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

在这个研究项目中，一个动态运输复合物的三维结构和分子相互作用将被表征为对药物发现有用的分辨率。四蛋白转运（TETRAF）复合物涉及四种人类蛋白质。四聚氰胺蛋白跨越细胞膜四次，并直接连接到细胞质中称为联筋蛋白的接头蛋白。我们将确定syntenin如何以磷酸化依赖的方式通过受体的细胞质尾部诱导特异性异二聚化。泛素蛋白在其c端附近有一个新的结合位点，它介导了这种四跨蛋白-syntenin复合物的再循环。tollip蛋白直接与磷酸肌醇脂相互作用，我们还发现了它独特的syntenin结合位点，揭示了一个以前未知的超分子复合物。我们提出，这种膜相关蛋白组装负责控制健康细胞内四联蛋白的控制，并有助于肿瘤细胞的迁移和侵袭。由于在感染、肿瘤形成和转移中起关键作用，靶向四联蛋白复合物提供了有希望的治疗机会。然而，我们对其潜在机制的理解仍处于起步阶段，尽管它与改善人类健康和福祉有关，但没有任何可用于任何TETRAF复合物的结构。这些多域靶点在生理条件下的溶液结构和构象动力学分析仍然具有挑战性，需要进一步的基础生化和分子研究来确定关键的相互作用和调控机制。我们将使用一种被称为核磁共振波谱的方法，使用我们国家设施的超导磁体，它可以用来检测分子复合体中数千个原子核中的每个原子核的独特信号。该方法提供了有关蛋白质在三维空间和从皮秒到秒的时间尺度范围内的形状、构象、运动和化学相互作用的前所未有的信息。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，从而提供四蛋白蛋白内化的机制。我们已经在syntenin和tollip中发现了结合脂质分子的新结合位点，并将使用自旋标记和计算机方法来验证和定义膜募集如何介导血浆和内吞室之间复合物的运输。与我们的合作者一起，我们将提供第一个全面的结构，功能和生化见解，了解这种组装如何在分子水平上起作用，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脂质和蛋白质配体结合的复合物的结构，这些复合物调节细胞中的四白蛋白活性，描述这些结合事件的动力学和结构决定因素，从长远来看，为设计抑制剂和突变提供合理的基础，用于体外和体内分析该系统，用于药物发现。

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.