Regulation of cullin-RING ligases by Nedd8

描述(申请人提供)：人类基因组有可能编码约350种不同的泛素连接酶，这些酶基于cullin环催化核心，使cullin环连接酶(CRL)成为已知的最大的酶超家族之一。为了与CRL的数量保持一致，这个超家族的成员参与了细胞和生物生物学的许多方面的调节-从营养感知到昼夜节律的控制。基于它们非同寻常的多样性和对生物学的深远影响，我们必须了解这些酶是如何工作的，它们是如何被控制的，以及它们可能如何被操纵以造福人类健康。CRLS的活性受泛素样肽NeddS的调节，NeddS共价连接到cullin亚基上。所有的库林人都经历了NEDS的附着(“去附着”)和移除(“去附着”)的循环。经过NeddS修饰的cullins组装成活性CRL，而未经修饰的cullins可以隔离成具有隔离因子CAND1的非活性复合体。在这个应用中，我提出了三个具体的目标来评估新的假说，即NeddS的附着如何刺激CRLS的泛素连接酶活性(目标1)，以及剔除和去脱染周期如何与CRLS的组装和活性耦合(目标2)。最后，我建议区分库林细胞如何从CAND1中解放出来的不同模型，以便它们能够使完整的、有功能的CRL的组装成核(目标3)。这些目标将通过应用生化重建、基于荧光的蛋白质相互作用测量(动态和平衡)以及分析突变的CRL亚基在体内的组装和修饰状态来实现。确定这些酶的作用机制和调节机制将使我们能够更深入地了解它们对人类调节生物学的贡献，以及它们在维持疾病(其中几种已知，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，对这类酶的作用机制和调节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的活性来缓解疾病症状或阻止疾病的生化过程。

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.