Novel mechanisms of ubiquitin signaling coordinating ribosome maturation and function

通过泛素化的细胞信号传导在真核生物几乎所有的生物过程中起着至关重要的作用。这个反应发生在三个连续的步骤中，涉及E3泛素连接酶的作用。此外，泛素化是可逆的，特异性蛋白酶，即去泛素酶（DUBs），去泛素化蛋白质底物。大量的泛素连接酶（约500个基因）和dub（约100个基因）调节着泛素介导的过程。虽然我们现在认识到泛素连接酶和dub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控。然而，先前的研究结果表明，这种DUB也存在于细胞质中。因此，我们试图研究USP16核细胞质运输是如何被调节的。在先前资助的NSERC资助下，我们证明了USP16在所有细胞周期阶段主要是细胞质。我们确定了负责USP16细胞质定位的核输出信号（NES）。我们还挑战了先前建立的发现，并进一步表明这种DUB不会在DNA损伤后在细胞核中积累。因此，与主流观点相反，我们的数据表明USP16是细胞质，并提示该DUB在细胞质中的新功能。事实上，我们和其他人提出的证据表明，USP16与核糖体有关，并调节其生物发生和功能。此外，我们还发现了几种可能调节USP16功能的翻译后修饰（包括泛素化和磷酸化）和相互作用伙伴。假设：USP16去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。这种DUB靶向多种底物，并受相互作用的伙伴和信号通路的调节。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相互作用伙伴对40S核糖体成熟的重要性。2)研究USP16翻译后修饰在调控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和药理学抑制剂将用于确定特定信号分子在调节USP16功能中的需求。我们将对USP16和其他相关蛋白的各种突变体进行表达，以确定它们对核糖体组成、成熟和功能的影响。这项研究不仅将揭示泛素系统在调节关键细胞过程（如蛋白质合成）中的重要性，而且还将有助于进一步了解细胞信号传导机制。

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.