Investigation into the role of DUB3/USP17 in Ras signalling

我们发现了一种新的调控Ras通路的调控机制，Ras通路是一种主要的细胞信号通路，参与细胞增殖和存活的控制。具体来说，我们已经证明了一种新的酶（DUB-3），我们首次报道对细胞增殖有重大影响，通过它对Ras转化酶1 （RCE1）的作用来实现这一点。为了使Ras正常激活，它经历了许多翻译后修饰，其中一个修饰是由RCE1完成的。以前，人们认为RCE1在细胞产生时是活跃的。我们现在已经证明，这种对蛋白酶的简单看法是不准确的，它的活性是由DUB-3控制的。我们已经在许多细胞类型中表明，RCE1是通过添加泛素来“标记”的。将泛素添加到蛋白质中有许多不同的效果，这取决于目标蛋白质的性质和泛素连接的特定性质。这些作用包括标记靶蛋白的降解、激活、失活或在细胞内重新定位。在RCE1的具体情况下，我们的数据表明，DUB-3去除泛素会抑制RCE1的活性，从而减弱其处理Ras的能力。我们的结果还表明DUB-3的作用是特异性的，因为其他去泛素化酶不能调节RCE1，并且DUB-3对增殖的作用依赖于RCE1。尽管有这样的见解，但这些令人兴奋的结果突出了许多需要进一步研究的问题。在本提案中，我们旨在评估DUB-3及其与RCE1的关系。目前，我们还不了解RCE1上存在的泛素连锁类型，以及它如何促进或维持其活性。在这里，我们提出了一些实验，这些实验将迅速揭示DUB-3控制的分子基础。此外，通过对细胞的显微镜分析，我们将看到DUB-3存在时RCE1的定位是如何受到影响的。最后，其他人已经表明Ras并不是唯一可以被RCE1修饰的蛋白质。事实上，许多关键的细胞信号蛋白家族都含有与Ras中相同的基序，并被RCE1识别和剪切。因此，我们将研究DUB-3对这些其他途径的影响，旨在深入了解存在于分子水平上的复杂相互作用，这些相互作用控制着具有不同作用的蛋白质，这些蛋白质都由RCE1处理。

We have uncovered a novel regulatory mechanism for the control of the Ras pathway, a major cell signalling pathway that is involved in the control of cell proliferation and survival. Specifically, we have shown that a novel enzyme (DUB-3), that we first reported to have a major impact on cell proliferation, achieves this via its action on Ras Converting Enzyme 1 (RCE1). In order for Ras to be activated properly, it undergoes a number of post-translational modifications, one of which is performed by RCE1. Previously it has been assumed that RCE1 is active upon its production by the cell. We have now shown that this simplistic view of the protease is inaccurate and that its activity is controlled by DUB-3. We have shown in a number of cell types that RCE1 is 'labelled' by the addition of ubiquitin. The addition of ubiquitin to proteins has many different effects, depending on the nature of the target protein and the specific nature of the ubiquitin linkage. These effects include labelling of the target protein for degradation, activation, inactivation or re-localisation within the cell. In the specific case of RCE1, our data has shown that removal of ubiquitin by DUB-3 inhibits the activity of RCE1 and therefore blunts its ability to process Ras. Our results also suggest that the action of DUB-3 is specific, in that other deubiquitinating enzymes cannot regulate RCE1 and that DUB-3 action upon proliferation is RCE1 dependant. Despite this insight, these exciting results highlight many questions which require further investigation. In this proposal we aim to evaluate DUB-3 and its relationship with RCE1. At this time we do not understand the type of ubiquitin linkage present on RCE1 and how this promotes or maintain its activity. Here we have presented a number of experiments that will rapidly uncover the molecular basis of this control by DUB-3. Furthermore, using microscopy analysis of cells we will visualise how RCE1 localisation is affected in the presence of DUB-3. Finally, others have shown that Ras is not the only protein that can be modified by RCE1. In fact, a number of key cell signalling protein families contain the same motif that is present in Ras and recognised and clipped by RCE1. Therefore, we will examine the effect of DUB-3 on these other pathways, aiming to gain insight into the undoubtedly complicated interplay that exists at the molecular level controlling proteins with varying roles that are all processed by RCE1.