Structure and Function of the Essential Cell Cycle Regulator Cdc34

项目摘要 细胞以调节的方式控制细胞周期进程的能力是基本的 对真核生物学的重要性。控制细胞周期的检查点的崩溃导致 对正常细胞功能的灾难性影响，并且是癌细胞的定义特征。细胞分裂周期 （Cdc 34）是一种泛素（Ub）结合酶（E2），在控制肿瘤进展中起重要作用。 在G1/S和G2/M检查点通过泛素化关键细胞周期调节蛋白调节细胞周期， 在规定的时间点发出蛋白酶体破坏的信号。E2酶，如Cdc 34， 在Ub共轭级联反应中，三个分子的顺序相互作用和活动 酶（E1、E2和E3）是靶蛋白泛素化所必需的。这个级联反应是由E1启动的， 它激活并将Ub转移到数十个不同的E2上。生成的E2~Ub中间体接下来相互作用 具有数百种Ub E3连接酶的库，其通过不同的方式催化Ub与靶蛋白的缀合， 机制等虽然在大多数情况下，同源E2/E3对负责调节特定的 生物学过程是未知的，RING E3连接酶的Skp，Cullin，F-box（SCF）亚家族是良好的。 建立与Cdc 34一起发挥作用，通过催化赖氨酸48- 靶蛋白上的连接的多聚泛素（polyUb）链，作为蛋白酶体破坏的信号。 尽管Cdc 34具有根本性的重要性，但关于Cdc 34在体内如何发挥其功能的许多关键问题仍然存在。 细胞包括：1）在Cdc 34-Ub中间体形成期间，Cdc 34如何被募集到E1，2）Cdc 34-Ub中间体形成期间， Cdc 34 ~（1b）随后被募集到SCF中，以及3）Cdc 34/SCF复合物如何特异性地催化 Lys 48连接的polyUb链，其发出靶蛋白的蛋白酶体破坏的信号，而不是其他的 polyUb链的类型。cdc 34具有许多独特的序列和结构特征， 其他E2对它的功能至关重要，尽管这些观察结果的结构基础尚不清楚。 这些特征包括接近活性位点的酸性环插入和大的C末端延伸， 这两者都是Lys 48连接的polyUb链特异性和最大酶活性所需的。 通过使用结构、生物化学/生物物理和基于细胞的方法，该提案旨在 建立E1/Cdc 34和Cdc 34/SCF相互作用中的分子识别规则（目标1）， 确定Cdc 34和SCF共同作用以特异性组装Lys 48的结构基础。 连接的polyUb链（目的2）。Ub信号传导是一个有效的癌症治疗干预靶点， FDA批准的靶向蛋白酶体的药物目前延长了多发性骨髓瘤的生命 患者对E1、Cdc 34和SCF如何共同控制细胞周期的分子理解 从拟议的研究中产生的结果可以为新癌症的发展提供一个平台， 治疗学

Project Summary The ability of the cell to control progression through the cell cycle in a regulated manner is of fundamental importance to eukaryotic biology. Breakdown of the checkpoints controlling the cell cycle results in catastrophic effects on normal cell function and is a defining feature of cancer cells. Cell division cycle 34 (Cdc34) is a ubiquitin (Ub) conjugating enzyme (E2) that plays an essential role in controlling progression of the cell cycle at both the G1/S and G2/M checkpoints by ubiquitinating key cell cycle regulatory proteins and signaling for their proteasomal destruction at defined timepoints. E2 enzymes such as Cdc34 are the central players in the Ub conjugation cascade in which the sequential interactions and activities of three enzymes (E1, E2, and E3) are required for ubiquitination of target proteins. This cascade is initiated by E1, which activates and transfers Ub to tens of different E2s. The resulting E2~Ub intermediates next interact with a repertoire of hundreds of Ub E3 ligases that catalyze Ub conjugation to target proteins by distinct mechanisms. While in most cases the cognate E2/E3 pair responsible for regulation of a particular biological process is unknown, the Skp, Cullin, F-box (SCF) subfamily of RING E3 ligases is well- established to function with Cdc34 to control cell cycle progression by catalyzing the extension of Lysine 48- linked polyubiquitin (polyUb) chains on target proteins that serve as a signal for proteasomal destruction. Despite its fundamental importance, many key questions remain about how Cdc34 performs its functions in the cell including: 1) how Cdc34 is recruited to E1 during formation of the Cdc34~Ub intermediate, 2) how Cdc34~Ub is subsequently recruited to SCF, and 3) how the Cdc34/SCF complex specifically catalyzes Lys48-linked polyUb chains that signal for proteasomal destruction of target proteins as opposed to other types of polyUb chains. Cdc34 harbors a number of unique sequence and structural features compared to other E2s that are crucial for its function, though the structural basis for these observations are unknown. These features include an acidic loop insertion proximal to the active site and a large C-terminal extension, both of which are required for Lys48-linked polyUb chain specificity and maximal enzymatic activity. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to establish the rules governing molecular recognition in E1/Cdc34 and Cdc34/SCF interactions (Aim 1) and to determine the structural basis by which Cdc34 and SCF function together to specifically assemble Lys48- linked polyUb chains (Aim 2). Ub signaling is a validated target for therapeutic intervention in cancer with FDA-approved medications targeting the proteasome currently extending the lives of multiple myeloma patients. A molecular understanding of how E1, Cdc34, and SCF work together to control the cell cycle resulting from the proposed studies could provide a platform for the development of novel cancer therapeutics.