E2-25K--MULTIUBIQUITINATION AND PROTEIN DEGRADATION

多泛素链与细胞蛋白的共价结合 代表了一种有效的降解性信号机制。多泛素化 蛋白质由一种特定的蛋白酶识别；目标蛋白质是 降解，而泛素则再生。泛素在体内的连续存在 多个泛素链通过涉及侧链的异肽键连接起来 Lys-48链和泛素C末端。哺乳动物的泛素- 结合酶E_2-25K具有较高的比活力 从分离的泛素中分离出多个泛素链，并具有很高的(氨基) 酸)序列与酵母E2s的一个亚家族的相似性，这些E2s发挥着 在蛋白质降解中起着至关重要的作用。全面的研究是 提出解决E2-25K起主导作用的假说 在哺乳动物细胞中的多泛素化和蛋白质降解。一个 将使用反向遗传方法(反义RNA)来干扰 E_2-25K的表达；对蛋白质周转率的影响； 细胞内自由多泛素链的水平将被确定。 E_2-25K基因的缺失分析表明其C-末端是一个 结合特异性的决定因素；我们将进一步测试这一点 通过评估嵌合体的结合特异性而提出的假说 E_2-25K C-末端融合到核心区的蛋白质 不同的雌二醇。这些结果将根据详细的 由X-射线晶体分析获得的结构信息 E2和催化的E2-泛素硫醇酯的类似物 中级的。拟议的研究结果可能确定E2-25K 作为一种泛素结合酶，在蛋白质中发挥全球功能 哺乳动物细胞的周转，而且有可能提供 E2结构之间的第一个直接连接(在任何系统中)，共轭 特异性和生物学功能。其他实验将解决 的结构-功能关系的后共轭方面 泛素本身。含有反应性基团的多泛素链 链表面上的定义位置将用于交联剂 泛素结合蛋白酶的研究。结果将解决 链与蛋白酶结合的全局模式，并应揭示 这种多亚单位酶的哪些成分参与了偶联反应 承认。

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.