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种不同的基于cullin-RING催化核心的泛素连接酶，使cullin-RING连接酶（CRL）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生物体生物学的许多方面-从营养感测到昼夜节律的控制。基于它们非凡的多样性和对生物学的深远影响，我们必须了解这些酶如何工作，如何控制它们，以及如何操纵它们以造福人类健康。CRL的活性由泛素样肽NeddS调节，NeddS共价连接到cullin亚基。所有cullin都经历NeddS的连接（“neddylation”）和去除（“deneddylation”）循环。被NeddS修饰的cullin被组装成活性CRL，而未修饰的cullin可以被称为CAND 1的螯合因子螯合成非活性复合物。在这个应用程序中，我提出了三个具体的目标，以评估新的假设如何连接NeddS刺激泛素连接酶活性的CRL（目的1），以及如何循环的cullin neddylation和deneddylation耦合到组装和活性的CRL（目的2）。最后，我建议区分不同的模型，cullin是如何从CAND 1解放出来，使他们能够有核组装一个完整的，功能性的CRL（目标3）。这些目标将通过应用生物化学重建，基于荧光的蛋白质相互作用的测量（动态和平衡），以及突变CRL亚基在体内的组装和修饰状态的分析的组合来实现。定义这些酶的作用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（其中一些是已知的，包括癌症和炎性疾病）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，这类酶的作用和调节机制的详细知识可能有助于开发基于小分子的治疗策略，用于调节CRL的活性，以减轻疾病症状或引起疾病的生化过程。

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.