位置将用于交联 用泛素结合蛋白酶进行的研究。 结果将解决 链与蛋白酶结合的全局模式，并应揭示 这种多亚基酶的哪些组分参与缀合物 识别.

Covalent attachment of multi-ubiquitin chains to cellular proteins represents a potent degradative signaling mechanism. Multi-ubiquitinated proteins are recognized by a specific protease; the target protein is degraded, while ubiquitin is regenerated. Successive ubiquitins in multi-ubiquitin chains are linked by isopeptide bonds involving the side chain of Lys-48 and the ubiquitin C-terminus. The mammalian ubiquitin- conjugating enzyme E2-25K has a high specific activity in synthesis of multi-ubiquitin chains from isolated ubiquitin, and has a high (amino acid) sequence similarity to a subfamily of yeast E2s which play an essential role in protein degradation. Comprehensive studies are proposed to address the hypothesis that E2-25K plays a predominant role in multi-ubiquitination and protein degradation in mammalian cells. A reverse genetic method (antisense RNA) will be used to interfere with the expression of E2-25K; the effects on protein turnover rates, and on the level of free multi-ubiquitin chains within cells, will be determined. Deletional analysis of E2-25K suggests that its C-terminus is a determinant of conjugative specificity; we will further test this hypothesis by evaluating the conjugative specificity of a chimeric protein in which the E2-25K C-terminus is fused to the core of a different E2. These results will be interpreted in the light of detailed structural information obtained by X-ray crystallographic analysis of the E2, and of an analog of the catalytic E2-ubiquitin thiol ester intermediate. The results of the proposed studies may identify E2-25K as a ubiquitin-conjugating enzyme which functions globally in protein turnover in mammalian cells, and moreover have potential to provide the first direct link (in any system) between E2 structure, conjugative specificity, and biological function. Other experiments will address post-conjugative aspects of the structure-function relationship in ubiquitin itself. Multi-ubiquitin chains bearing reactive groups at defined positions on the chain surface will be used in cross-linking studies with the ubiquitin conjugate protease. The results will address the global mode of binding of chains to the protease, and should reveal which components of this multi-subunit enzyme are involved in conjugate recognition.