Regulatory Mechanism of Cullin-RING Ubiquitin Ligases
Cullin-RING 泛素连接酶的调控机制
基本信息
- 批准号:10027904
- 负责人:
- 金额:$ 37.61万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-09-01 至 2025-06-30
- 项目状态:未结题
- 来源:
- 关键词:ArabidopsisBiochemistryBiologicalBiological AssayBiophysicsCell modelCellsClustered Regularly Interspaced Short Palindromic RepeatsCullin ProteinsDefectDevelopmentDiagnosisDiseaseEnsureEnvironmentEnzymesEukaryotic CellFamilyFoundationsFunctional disorderGuanine Nucleotide Exchange FactorsHomologous GeneHumanIL27RA geneImmunoprecipitationImpairmentIn VitroKineticsLeadLigaseMalignant NeoplasmsMass Spectrum AnalysisMetabolic DiseasesMolecular GeneticsMuscular DystrophiesNerve DegenerationOrganismPathogenesisPerformancePharmaceutical PreparationsPlant ModelPlayPreventionPrevention strategyProcessProteinsProteomeProteomicsRecyclingReportingResearchRoleSystemTechniquesThalidomideTimeUbiquitinUbiquitinationUpdateWorkanticancer activitybasecell typegenetic regulatory proteingenome editinghuman diseaseinsightmathematical modelmembermuscle degenerationnovelnovel strategiesprotein complexreceptorrecruitscaffoldubiquitin ligaseubiquitin-protein ligase
项目摘要
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.
抽象的
泛素化是泛素或泛素链的翻译后附着,控制稳定性、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、肌肉和神经疾病
退化。泛素化过程的核心是 E3 连接酶,它将泛素和靶标
蛋白质结合在一起,并使泛素转移至其靶标。我的实验室调查了最大的家族
E3 连接酶,称为 Cullin-RING 连接酶 (CRL)。这些酶是模块化蛋白质复合物,具有
共同的cullin支架和可互换的底物受体,为CRL-招募特定的靶蛋白
依赖性泛素化和随后的降解。人类细胞中存在七种 cullin (Cul1-7),每一种
与不同组的底物受体相互作用,产生约 250 个 CRL。我们使用多种方法,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来研究如何
CRL 的工作原理、它们的活动如何受到调节以及它们在细胞和生物体中发挥哪些关键作用。鉴于
大量的底物受体竞争进入同一个cullin,我们目前的研究重点是
揭示如何控制不同 CRL 的细胞库以确保各种 CRL 泛素化
在正确的时间使用基质。使用基于 Cul1 的 CRL1,我们之前报道过 CRL1 不断经历
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。在这个高度动态的过程中,一个关键的角色是
Cand1,一种蛋白质交换因子,可促进与其相关的底物受体的交换
Cul1 核心。消除 Cand1 活性会导致人体细胞中 CRL1 底物的降解受损,
多细胞生物的严重发育缺陷。在此应用中,我们问,动态如何?
其他受监管的 CRL? Cand2(人类细胞中 Cand1 的同源物)在调节 CRL 中发挥什么作用?
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 和 CRL 的动力学参数
Cand1/2 相互作用。我们将应用更新的定量免疫沉淀-质谱分析来
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 等技术使用培养的人类细胞和模型来检查 Cand1/2 的生物学作用
植物拟南芥作为我们的实验系统。我们将继续开发 CRL 数学模型
组装和活动,以帮助了解不同细胞类型或变化的细胞中的 CRL 网络
环境。我们在理解 CRL 监管机制方面所做的努力将有助于剖析 CRL 的性能
这些 E3 连接酶存在于正常、患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。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Xing Liu其他文献
Xing Liu的其他文献
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{{ truncateString('Xing Liu', 18)}}的其他基金
Regulatory Mechanism of Cullin-RING Ubiquitin Ligases
Cullin-RING 泛素连接酶的调控机制
- 批准号:
10651863 - 财政年份:2020
- 资助金额:
$ 37.61万 - 项目类别:
Regulatory Mechanism of Cullin-RING Ubiquitin Ligases
Cullin-RING 泛素连接酶的调控机制
- 批准号:
10436384 - 财政年份:2020
- 资助金额:
$ 37.61万 - 项目类别:
Regulatory Mechanism of Cullin-RING Ubiquitin Ligases
Cullin-RING 泛素连接酶的调控机制
- 批准号:
10241504 - 财政年份:2020
- 资助金额:
$ 37.61万 - 项目类别:
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