Regulation of cullin-RING ligases by Nedd8

描述（由申请人提供）：人细胞表达数十种SCF泛素连接酶。这些酶通过将泛素连接到细胞内蛋白质上来调节广泛的细胞和生物体生物学，这通常在26 S蛋白酶体手中的修饰蛋白质的降解中达到高潮。尽管SCF酶的重要性，我们仍然不知道它们是如何工作的。此外，关于如何对它们进行监管，仍有许多问题需要了解。一种特定的SCF，SCFCdc 4，是整个cullin-RING连接酶（CRL）家族的原型，并且对这种和其他SCF酶的研究提供了用于理解在人类细胞中表达的数百种CRL的模板。在人类细胞中表达的数十种不同SCF复合物中的每一种都具有不同的底物受体（例如，在SCFCdc 4的情况下为Cdc 4），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Cul 1和RING结构域亚基Rbx 1/Roc 1/Hrt 1组成的共同催化核心竞争组装。据认为，FBPs与Cul 1-Rbx 1模块处于动态平衡，这使得细胞能够调节其SCF复合物的库以满足需求。然而，不知道这是如何发生的，甚至不知道是否会发生。在这个应用程序中，我建议解决关键的SCF机制和监管悬而未决的问题。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测试我们的假设，即存在一个迄今未检测到的，底物引发的SCF的构象变化，我们最近的观察表明是泛素化反应的限速步骤。在第二个目标中，我们将利用目标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性质，这种相互作用必然是细胞内存在的任何动态平衡的基础。在第三个目标中，我建议开发一套新的方法的基础上定量多维质谱技术来监测动态的SCF复合物活细胞内。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复合物的库的快照，并监测该库在未受干扰的细胞中或响应药物或遗传操作时如何随时间变化。总之，这里提出的目标将产生重要的新的见解SCF复合物如何进行泛素结合和SCF复合物的细胞库是如何控制的。鉴于一种特定的CRL是重要抗癌药物thalomid的靶点，而整个CRL家族是目前正在人体临床试验中的第二种抗癌药物（MLN 4924）的靶点，因此有关SCF机制和调控的知识具有很好的潜力，可以转化为抗癌新药的开发。

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.