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

阐明 SHP-2 磷酸酶定位和活性的机制

• 批准号: BB/I013865/1
• 负责人: Michael Overduin
• 金额: $ 49.01万
• 依托单位: University of Birmingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI013865%2F1
• 关键词: Elucidation; mechanism; SHP; phosphatase; localisation

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.

在该研究项目中，人类 SHP-2 蛋白的三维解决方案结构、分子相互作用和域间灵活性将以理解细胞信号传导和为药物发现提供信息所需的分辨率水平进行表征。我们重点研究信号酶的详细机制，该信号酶充当蛋白质酪氨酸磷酸酶，并且已发现其中的突变会导致努南综合症。这是最重要的磷酸酶靶点之一，全世界每 1-2000 名活产儿中就有 1 人患有这种突变，并且也与白血病和实体瘤直接相关。分子机制围绕两个 SH2 结构域和附着的磷酸酶，该酶可去除底物蛋白酪氨酸残基上的磷酸盐，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通路的正转导器。已知与疾病有关的突变通常会过度激活SHP2并导致细胞转化，但确切的机制尚不清楚，因为它涉及局部和整体结构和灵活性的变化，因此确实需要进一步的研究来为准确的诊断和设计提供信息特定和定制的代理。像 SHP-2 这样的磷酸酶仍然是药物发现和分子分析的挑战性目标，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，使它们更适合实验研究并揭示其机制预测水平。我们的分析依赖于一种称为核磁共振波谱的方法，该方法可用于检测 SHP-2 和配体分子中数千个单个原子核的独特信号。该方法提供了关于三维空间中蛋白质在皮秒到秒的一系列时间尺度上的形状、构象、运动和化学相互作用的前所未有的信息，并将为理解蛋白质在细胞和细胞中的行为提供有价值的见解。新药发现方法的适应性。我们已经获得了 SHP2 催化和调控串联 SH2 结构域的有前途的光谱，并且刚刚开始分配其 NMR 信号，以识别新的推定配体并绘制前所未有的结合位点。我们现在最有能力帮助提供对其解决机制的全面理解，这比任何其他人类 PTP 目标可用的解决机制更好。我们预测了一个独特的位点，可以将 SHP-2 引导至质膜位点，并将使用自旋标签、双层和计算模型来定义和验证所提出的膜结合机制的性质。我们将研究与定位和改变 SHP-2 信号活性的受体配体的相互作用，并致力于发现全新类型的抑制剂。我们将与我们的合作者一起开发更全面的 SHP-2 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方式，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，更深入地了解灵活性在这些结合事件中的作用，并为设计用于体外和体内分析的新型抑制剂和转基因模型，它们共同有助于释放 SHP-2 的治疗潜力，并在磷酸酶超家族中具有更广泛的应用。

项目成果

Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.

• DOI: 10.1074/jbc.m114.561787
• 发表时间: 2014-08-22
• 期刊: The Journal of biological chemistry
• 作者: Lenoir M;Sugawara M;Kaur J;Ball LJ;Overduin M
• 通讯作者: Overduin M

Structural Mechanisms and Drug Discovery Prospects of Rho GTPases.

• DOI: 10.3390/cells5020026
• 发表时间: 2016-06-13
• 期刊: Cells
• 影响因子: 6
• 作者: Smithers CC;Overduin M
• 通讯作者: Overduin M

Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting.

• DOI: 10.1038/s41467-018-03370-1
• 发表时间: 2018-03-08
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Lenoir M;Ustunel C;Rajesh S;Kaur J;Moreau D;Gruenberg J;Overduin M
• 通讯作者: Overduin M

Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection.

• DOI: 10.1074/jbc.m116.722066
• 发表时间: 2016-04-22
• 期刊: The Journal of biological chemistry
• 作者: Salim M;Knowles TJ;Hart R;Mohammed F;Woodward MJ;Willcox CR;Overduin M;Hayday AC;Willcox BE
• 通讯作者: Willcox BE

Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention (vol 6, pg 1423, 2014)

靶向蛋白酪氨酸磷酸酶 SHP2 进行治疗干预（第 6 卷，第 1423 页，2014 年）

• 发表时间: 2014
• 期刊: FUTURE MEDICINAL CHEMISTRY
• 影响因子: 4.2
• 作者: Butterworth Sam