Novel mechanisms of ubiquitin signaling coordinating ribosome maturation and function

泛素信号协调核糖体成熟和功能的新机制

• 批准号: RGPIN-2021-04110
• 负责人: Affar, ElBachir
• 金额: $ 4.23万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742879
• 关键词: Novel; mechanisms; ubiquitin; signaling; coordinating

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.

通过泛素化的细胞信号传导在真核生物的几乎所有生物过程中都发挥着关键作用。该反应分三个连续步骤发生，涉及 E3 泛素连接酶的作用。此外，泛素化是可逆的、特异性的蛋白酶，即去泛素化酶（DUB），去泛素化蛋白质底物。大量泛素连接酶（> 500 个基因）和 DUB（约 100 个基因）调节泛素介导的过程。尽管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不完全清楚。 USP16 (UBP-M) 最初涉及染色质相关过程的调节。然而，之前的研究结果证明这种 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受到调节的。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主要位于细胞质中。我们确定了负责 USP16 细胞质定位的核输出信号 (NES)。我们还对之前的研究结果提出了质疑，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，与普遍观点相反，我们的数据表明 USP16 是细胞质的，并表明该 DUB 在细胞质中具有新功能。事实上，我们和其他人提供的证据表明 USP16 与核糖体相关并调节其生物发生和功能。此外，我们还发现了 USP16 的几种翻译后修饰（包括泛素化和磷酸化）和可能调节其功能的相互作用伙伴。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，这是细胞正常生长和增殖所必需的。该 DUB 靶向多种底物，并受到相互作用的伙伴和信号通路的调节。 具体目标：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 和药理学抑制剂将用于确定调节 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。这项研究不仅将揭示泛素系统在调节蛋白质合成等关键细胞过程中的重要性，还将有助于进一步了解细胞信号传导机制。