Regulatory Mechanism of Cullin-RING Ubiquitin Ligases

摘要 泛素化，即泛素或泛素链的翻译后连接，控制着蛋白质的稳定性、相互作用 或真核细胞中许多关键调节蛋白的活性。因此，蛋白质的错误调节 泛素化可导致多种人类疾病，如代谢紊乱、癌症、肌肉和神经疾病， 退化泛素化过程的核心是E3连接酶，它将泛素和靶蛋白 蛋白质一起，并使泛素转移到其目标。我的实验室调查了 E3连接酶，称为Cullin-RING连接酶（CRL）。这些酶是模块化的蛋白质复合物，具有 一个共同的cullin支架和一个可互换的底物受体，为CRL募集特异性靶蛋白， 依赖的泛素化和随后的降解。人类细胞中存在七种cullin（Cul 1 -7），每一种都 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，以研究如何 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样的关键角色。鉴于 大量的底物受体竞争进入相同的cullin，我们目前的研究重点是 揭示不同CRL的细胞库是如何被控制的，以确保各种CRL的泛素化 在正确的时间使用基板。使用基于Cul 1的CRL 1，我们以前报道过CRL 1不断经历 组装和拆卸的循环，这允许Cul 1的快速回收和新CRL的及时形成 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， Cand 1，一种促进与其相关的底物受体交换的蛋白质交换因子 Cul 1核心。Cand 1活性的消除导致人细胞中CRL 1底物的降解受损， 多细胞生物的严重发育缺陷。在这个应用中，我们问， 其他CRL规范？Cand 2是人类细胞中Cand 1的同源物，在调节CRL中起什么作用？ 这种进化上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的动力学参数， Cand 1/2交互。我们将应用我们更新的定量免疫沉淀-质谱分析， 描述Cand 1和Cand 2对cullin相关蛋白质组的影响。我们将使用基因组编辑技术 CRISPR等技术来研究Cand 1/2的生物学作用，使用培养的人类细胞和模型 植物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 组装和活动，以帮助了解CRL网络在不同的细胞类型或在不断变化的细胞 环境我们在理解调节CRL的机制方面所做的努力将有助于剖析 这些E3连接酶在正常、患病和药物处理的细胞中，为预防， 诊断和治疗人类疾病。

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.