Regulation of cullin-RING ligases by Nedd8

Nedd8 对 cullin-RING 连接酶的调节

• 批准号: 8460158
• 负责人: RAYMOND J DESHAIES
• 金额: $ 24.7万
• 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/8460158
• 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到细胞内蛋白质上来调节广泛的细胞和生物体生物学，这通常最终导致修饰的蛋白质在 26S 蛋白酶体手中降解。尽管 SCF 酶很重要，但我们仍然对它们的工作原理知之甚少。此外，关于它们的监管方式还有很多东西需要了解。一种特殊的 SCF SCFCdc4 是整个 cullin-RING 连接酶 (CRL) 家族的原型，对此酶和其他 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。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），这使其能够结合一组独特的底物。被称为 F-box 蛋白 (FBP) 的底物受体与由 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，这使得细胞能够调节其 SCF 复合物库以满足需求。然而，尚不清楚这种情况是如何发生的，甚至是否会发生。在此申请中，我建议解决有关供应链金融机制和监管的关键未解答问题。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检验我们的假设，即 SCF 中存在迄今为止未被检测到的、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 的工作过程中生成的荧光标记的 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特性，这种相互作用必然是细胞内存在的任何动态平衡的基础。第三个目标是，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。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，并监测该库在未受干扰的细胞中如何随时间变化或对药物或基因操作的反应。总之，这里提出的目标将为 SCF 复合物如何进行泛素缀合以及 SCF 复合物的细胞库如何控制提供重要的新见解。鉴于一种特定的 CRL 是重要的抗癌药物沙洛米德的靶点，而整个 CRL 家族是目前正在进行人体临床测试的第二种抗癌药物 (MLN4924) 的靶点，因此获得的有关 SCF 机制和调节的知识具有极好的潜力，可以转化为抗癌新药的开发。