Ubiquitin-like Protein Transfer Cascades

描述(由申请人提供)：泛素样蛋白(UBL)的共价连接，如泛素(Ub)、NEDD8和SUMO是真核蛋白质调控的主要形式。UBL修饰了大量蛋白质，以各种方式改变了它们的功能。UBL修饰会影响靶标的半衰期、亚细胞定位、酶活性或与蛋白质或DNA伙伴相互作用的能力。因此，UBL调控着许多生物过程，如细胞周期、信号转导、细胞凋亡、免疫反应、自噬和发育。UBL通路的缺陷与疾病广泛相关，包括癌症、发育障碍、高血压、神经退行性疾病和恶病质。我们建议将我们在UBL接合方面的专业知识扩展到两个最大的E3家族：Ring(在人类中预测的真正有趣的新基因-570)和Hect(与在人类中预测的E6AP C-Terminus-28同源)。在环E3中，最大的一类是模多亚单位库林环(CRL)家族。CRLS与不同的E2相继发挥作用，以修饰不同的靶点：首先，环域与NEDD8 E2结合，并通过NEDD8的自我修饰激活cullin亚基。然后，CRL绑定作为要传输到目标的Ub的源的Ub加载的E2。Hect E3利用一种不同的机制，其中Hect结构域催化半胱氨酸通过硫酯连接的中间体直接参与。首先，Hect结构域与硫酯连接的E_2~Ub复合体结合，Ub从E_2半胱氨酸转移到Hect结构域催化的半胱氨酸。最终，将Ub从Hect E3 Cys转移到目标或Ub Lys。我们提出了一个专注于结构生物学和生物化学的研究计划，以了解CRL E3(目标1)和Hect E3(目标2)的潜在功能机制。 公共卫生相关性：泛素样蛋白(UBL)结合调节许多生物过程，包括细胞分裂、免疫反应、发育和信号转导，UBL途径的缺陷广泛与癌症、神经退行性疾病、发育障碍、心脏病(如高血压)以及病毒和逆转录病毒感染有关。蛋白酶体抑制剂Bortezomib(VelcadeTM)最近被批准用于治疗多发性骨髓瘤，突显了针对泛素和UBL途径中的酶的治疗潜力，并强调了了解这些酶的详细机制和特异性的重要性。因此，我们预计，所提出的研究揭示的酶转移UBL的机制的知识将对许多人类疾病具有广泛的意义，就像对蛋白激酶的研究影响了我们对信号通路及其在疾病中的作用的知识一样。

DESCRIPTION (provided by applicant): Covalent attachment of ubiquitin-like proteins (UBLs) such as ubiquitin (Ub), NEDD8, and SUMO is a predominant form of eukaryotic protein regulation. UBLs modify a vast number of proteins, altering their functions in a variety of ways. UBL modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, UBLs regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in UBL pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on UBL conjugation to the two largest E3 families: RING (Really Interesting New Gene - 570 predicted in humans) and HECT (Homologous to E6AP C-Terminus - 28 predicted in humans). Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds a NEDD8 E2, and the cullin subunit is activated by self-modification with NEDD8. Then a CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. HECT E3s utilize a distinct mechanism, in which a HECT domain catalytic Cys participates directly via a thioester-linked intermediate. First, the HECT domain binds a thioester-linked E2~Ub complex, and Ub is transferred from the E2 Cys to the HECT domain catalytic Cys. Ultimately Ub is transferred from the HECT E3 Cys to a target or Ub Lys. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying functions of CRL E3s (Aim 1) and HECT E3s (Aim 2). PUBLIC HEALTH RELEVANCE: Ubiquitin-like protein (UBL) conjugation regulates many biological processes, including cell division, the immune response, development and signal transduction, and defects in UBL pathways have been widely associated with cancers, neurodegenerative disorders, developmental disorders, heart diseases (e.g., high blood pressure), and viral and retroviral infections. The recent approval of the proteasome inhibitor Bortezomib (VelcadeTM) for treatment of multiple myeloma underscores the therapeutic potential for targeting enzymes in the ubiquitin, and UBL, pathways, and highlights the importance of understanding the detailed mechanisms and specificities of these enzymes. Thus, we anticipate that knowledge of the mechanisms by which enzymes transfer UBLs as revealed by the proposed studies will be of broad significance to many human diseases, much like studies of protein kinases have influenced our knowledge of signaling pathways and their roles in diseases.