Novel mechanisms of ubiquitin signaling coordinating ribosome maturation and function

通过泛素化的细胞信号在真核生物的几乎所有生物过程中都发挥着关键作用。该反应分三步进行，涉及E3泛素连接酶的作用。此外，泛素化是可逆的和特定的蛋白酶，即去泛素酶(DUBS)，去泛素化蛋白底物。大量的泛素连接酶(&gt；500个基因)和DUBS(~100个基因)调控泛素介导的过程。虽然我们现在认识到泛素连接酶和DUBS的重要性，但它们的生物学功能和作用机制仍然不完全清楚。USP16(UBP-M)最初参与染色质相关过程的调节。然而，以前的发现提供了证据，证明该Dub也存在于细胞质中。因此，我们试图研究USP16核质转运是如何受到调控的。利用之前资助的NSERC拨款，我们证明了Usp16在所有细胞周期阶段都是主要的细胞质。我们鉴定了与Usp16细胞质定位有关的核输出信号(NES)。我们还挑战了先前确定的发现，并进一步表明，在DNA损伤后，这种复制不会在细胞核中积聚。因此，与流行的观点相反，我们的数据表明Usp16是一种细胞质，并提示该Dub在细胞质中具有新的功能。事实上，我们和其他人提出的证据表明，Usp16与核糖体相关，并调控它们的生物发生和功能。此外，我们确定了Usp16的几个翻译后修饰(包括泛素化和磷酸化)以及可能调节其功能的相互作用伙伴。假设：Usp16脱泛素酶在细胞质中发挥作用，协调核糖体成熟和其他细胞过程，这是正常细胞生长和增殖所必需的。该DUB以多种底物为靶标，受相互作用的伙伴和信号通路调节。具体目的：为了阐明Usp16在核糖体成熟和其他细胞过程中的作用，我们计划：1)确定Usp16相互作用的伙伴对40s核糖体成熟的重要性。2)研究Usp16翻译后修饰在调节40S核糖体成熟中的作用。3)鉴定细胞质中Usp16底物的全谱这些研究将使用模型细胞系和原代细胞来完成。RNAi和药物抑制剂将被用来确定调节Usp16功能的特定信号分子的需求。将进行Usp16和其他相关蛋白的各种突变体的表达，以确定它们对核糖体组成、成熟和功能的影响。这项研究不仅将为泛素系统在调节蛋白质合成等关键细胞过程中的重要性提供新的线索，而且将有助于我们进一步了解细胞信号转导的机制。

Cell signaling through ubiquitination plays critical roles in virtually all biological processes of eukaryotic organisms. This reaction occurs in three sequential steps involving the action of E3 ubiquitin ligases. Moreover, ubiquitination is reversible and specific proteases, i.e. deubiquitinases (DUBs), deubiquitinate protein substrates. A vast repertoire of ubiquitin ligases (>500 genes) and DUBs (~100 genes) regulate ubiquitin-mediated processes. Although, we now appreciate the importance of ubiquitin ligases and DUBs, their biological functions and mechanisms of action remain incompletely understood. USP16 (UBP-M) was initially implicated in the regulation of chromatin-associated processes. However, previous findings provided evidence that this DUB is also present in the cytoplasm. Thus, we sought to investigate how USP16 nucleocytoplasmic trafficking is regulated. With the previously funded NSERC grant, we demonstrated that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for USP16 cytoplasm localization. We also challenged previously established findings and further show that this DUB does not accumulate in the nucleus following DNA damage. Thus, in contrast to the prevailing view, our data indicate that USP16 is a cytoplasmic and suggest new functions of this DUB in the cytoplasm. Indeed, we and others present evidence that USP16 is associated with ribosomes and regulate their biogenesis and function. Moreover, we identified several post-translational modifications (including ubiquitination and phosphorylation) and interacting partners of USP16 that might regulate its function. HYPOTHESIS: USP16 deubiquitinase functions in the cytoplasm to orchestrate ribosome maturation and other cellular processes, and this is required for proper cell growth and proliferation. This DUB targets multiple substrates and is regulated by interacting partners and signaling pathways. SPECIFIC AIMS: To elucidate the roles of USP16 in ribosome maturation and other cellular process, we plan to: 1) Determine the importance of USP16-interacting partners for 40S ribosome maturation. 2) Investigate the roles of USP16 post-translational modifications in regulating 40S ribosome maturation. 3) Identify the full spectrum of USP16 substrates in the cytoplasm These studies will be accomplished using model cell lines as well as primary cells. RNAi and pharmacological inhibitors will be used to determine the requirement of specific signaling molecules in regulating USP16 function. Expression of various mutants of USP16 and other relevant proteins will be conducted to determine their effects on ribosome composition, maturation and function. This investigation will not only shed new light on the importance of the ubiquitin system in regulating critical cellular processes such as protein synthesis, but will also help in furthering our understanding of the mechanisms of cell signaling.