Characterising the molecular mode of Ras-mediated Cdc42 activation from a mechanical viewpoint

RAS是一组保守的小分子GTP酶，参与细胞的增殖、分化和存活。大约30%的癌症都有致癌的RAS突变，这一事实突显了RAS蛋白的生理重要性。由于RAS分子很小，其“光滑的”表面通常不容易与传统的药物设计方法兼容。因此，近年来的研究重点集中在针对Ras-分子下游的效应分子，如PI3K和MAPK。然而，抑制这些蛋白并没有产生人们所希望的成功--抑制一个效应器的作用微乎其微，同时抑制PI3K和MAPK会产生有害的副作用。此外，在致癌的Kras.G12D小鼠模型中，尽管观察到肿瘤易感性，但在分离的小鼠胚胎成纤维细胞(MEF)中，PI3K和ERK(MAPK)信号通路在生长因子刺激下发生衰减，与野生型MEF相似。因此，在生理条件下，PI3K或MAPK通路可能不是主要的致病信号通路，因此需要将焦点转移到另一条通路上。我们的重点放在一条导致CDC42激活的途径上(肌动蛋白细胞骨架重组中的一种小GTP酶积分)，这似乎也是RAS介导的组织培养细胞致癌转化和裸鼠系统肿瘤发展的必要条件。我们的目的是刻画和揭示RAS介导的CDC42激活的分子和力学模式。为此，该项目包括三个部分：(1)鉴定负责CDC42激活的RAS残基，(2)研究这些RAS残基如何促进CDC42激活的机制，以及(3)开发结合和掩蔽这些关键RAS残基的小分子以阻断CDC42的激活。通过揭示RAS与CDC42激活剂之间的相互作用表面并进一步了解CDC42是如何被激活的(通过准确地识别所需的关键残基)，可以形成未来潜在的新型抗癌药物的基础。

Ras belongs to a conserved group of small GTPases involved in cell proliferation, differentiation and survival. Physiological importance of Ras proteins is underlined by the fact that approximately 30% of all cancers have oncogenic Ras mutations. As a molecule Ras is small and its "smooth" surface is often not readily compatible with conventional drug design approaches. Therefore the focus in recent years has fallen onto specifically targeting the downstream effectors of Ras - molecules such as PI3K and MAPK. However inhibiting these proteins have not yielded the success that had been hoped - inhibition of one effector brings about minimal effect and simultaneous inhibition of PI3K and MAPK have resulted in deleterious side effects. Furthermore, in an oncogenic Kras.G12D mouse model, although tumor susceptibility was observed, attenuation of PI3K and ERK (MAPK) pathways occurs upon growth factor stimulation in the isolated mouse embryonic fibroblasts (MEFs), in a similar way as in the wildtype MEFs. Therefore, in physiological conditions, PI3K or MAPK pathway may not be the major causal signaling pathway.Therefore the focus needs to move onto another pathway. Our focus is set on a pathway which leads to Cdc42 activation (a small GTPase integral in actin cytoskeleton reassembly), which appears to also be a requirement for oncogenic Ras-mediated transformation of tissue culture cells and tumor development in the nude mouse system. Our aim is to characterise and reveal the molecular and mechanical mode of Ras-mediated Cdc42 activation. To this end, the project consists of three parts: (1) identification of Ras residues responsible for Cdc42 activation, (2) examination of the mechanism how these Ras residues facilitate Cdc42 activation and (3) development of small molecules that bind and mask these key Ras residues in order to block the Cdc42 activation.By revealing the interaction surface between Ras and Cdc42 activators and further understanding how Cdc42 is activated (by precisely identifying the critical residues required), the basis of potentially novel anti-cancer drugs for the future can be formed.