Regulation of cullin-RING ligases by Nedd8

Nedd8 对 cullin-RING 连接酶的调节

• 批准号: 8109197
• 负责人: RAYMOND J DESHAIES
• 金额: $ 25.6万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8109197
• 关键词: 26S proteasome; Adaptor Signaling Protein; Address; Antineoplastic Agents; Behavior; Binding; Binding Proteins; Biological Assay; Biological Process; Biology; Catalytic Domain; Cell division; Cells; Circadian Rhythms; Complex; Coupled; Cullin Proteins; Cycloheximide; Development; Dissociation; Energy Transfer; Enzymes; Equilibrium; F-Box Proteins; Family; Family Study; Fluorescence; Foundations; Funding; Glucose; Grant; Hand; Human; Human Biology; Investigation; Kinetics; Knowledge; Label; Learning; Life; Ligase; MG132; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methionine; Methods; Modeling; Monitor; Mutation; Nature; Optical Methods; Perception; Pharmaceutical Preparations; Phase; Plant Roots; Play; Property; Protein Binding; Proteins; Reaction; Regulation; Role; SKP Cullin F-Box Protein Ligases; Substrate Specificity; Techniques; Testing; Time; Translating; Ubiquitin; Ubiquitination; Work; base; fighting; genetic manipulation; in vivo; inhibitor/antagonist; insight; meetings; member; mutant; novel; novel therapeutics; operation; prevent; protein degradation; prototype; receptor; research clinical testing; research study; response; success; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): Human cells express dozens of SCF ubiquitin ligases. These enzymes regulate a broad swath of cell and organismal biology by attaching ubiquitin to intracellular proteins, which often culminates in degradation of the modified protein at the hands of the 26S proteasome. Despite the importance of SCF enzymes, there is much that we still do not know about how they work. In addition, there remains much to learn about how they are regulated. One particular SCF, SCFCdc4, is the archetype for the entire cullin- RING ligase (CRL) family and studies on this and other SCF enzymes have provided a template for understanding the hundreds of CRLs expressed in human cells. Each of the dozens of different SCF complexes expressed in human cells has a distinct substrate receptor (e.g. Cdc4 in the case of SCFCdc4) which enables it to bind a unique set of substrates. The substrate receptors, known as F-box proteins (FBPs) compete for assembly with a common catalytic core composed of Cul1 and the RING domain subunit Rbx1/Roc1/Hrt1. It is thought that the FBPs are in dynamic equilibrium with the Cul1-Rbx1 module, which enables the cell to modulate its repertoire of SCF complexes to meet demand. However it is not known how or even whether this occurs. In this application, I propose to address key unanswered questions about SCF mechanism and regulation. The proposed work is divided into three specific aims. In the first aim we will use optical methods to test our hypothesis that there exists a heretofore undetected, substrate-triggered conformational change in SCF that our recent observations suggest is the rate-limiting step of the ubiquitination reaction. In the second aim, we will exploit fluorescently-tagged SCF subunits generated during the course of work on aim 1 to investigate the dynamic nature of the interaction between the substrate binding module and the catalytic core. These experiments will reveal the intrinsic properties of this interaction that must underlie any dynamic equilibrium that exists within cells. In the third aim, I propose to develop a set of novel methods based on quantitative multidimensional mass spectrometry techniques to monitor dynamics of SCF complexes within living cells. The methods developed in aim 3 will enable us to get a snapshot of the repertoire of SCF complexes in a cell at any given time and monitor how that repertoire changes over time in unperturbed cells or in response to drugs or genetic manipulations. Together, the aims proposed here will yield important new insights into how SCF complexes carry out ubiquitin conjugation and how the cellular repertoire of SCF complexes is controlled. Given that one particular CRL is the target of the important anti-cancer drug thalomid and the entire family of CRLs is targeted by a second anti-cancer drug (MLN4924) that is currently in human clinical testing, knowledge gained about the mechanism and regulation of SCF has excellent potential to be translated into development of new drugs to fight cancer. PUBLIC HEALTH RELEVANCE: Cullin-RING ubiquitin ligases (CRLs) comprise a family of enzymes that play major roles in human biology. Mutations in CRL components such as Fbw7/hCdc4 are the root cause of a significant fraction of human cancers and one particular CRL, CRL4CRBN is the target of the important anti-cancer drug thalomid. My lab co-discovered SCFCdc4, which is the archetype for all CRL enzymes. We have studied this enzyme in depth to provide a template for understanding how it and other CRLs work and how their activities are controlled. In this application I propose new investigations into how SCFCdc4 and other SCF enzymes carry out their core function of transferring ubiquitin to other proteins. I also propose experiments to investigate the mechanisms that enable cells to maintain a properly balanced repertoire of dozens of different SCF complexes. Given prior successes in developing drugs to target CRL4CRBN and the entire CRL family, the work proposed here has excellent potential to serve as a foundation for the development of new therapeutics.

描述(申请人提供)：人类细胞表达数十种干细胞因子泛素连接酶。这些酶通过将泛素附着到细胞内的蛋白质上来调节广泛的细胞和生物生物学，这通常会导致26S蛋白酶体手中修饰的蛋白质降解。尽管SCF酶很重要，但我们仍然不知道它们是如何工作的。此外，关于它们是如何受到监管的，还有很多需要了解的地方。一个特殊的SCF，SCFCDc4，是整个剔除环连合酶(CRL)家族的原型，对该酶和其他SCF酶的研究为理解在人类细胞中表达的数百个CRL提供了模板。在人类细胞中表达的几十种不同的SCF复合体中的每一种都有不同的底物受体(例如SCFCDC4)，这使得它能够结合一组独特的底物。底物受体，称为F-box蛋白(FBPs)，与由cul1和环结构域亚基Rbx1/Roc1/Hrt1组成的共同催化核心竞争组装。FBP与Cul1-Rbx1模块处于动态平衡状态，使细胞能够调节其SCF复合体以满足需求。然而，目前还不知道这种情况是如何发生的，甚至是否会发生。在本申请中，我建议解决关于SCF机制和监管的关键悬而未决的问题。拟议的工作分为三个具体目标。在第一个目标中，我们将使用光学方法来验证我们的假设，即在SCF中存在迄今未被检测到的底物触发的构象变化，我们最近的观察表明，这是泛素化反应的限速步骤。在第二个目标中，我们将利用在Aim 1的工作过程中产生的荧光标记的SCF亚基来研究底物结合模块和催化核心之间相互作用的动态性质。这些实验将揭示这种相互作用的内在属性，这些属性肯定是细胞内存在的任何动态平衡的基础。在第三个目标中，我建议开发一套基于定量多维质谱技术的新方法来监测活细胞内SCF复合体的动力学。在AIM 3中开发的方法将使我们能够获得细胞中SCF复合体在任何给定时间的快照，并监测该复合体如何在未受干扰的细胞中随时间变化或对药物或基因操作的反应。总之，这里提出的目标将对SCF复合体如何进行泛素结合以及SCF复合体的细胞谱如何控制产生重要的新见解。鉴于一种特定的CRL是重要的抗癌药物Thalomid的靶标，而CRL的整个家族都是另一种抗癌药物(MLN4924)的靶标，该药物目前正在进行人体临床试验，因此关于SCF机制和调控的知识具有极大的潜力转化为抗癌新药的开发。 与公共卫生相关：库林环泛素连接酶(CRL)由一系列在人类生物学中发挥重要作用的酶组成。CRL成分的突变，如Fbw7/hCDC4，是相当一部分人类癌症的根本原因，其中一种特殊的CRL，CRL4CRBN是重要的抗癌药物泰罗米特的靶点。我的实验室共同发现了SCFCDC4，它是所有CRL酶的原型。我们深入研究了这种酶，为了解它和其他CRL的工作原理以及它们的活性是如何控制的提供了一个模板。在这项应用中，我提议对SCFCDC4和其他SCF酶如何执行将泛素转移到其他蛋白质的核心功能进行新的研究。我还建议进行实验，以研究使细胞能够维持数十种不同SCF复合体的适当平衡的机制。鉴于以前针对CRL4CRBN和整个CRL家族的药物开发的成功，这里提出的工作具有极大的潜力，可以作为新疗法开发的基础。