Molecular basis for the trafficking of transmembrane proteins through Ubiquitin, Syntenin-1 and Tollip complexes

通过泛素、Syntenin-1 和 Tollip 复合物运输跨膜蛋白的分子基础

• 批准号: BB/K019686/1
• 负责人: Michael Overduin
• 金额: $ 55.25万
• 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: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK019686%2F1
• 关键词: Molecular; basis; trafficking; transmembrane; proteins

In this research project the three dimensional structures and molecular interactions of a dynamic trafficking complex that internalizes transmembrane proteins will be characterized at a resolution that is useful for drug discovery. The tetraspanin trafficking (TETRAF) complex involves four human proteins. Tetraspanin proteins span the membrane four times, and link directly to a cytosplasmic adaptor protein called syntenin. We will determine how syntenin induces specific hetero-dimerization of receptors via their cytoplasmic tails in a phosphorylation-dependent manner. The recycling of this tetraspanin-syntenin complex is mediated by the ubiquitin protein, which exhibits a novel binding site near its C-terminus. The tollip protein interacts directly with phosphoinositide lipids, and we have additionally discovered its unique binding site for syntenin, revealing a previously unknown supermolecular complex. We propose that this membrane-associated protein assembly is responsible for controlling how tetraspanins are controlled within healthy cells and contributes to the migration and invasiveness of tumour cells. The targeting of the tetraspanin complexes presents promising therapeutic opportunities due to the key roles played in infection, tumour formation and metastasis. Our understanding of the underlying mechanisms however remains in its infancy, with no structures available for any TETRAF complexes despite its relevance to improving human health and well being. These multidomain targets remain challenging for analysis in terms of their solution structures and conformational dynamics under physiological conditions, warranting further fundamental biochemical and molecular research to define the key interactions and regulatory mechanisms. We will apply a method known as nuclear magnetic resonance spectroscopy using our national facility's superconducting magnets, which can be used to detect a unique signal for each of the thousands of atomic nuclei in the molecular complex. 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. We have obtained resolved spectra of all four proteins that form the TETRAF complex, and plan to extend these to elucidate the molecular structures and functions, thus providing a mechanism for tetraspanin internalization. We have identified novel binding sites in syntenin and tollip that bind lipid molecules, and will use spin labels and computer methods to validate and define how membrane recruitment mediates traffic of the complex between plasma and endocytic compartments. Together with our collaborators we will provide the first comprehensive structural, functional and biochemical insights into how this assembly acts at a molecular level, allowing us to much more accurately manipulate its behaviour in vitro and in vivo. The endpoints of the project include structures of the complexes bound to lipid and protein ligands that regulate tetraspanin activity in cells, a description of the dynamics and structural determinants of these binding events, and, in the long term, a rational basis for designing inhibitors and mutations for in vitro and in vivo analysis of this system for drug discovery.

在该研究项目中，将以对药物发现有用的分辨率来表征内化跨膜蛋白的动态运输复合物的三维结构和分子相互作用。四跨膜蛋白运输 (TETRAF) 复合物涉及四种人类蛋白质。四跨膜蛋白跨越膜四次，并直接与称为 Syntenin 的细胞质接头蛋白连接。我们将确定连线蛋白如何通过其细胞质尾部以磷酸化依赖性方式诱导受体的特异性异二聚化。这种四跨膜蛋白-syntenin 复合物的回收是由泛素蛋白介导的，该蛋白在其 C 末端附近表现出一个新的结合位点。 Tollip 蛋白直接与磷酸肌醇脂质相互作用，我们还发现了其独特的 Syntenin 结合位点，揭示了一种以前未知的超分子复合物。我们认为这种膜相关蛋白组装负责控制健康细胞内四跨膜蛋白的控制方式，并有助于肿瘤细胞的迁移和侵袭。由于四跨膜蛋白复合物在感染、肿瘤形成和转移中发挥的关键作用，靶向四跨膜蛋白复合物提供了有希望的治疗机会。然而，我们对潜在机制的理解仍处于起步阶段，尽管 TETRAF 复合物与改善人类健康和福祉相关，但还没有任何可用的结构。这些多域靶标在生理条件下的溶液结构和构象动力学方面仍然具有挑战性，需要进一步的基础生化和分子研究来定义关键的相互作用和调节机制。我们将使用我们国家设施的超导磁体应用一种称为核磁共振波谱的方法，该方法可用于检测分子复合物中数千个原子核中每一个的独特信号。该方法提供了关于三维空间中蛋白质的形状、构象、运动和化学相互作用以及从皮秒到秒的一系列时间尺度的前所未有的信息。我们已经获得了形成 TETRAF 复合物的所有四种蛋白质的解析光谱，并计划扩展这些光谱以阐明分子结构和功能，从而提供四跨膜蛋白内化的机制。我们已经在 Syntenin 和 tollip 中发现了与脂质分子结合的新结合位点，并将使用自旋标签和计算机方法来验证和定义膜募集如何介导血浆和内吞区室之间复合物的运输。我们将与我们的合作者一起提供关于该组装体如何在分子水平上发挥作用的第一个全面的结构、功能和生化见解，使我们能够更准确地操纵其在体外和体内的行为。该项目的终点包括与调节细胞内四跨膜蛋白活性的脂质和蛋白质配体结合的复合物的结构、这些结合事件的动力学和结构决定因素的描述，以及从长远来看，设计抑制剂和药物的合理基础。对该系统进行体外和体内分析以进行药物发现的突变。

项目成果

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

• DOI: 10.1038/srep32337
• 发表时间: 2016-08-31
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Gräßel L;Fast LA;Scheffer KD;Boukhallouk F;Spoden GA;Tenzer S;Boller K;Bago R;Rajesh S;Overduin M;Berditchevski F;Florin L
• 通讯作者: Florin L