Investigation into the role of DUB3/USP17 in Ras signalling

我们发现了一种控制RAS通路的新的调控机制，RAS通路是一种主要的细胞信号通路，参与控制细胞的增殖和存活。具体地说，我们已经证明了一种新的酶(DUB-3)，我们第一次报道对细胞增殖有重要影响，通过它对RAS转换酶1(RCE1)的作用来实现这一点。为了使RAS被正确激活，它经历了许多翻译后修改，其中之一是由Rce1执行的。以前一直假设Rce1在细胞产生时是活跃的。我们现在已经证明，这种对蛋白酶的简化观点是不准确的，它的活性是由DUB-3控制的。我们已经在许多细胞类型中证明了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。这些作用包括标记目标蛋白以进行降解、激活、失活或在细胞内重新定位。在Rce1的特定情况下，我们的数据表明，DUB-3去除泛素会抑制Rce1的活性，从而削弱其处理RAS的能力。我们的结果还表明，DUB-3的作用是特异的，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，这些令人兴奋的结果突出了许多需要进一步研究的问题。在这项提案中，我们旨在评估DUB-3及其与RCE1的关系。目前，我们还不清楚Rce1上存在的泛素连接的类型，以及这如何促进或维持其活性。在这里，我们已经提出了一些实验，将迅速揭示这种控制的DUB-3的分子基础。此外，使用细胞的显微镜分析，我们将可视化Rce1的定位是如何在DUB-3的存在下受到影响的。最后，其他研究表明，RAS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包含与RAS中存在的相同的基序，并被Rce1识别和剪裁。因此，我们将研究Dub-3在这些其他途径上的作用，旨在深入了解分子水平上存在的无疑复杂的相互作用，这些相互作用控制着都由Rce1处理的具有不同角色的蛋白质。

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.