Regulatory Mechanism of Cullin-RING Ubiquitin Ligases

摘要 泛素化，泛素或泛素链的翻译后连接，控制着稳定性、相互作用 或真核细胞中众多关键调控蛋白的活性。因此，蛋白质的失调 泛素化可导致多种人类疾病，如代谢紊乱、癌症、肌肉和神经 退化。泛素化过程的核心是E3连接酶，它带来泛素和靶标 蛋白质结合在一起，并使泛素能够转移到它的目标。我的实验室调查了最大的家族 E3连接酶，称为库林环连接酶(CRL)。这些酶是模块化的蛋白质复合体，具有 常见的cullin支架和一种可互换的底物受体，为CRL招募特定的靶蛋白- 依赖泛素化和随后的降解。人类细胞中存在7个库林(cul1-7)，每个库林 与不同的底物受体相互作用，产生约250个CRL。我们使用各种方法，包括 生物化学、生物物理学、分子遗传学、定量蛋白质组学和数学建模，以研究 CRL的工作原理，它们的活动是如何被调节的，以及它们在细胞和生物体中扮演着什么关键角色。考虑到 大量的底物受体竞争获得相同的cullin，我们目前的研究重点是 揭示不同CRL的细胞库如何受到控制，以确保各种CRL的泛素化 在正确的时间进行衬底。使用基于cul1的CRL1，我们之前报告了CRL1不断地经历 组装和拆卸循环，允许快速回收cul1并及时形成新的CRL 当它们的目标蛋白出现并需要泛素化时。在这一高度动态的过程中，一个关键的参与者是 Cand1，一种蛋白质交换因子，促进与其相关的底物受体的交换 CUL1核心。消除Cand1活性会导致CRL1底物在人类细胞中的降解受损 多细胞生物体的严重发育缺陷。在此应用程序中，我们问， 其他CRL受监管吗？Cand2是人类细胞中Cand1的同系物，在调节CRL方面扮演什么角色？ 这种进化保守的动态交换机制为CRL系统提供了什么优势？ 为了回答这些问题，我们将使用体外生物物理测定来量化CRL和CRL的动力学参数 Cand1/2交互作用。我们将把我们最新的定量免疫沉淀-质谱分析应用于 描述Cand1和Cand2对cullin相关蛋白质组的影响。我们将采用基因组编辑 CRISPR等技术，利用培养的人类细胞和模型检测Cand1/2的生物学作用 种植拟南芥作为我们的实验系统。我们将继续开发CRL的数学模型 组装和活性，以帮助了解不同细胞类型或变化的细胞中的CRL网络 环境。我们在了解CRL监管机制方面的努力将有助于剖析 这些E3连接酶在正常、疾病和药物处理的细胞中，为预防提供了新的见解， 人类疾病的诊断和治疗。

ABSTRACT Ubiquitination, the post-translational attachment of ubiquitin or ubiquitin chains, controls the stability, interaction or activity of numerous key regulatory proteins in eukaryotic cells. Consequently, misregulation in protein ubiquitination can result in various human diseases, such as metabolic disorders, cancers, muscle and nerve degeneration. At the core of the ubiquitination process is the E3 ligase, which brings ubiquitin and the target protein together, and enables the transfer of the ubiquitin to its target. My lab investigates the largest family of E3 ligases, known as Cullin-RING ligases (CRLs). These enzymes are modular protein complexes, featuring a common cullin scaffold and an interchangeable substrate receptor that recruits specific target proteins for CRL- dependent ubiquitination and subsequent degradation. Seven cullins (Cul1-7) exist in human cells, each of which interacts with different sets of substrate receptors, yielding ~250 CRLs. We use a variety of approaches including biochemistry, biophysics, molecular genetics, quantitative proteomics, and mathematical modeling to study how CRLs work, how their activities are regulated, and what critical roles they play in cells and organisms. Given that a large number of substrate receptors compete for access to the same cullin, our current research focus is to uncover how the cellular repertoire of diverse CRLs is controlled to ensure ubiquitination of various CRL substrates at the right time. Using Cul1 based CRL1, we previously reported that CRL1s constantly undergo cycles of assembly and disassembly, which allows rapid recycling of Cul1 and timely formation of new CRLs when their target proteins emerge and demand ubiquitination. A crucial player in this highly dynamic process is Cand1, a protein exchange factor that promotes the exchange of substrate receptors associated with the same Cul1 core. Eliminating the Cand1 activity leads to impaired degradation of CRL1 substrates in human cells and severe developmental defects in multicellular organisms. In this application, we ask, how are the dynamics of other CRLs regulated? What role does Cand2, a homologue of Cand1 in human cells, play in regulating CRLs? What advantage does this evolutionarily conserved dynamic exchange mechanism provide for the CRL system? To answer these questions, we will use in vitro biophysical assays to quantify kinetic parameters for CRL and Cand1/2 interactions. We will apply our updated quantitative immunoprecipitation-mass spectrometry assay to characterize the impact of Cand1 and Cand2 on the cullin-associated proteome. We will employ genome-editing techniques such as CRISPR to examine the biological role of Cand1/2, using cultured human cells and the model plant Arabidopsis as our experimental systems. We will continue developing our mathematical model of CRL assembly and activity, to help understand the CRL network in different cell types or under changing cellular environment. Our efforts in understanding mechanisms regulating CRLs will help dissect the performance of these E3 ligases in normal, diseased, and drug treated cells, providing novel insights for the prevention, diagnosis, and treatment of human diseases.