Elucidation of the mechanism of SHP-2 phosphatase localisation and activity

在这项研究项目中，人类SHP-2蛋白的三维溶液结构，分子相互作用和结构域间的灵活性将在理解细胞信号传导和药物发现所需的分辨率水平上进行表征。我们专注于详细的机制，信号酶作为一种蛋白酪氨酸磷酸酶，其中突变已被发现导致努南综合征。这是最重要的磷酸酶靶点之一，全世界每1-2千例活产中就有1例发生突变，并且还直接涉及白血病和实体瘤。分子机制围绕两个SH 2结构域和附着的磷酸酶，其从底物蛋白质的酪氨酸残基上去除磷酸盐，并调节分子内和分子间相互作用的系统。磷酸酶结构域是控制细胞生长和分化的信号传导途径的正性转导子。已知参与疾病的突变通常会过度激活SHP 2并导致细胞转化，但确切的机制尚不清楚，因为它涉及局部和全局结构和灵活性的变化，因此真实的需要进一步研究以提供准确的诊断和设计特异性和定制的药物。像SHP-2这样的磷酸酶仍然是药物发现和分子分析的具有挑战性的目标，特别是在生理条件下解析其溶液结构和构象动力学方面，需要进一步的基础研究和技术发展，使其更适合实验研究，并在预测水平上揭示其机制。我们的分析依赖于一种称为核磁共振光谱的方法，该方法可用于检测SHP-2和配体分子中数千个原子核的独特信号。该方法提供了一个前所未有的水平的信息的形状，构象，运动和化学相互作用的蛋白质在三维空间和时间范围从皮秒到秒，并将产生有价值的见解，了解蛋白质在细胞中的行为和顺应性的新药物发现的方法。我们已经获得了有前途的光谱SHP 2的催化和调节串联SH 2域，并刚刚开始分配其NMR信号，以确定新的推定配体和映射前所未有的结合位点。我们现在处于最佳位置，可以帮助提供对其解决机制的全面理解，比任何其他人类PTP目标都要好。我们已经预测了一个独特的网站，可以直接SHP-2的质膜网站，并将使用自旋标签，双层和计算建模来定义和验证拟议的膜结合机制的性质。我们将研究与受体配体的相互作用，这些配体定位和改变SHP-2的信号活性，并正在探索全新的抑制剂。与我们的合作者一起，我们将为SHP-2开发一种更全面的分子机制，包括结构，功能动力学和化学维度，解释这种磷酸酶在生理环境中的行为，使我们能够更准确地操纵其在细胞和体内的行为。该项目将提供与脂质胶束和配体结合的蛋白质的溶液结构，这些配体负责定位和调节细胞中野生型和突变状态的活性，更深入地了解这些结合事件中灵活性的作用，以及设计用于体外和体内分析的新型抑制剂和转基因模型的合理基础，这些模型一起可以帮助释放SHP的治疗潜力，2，在磷酸酶超家族中具有更广泛的应用。

In this research project the three dimensional solution structures, molecular interactions and interdomain flexibility of the human SHP-2 protein will be characterized at levels of resolution needed for understanding cell signaling and informing drug discovery. We focus on the detailed mechanisms of a signalling enzyme which acts as a protein tyrosine phosphatase and in which mutations have been found to cause Noonan's syndrome. This is one of the most important phosphatase targets, with mutations afflicting 1 in 1-2 thousand live births worldwide, and has also been directly implicated in leukaemias and solid tumours. The molecular mechanism revolves around the two SH2 domain and the attached phosphatase enzyme, which removes phosphates from substrate proteins on tyrosine residues and regulated a system of intra- and inter-molecular interactions. The phosphatase domain is a positive transducer of signaling pathways which control cell growth and differentiation. Mutations known to be involved in disease generally hyperactivate SHP2 and lead to cell transformation, but the exact mechanism are unclear as it involves changes in local and global structure and flexibility, hence there is a real need for further research to inform accurate diagnosis and the design of specific and tailored agents. Phosphatase like SHP-2 remain challenging targets for drug discovery and molecular analysis, particularly in terms of resolving their solution structures and conformational dynamics under physiological conditions, warranting further fundamental research and technological developments to render them more amenable to experimental investigation and uncover their mechanisms at a predictive level. Our analysis relies on a method known as nuclear magnetic resonance spectroscopy, which can be used to detect a unique signal for the thousands of individual atomic nuclei in the SHP-2 and ligand molecules. The method provides an unprecedented level of information about the shape, conformation, motions and chemical interactivity of a protein in three dimensional space and over a range of timescales from picoseconds to seconds, and will yield a valuable insights for understanding the proteins behavious in cells and amenability for new drug discovery approaches. We have obtained promising spectra of SHP2's catalytic and regulatory tandem SH2 domains, and have just begun to assign its NMR signals, to identify novel putative ligands and map unprecedented binding sites. We are now best positioned to help provide a comprehensive understanding of its solution mechanisms, better than that which is available for any other human PTP target. We have predicted a unique site that could direct SHP-2 to plasma membrane sites, and will use spin labels, bilayers and computational modeling to define and validate the nature of the proposed membrane binding mechanism. We will study the interactions with receptor ligands that locallize and alter SHP-2's signaling activity, and are on the road to discover completely new classes of inhibitors. Together with our collaborators we will develop a much more comprehensive molecular mechanism for SHP-2 which includes structural, functional dynamical and chemical dimensions, explaining how this phosphatase behaves in physiological contexts, allowing us to more accurately manipulate its behaviour in cells and in vivo. This project will deliver solution structures of the protein bound to the lipid micelles and ligands responsible for localising and regulating activity of wild-type and mutated states in cells, a deeper understanding of the role of flexibility in these binding events, and a rational basis for designing novel inhibitors and transgenic models for in vitro and in vivo analysis which together could help to unlock the therapeutic potential of SHP-2 and has broader applications across phosphatase superfamily.