Structure and Function of the Essential Cell Cycle Regulator Cdc34

项目摘要 细胞以一种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，是癌细胞的一个重要特征。细胞分裂周期34 (CDC34)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检查点的细胞周期通过泛化关键的细胞周期调节蛋白和 在规定的时间点发出它们的蛋白酶体被破坏的信号。像CDC34这样的E2酶是 Ub共轭过程中的中心角色，其中三个成员的顺序相互作用和活动 酶(E1、E2和E3)是目标蛋白泛素化所必需的。这个级联是由E1发起的， 它激活Ub并将其传输到数十个不同的E2。由此产生的E2~Ub中间体下一步相互作用 具有数百种Ub E3连接酶，这些连接酶通过不同的方式催化Ub与靶蛋白的结合 机械装置。而在大多数情况下，同源的E2/E3对负责调节特定的 生物学过程未知，环E3连接酶的Skp，cullin，F-box(SCF)亚家族是很好的. 与CDC34一起作用，通过催化赖氨酸48的延长来控制细胞周期进展- 连接的多泛素(PolyUb)链位于靶蛋白上，作为蛋白酶体破坏的信号。 尽管它具有根本重要性，但关于CDC34如何在 该细胞包括：1)在形成CDC34~Ub中间体的过程中，CDC34如何被招募到E1中；2)如何 Cdc34~Ub随后被募集到SCF中，以及3)Cdc34/SCF复合体如何特异性地催化 Lys48连接的PolyUb链，与其他蛋白酶体相反，发出蛋白酶体破坏的信号 PolyUb链的类型。CDC34具有许多独特的序列和结构特征，与 其他对其功能至关重要的E2，尽管这些观察到的结构基础尚不清楚。 这些特征包括活性部位附近的酸性环插入和大的C-末端延伸， 这两者都是Lys48连接的PolyUb链特异性和最大酶活性所必需的。 通过使用结构、生化/生物物理和基于细胞的方法，这项提议旨在 建立在E1/CDC34和CDC34/SCF相互作用中的分子识别规则(目标1)和 确定CDC34和SCF共同作用以专门组装Lys48-的结构基础 链接的PolyUb链(目标2)。UB信号是癌症治疗干预的有效靶点 FDA批准的针对蛋白酶体的药物目前延长了多发性骨髓瘤的生命 病人。对E1、CDC34和SCF如何协同作用控制细胞周期的分子理解 所提出的研究结果可能为新型癌症的开发提供平台 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.