Ubiquitin-Conjugating Enzymes − Elucidating the Regulatory Mechanisms of Ubiquitin Chain Assembly

泛素结合酶 â 阐明泛素链组装的调控机制

• 批准号: 462017488
• 负责人: Professor Dr. Marco Trujillo Linke
• 金额: --
• 依托单位: Lehrstuhl für Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/462017488?language=en
• 关键词: Ubiquitin; Conjugating; Enzymes; Elucidating; Regulatory

Ubiquitination is a central process intricately involved in the regulation of most cellular processes. The E2 ubiquitin-conjugating enzymes (UBCs) are key mediators of ubiquitin chain assembly by coordinating a multitude of interactions that include ubiquitin itself, the E1 ubiquitin-activating enzyme and an E3 ubiquitin ligase. Moreover, they are largely responsible for the type of chain built, endowing the modified protein with different fates, including changes in activity, localization, as well as degradation. Ubiquitin attachment is responsive to cellular cues, and orchestrated by an intertwined cross-talk with kinase-mediated signaling pathways. However, in spite of the E2’s central importance in ubiquitination, there is scarce information on mechanisms regulating their activity. UBC35 and its close homologue UBC36, are the main sources of Lys63-linked ubiquitin chains in Arabidopsis, which is the second most abundant type of ubiquitin chain. Lys63-linked ubiquitin chains play key roles in the plasma membrane to vacuole transport of integral membrane proteins, by acting as a signal that coordinates various steps of vesicle traffic. By doing so, it contributes to the regulation of protein levels at the plasma membrane, and to the general maintenance of cellular homeostasis. We showed that the pairing between UBC35 and the E3 ligase PUB22 is enhanced in response to the activation of the immune response. UBC35, and the closely related UBC36, are potentially phosphorylated in various residues located on surfaces known to participate in the interaction with the E1 and the E3. Further, UBC35-interacting UEV1a-UEV1d are phosphorylated in N-terminal residues conserved in UBC35/36. Thus, the multitude of interactions and modification converging on the UBC35/36 duo, are indicative of a multi-tiered regulation and reflect their pivotal function as a central signaling hub through the generation of Lys63-linked chains. In this project, we aim to elucidate the mechanisms regulating Lys63-linked chain assembly by (i) confirming and identifying phosphorylation sites on UBC35/36, (ii) dissecting the impact of phosphorylation on ubiquitination activity, as well as on the interaction with known reaction partners, including ubiquitin-activating enzyme (E1), UEVs and E3s. In addition, (iii) we aim to identify new interacting proteins with a focus on kinases. (iv) Finally, we will functionally characterize the relationship of UEVs and UBC35/36 and analyse their subcellular localization. The expected outcome is to understand how cellular signaling impinges of the assembly of Lys63-linked chains by UBC35/36, enabling cells to react to challenges such as infection, as well as intrinsic cues during physiological processes.

泛素化是一个复杂的中央过程，参与大多数细胞过程的调节。E2泛素结合酶（UBC）是泛素链组装的关键介质，通过协调多种相互作用，包括泛素本身，E1泛素激活酶和E3泛素连接酶。此外，它们在很大程度上决定了所构建的链的类型，赋予修饰的蛋白质不同的命运，包括活性、定位以及降解的变化。泛素附着对细胞信号有反应，并通过与激酶介导的信号通路的相互交织的串扰来协调。然而，尽管E2的核心重要性，在泛素化，有很少的信息调节其活动的机制。UBC 35及其同源物UBC 36是拟南芥中Lys 63连接的泛素链的主要来源，其是第二丰富的泛素链类型。Lys 63连接的泛素链在质膜到液泡的膜蛋白运输中起着关键作用，作为一个信号，协调囊泡运输的各个步骤。通过这样做，它有助于调节质膜上的蛋白质水平，并有助于细胞内稳态的一般维持。我们发现，UBC 35和E3连接酶PUB 22之间的配对在免疫应答的激活中增强。UBC 35和密切相关的UBC 36在位于已知参与与E1和E3相互作用的表面上的各种残基中潜在地磷酸化。此外，UBC 35-相互作用UEV 1a-UEV 1d在UBC 35/36中保守的N-末端残基中被磷酸化。因此，聚集在UBC 35/36二聚体上的大量相互作用和修饰指示多层调节，并反映了它们作为通过产生Lys 63连接的链的中央信号传导枢纽的关键功能。在这个项目中，我们的目标是阐明调节Lys 63连接的链组装的机制，（i）确认和鉴定磷酸化位点的UBC 35/36，（ii）解剖的影响，磷酸化的泛素化活性，以及与已知的反应伙伴，包括泛素激活酶（E1），UEVs和E3的相互作用。此外，（iii）我们的目标是确定新的相互作用的蛋白质，重点是激酶。(iv)最后，我们将在功能上描述UEVs和UBC 35/36的关系，并分析它们的亚细胞定位。预期的结果是了解细胞信号如何影响UBC 35/36的Lys 63连接链的组装，使细胞能够对感染等挑战以及生理过程中的内在线索做出反应。