Regulation of cullin-RING ligases by Nedd8

描述（由申请人提供）：人类基因组有可能编码约350种基于cullin-RING催化核心的不同泛素连接酶，使cullin-RING连接酶（CRLs）成为已知最大的酶超家族之一。与大量的crl保持一致，这个超家族的成员参与调节细胞和有机体生物学的许多方面-从营养感知到昼夜节律的控制。基于它们非凡的多样性和对生物学的深远影响，重要的是我们要了解这些酶是如何工作的，它们是如何被控制的，以及如何为了人类健康而操纵它们。CRLs的活性受泛素样肽（NeddS）的调控，该肽共价附着在cullin亚基上。所有的cullins都经历了附着（“去角质”）和去除（“去角质”）的循环。经NeddS修饰的Cullins被组装成活性的CRLs，而未经修饰的Cullins可以通过一种名为CAND1的隔离因子被隔离成无活性的复合物。在本申请中，我提出了三个特定目标来评估新的假设，即NeddS的附着如何刺激crl的泛素连接酶活性（目的1），以及cullin类甲酰基化和去甲酰基化的周期如何与crl的组装和活性耦合（目的2）。最后，我建议区分cullins如何从CAND1中解放出来的不同模型，以便它们能够形成完整的、功能性CRL的组装（目标3）。这些目标将通过应用生物化学重组、基于荧光的蛋白质相互作用测量（动态和平衡）以及体内突变CRL亚基的组装和修饰状态分析的组合来实现。确定这些酶的作用和调控机制将使我们能够更深入地了解它们对人类调控生物学的贡献，以及它们在维持疾病（其中几种已知的疾病，包括癌症和炎症性疾病）中的作用，这些疾病的进展依赖于一个或多个CRL的功能。此外，对这类酶的作用和调控机制的详细了解可能有助于开发基于小分子的治疗策略，以调节crl的活性，以减轻疾病症状或阻止疾病基础的生化过程。

DESCRIPTION (provided by applicant): The human genome has the potential to encode ~350 different ubiquitin ligase enzymes that are based on a cullin-RING catalytic core, making the cullin-RING ligases (CRLs) one of the largest known superfamilies of enzymes. In keeping with the large number of CRLs, members of this superfamily have been implicated in regulating many aspects of cell and organismal biology - ranging from nutrient sensing to control of circadian rhythms. Based on their extraordinary diversity and profound impact on biology, it is important that we understand how these enzymes work, how they are controlled, and how they might be manipulated for the benefit of human health. The activity of CRLs is regulated by the ubiquitin-like peptide, NeddS, which is covalently attached to the cullin subunit. All cullins are subjected to cycles of attachment ('neddylation') and removal ('deneddylation') of NeddS. Cullins modified by NeddS are assembled into active CRLs, whereas unmodified cullins can become sequestered into inactive complexes with a sequestration factor named CAND1. In this application, I propose three Specific Aims to evaluate novel hypotheses for how attachment of NeddS stimulates the ubiquitin ligase activity of CRLs (Aim 1), and how the cycles of cullin neddylation and deneddylation are coupled to the assembly and activity of CRLs (Aim 2). Finally, I propose to discriminate between different models for how cullins are emancipated from CAND1 so that they can nucleate assembly of an intact, functional CRL (Aim 3). These Aims will be pursued by applying a combination of biochemical reconstitution, fluorescence-based measurement of protein interactions (both dynamically and at equilibrium), and analysis of the assembly and modification state of mutant CRL subunits in vivo. Defining mechanisms of action and regulation for these enzymes will enable us to understand in greater depth their contribution to human regulatory biology as well their role in sustaining diseases (of which several are known, including cancers and inflammatory diseases) whose progression relies on the function of one or more CRL. Moreover, detailed knowledge of the mechanism of action and regulation of this class of enzymes may assist in the development of small molecule-based therapeutic strategies for modulating the activity of CRLs to alleviate symptoms of disease or stem biochemical processes that underlie disease.