Deubiquitinases in Cell Cycle Control

细胞周期调控中的泛素酶 项目摘要 泛素信号转导几乎涉及细胞生理学的所有方面，并与衰老和疾病有关。 多聚泛素链与底物的共价结合触发了它们被蛋白酶体降解， 以及其他各种细胞结果。泛素化是通过泛素的酶级联反应进行的 活化剂（E1）、缀合物（E2）和连接酶（E3）。在正常的细胞周期中，泛素系统起着重要的作用。 在重塑蛋白质景观中起着重要和保守的作用。泛素底物由E3决定 连接酶和我们对泛素信号传导的许多理解都集中在同一性，底物和 E3的机制。然而，泛素化是可逆的，并且泛素通过催化作用从底物上除去， 称为DUB（去泛素化酶）的蛋白酶。尽管它们在塑造蛋白质组中起着关键作用， 已知DUB在细胞周期进程中的身份、底物和机制， E3对应物。然而，E3和DUBs的失调改变了细胞周期进程， 对基因组完整性的有害影响。此外，E3和DUB都可以在病理学中受到干扰， 癌症，有助于疾病的生物化学和表型特征。因此，定义身份，底物 DUBs在细胞周期中的作用和机制对于理解正常细胞增殖和基因组 稳定得到维护和协调。我们假设DUB对细胞周期进程至关重要， 染色体的稳定性，并同样重要，因为他们的E3对应。我们解决这个假设在三个 特定目标，联合收割机互补技术，并集中在DUBs在主要细胞周期中的作用 过渡。在目的1和2中，我们研究了Cezanne/OTUD 7 B，一种卵巢肿瘤家族去泛素化酶， 最近证实的是细胞周期调节和控制M到G1的转变。在目标1中，我们 使用蛋白质组学方法确定Cezanne的底物，定义DUB底物的机制 相互作用，以及塞尚在M/G1底物降解中的作用。在目标2中，我们将扩展这一点 分析以确定塞尚本身是如何调节的，无论是在其丰度和活性水平上，然后 确定这些调节系统如何影响其在细胞分裂中的作用。最后，在目标3中，我们确定 DUB在第二个主要细胞周期转换中的作用，G1/S。G1/S边界是增殖的主要障碍 在正常和癌细胞周期中，并且严重依赖于泛素信号传导。然而，人们对DUBs知之甚少 参与G1/S。我们将使用计算方法和功能丧失筛选来识别， 调查控制G1/S的DUB。总的来说，这项提案将填补细胞周期中的重大知识空白， 泛素和DUB领域，涉及DUB在增殖和基因组维持中的作用和机制。 我的实验室在解决这些问题方面具有独特的优势，我们之前的工作就说明了这一点，包括全球 分析泛素化网络，详细分析特定的泛素途径及其在细胞周期中的作用， 以及细胞周期DUBs的功能和酶学的测定。

DEUBIQUITINASES IN CELL CYCLE CONTROL Project Summary Ubiquitin signaling contributes to virtually all aspects of cell physiology and is implicated in aging and disease. The covalent conjugation of polyubiquitin chains onto substrates triggers their degradation by the proteasome, as well as various other cellular outcomes. Ubiquitination is carried out by an enzymatic cascade of ubiquitin activators (E1), conjugators (E2) and ligases (E3). During normal cell cycles the ubiquitin system plays an essential and conserved role in remodeling the protein landscape. Ubiquitin substrates are determined by E3 ligases and much of our understanding of ubiquitin signaling has focused on the identity, substrates and mechanisms of E3s. However, ubiquitination is reversible, and ubiquitin is removed from substrates by catalytic proteases termed DUBs (deubiquitinases). Despite their critical role in sculpting the proteome, much less is known about the identity, substrates and mechanisms of DUBs in cell cycle progression, when compared to their E3 counterparts. Nevertheless, dysregulation of both E3s and DUBs alters cell cycle progression and has deleterious effects on genome integrity. Moreover, both E3s and DUBs can be perturbed in pathologies such as cancer, contributing to the biochemical and phenotypic features of disease. Thus, defining the identity, substrates and mechanisms of DUBs in cell cycle is essential to understanding how normal cell proliferation and genome stability are maintained and coordinated. We hypothesize that DUBs are essential for cell cycle progression and chromosome stability and are equally important as their E3 counterparts. We address this hypothesis in three specific aims, that combine complementary techniques, and which focus on the role of DUBs in major cell cycle transitions. In Aims 1 and 2 we investigate Cezanne/OTUD7B, an ovarian tumor family deubiquitinase, that we recently demonstrated is cell cycle regulated and which controls the M to G1 transition. In Aim 1, we will determine substrates for Cezanne using proteomics approaches, define mechanisms of DUB-substrate interactions, and the role of Cezanne in the degradation of substrates at M/G1. In Aim 2, we will expand this analysis to determine how Cezanne is itself regulated, both at the level of its abundance and activity, and then determine how these regulatory systems influence its role in cell division. Finally, in Aim 3, we determine the role of DUBs in a second major cell cycle transition, G1/S. The G1/S boundary is a major barrier to proliferation in normal and cancer cell cycles and relies heavily on ubiuqitin signaling. However, little is known about DUBs involved in G1/S. We will use computational approaches and loss-of-function screens, to identify and then investigate DUBs that control G1/S. Collectively, this proposal will fill significant knowledge gaps in the cell cycle, ubiquitin and DUB fields, related to roles and mechanisms of DUBs in proliferation and genome maintenance. My lab is uniquely positioned to address these questions, illustrated by our prior work, that includes global analysis of ubiquitination networks, detailed analysis of specific ubiquitin pathways and their role in cell cycle, and determination of the function and enzymology of cell cycle DUBs.