Regulation of cullin-RING ligases by Nedd8

Nedd8 对 cullin-RING 连接酶的调节

• 批准号: 8265620
• 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/8265620
• 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.

描述（由申请人提供）：人类细胞表达数十种SCF泛素连接酶。这些酶通过将泛素附着在细胞内蛋白质上来调节广泛的细胞和有机体生物学，这通常以26S蛋白酶体降解修饰后的蛋白质为高潮。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，关于它们是如何被监管的，还有很多东西需要学习。一种特殊的SCF， SCFCdc4，是整个cullin- RING连接酶（CRL）家族的原型，对它和其他SCF酶的研究为理解人类细胞中表达的数百种CRL提供了模板。在人类细胞中表达的数十种不同的SCF复合物中的每一种都具有不同的底物受体（例如，SCFCdc4中的Cdc4），这使得它能够结合一组独特的底物。底物受体，被称为F-box蛋白（fbp），与由Cul1和RING结构域亚基Rbx1/Roc1/Hrt1组成的共同催化核心竞争组装。据认为，fbp与Cul1-Rbx1模块处于动态平衡状态，这使得细胞能够调节其SCF复合物的曲目以满足需求。然而，目前尚不清楚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机制和监管的关键未解问题。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，我们将利用在目标1的工作过程中产生的荧光标记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特性，这种特性必然是存在于细胞内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复合物的动态。在目标3中开发的方法将使我们能够在任何给定时间获得细胞中SCF复合物库的快照，并监测该库如何随时间在未受干扰的细胞中或对药物或遗传操作的反应而变化。总之，本文提出的目标将对SCF复合物如何进行泛素结合以及SCF复合物的细胞库如何被控制产生重要的新见解。考虑到一个特定的CRL是重要的抗癌药物thalomid的靶点，而整个CRL家族是目前正在进行人体临床试验的第二种抗癌药物（MLN4924）的靶点，关于SCF的机制和调控的知识具有极好的潜力，可以转化为开发新的抗癌药物。