Biophysical characterisation of tsg-6-ligand interactions: using dpi to investigate the interactome of a multi-functional inflammatory mediator

TSG-6是一种多功能、炎症诱导的蛋白质，在炎症期间保护/稳定组织&它在哺乳动物排卵/受精过程中也起着关键作用；例如，它可以保护软骨和骨骼免受破坏&我们正在进行研究，看看它是否有助于治疗关节炎和骨质疏松症。TSG-6通过与一系列蛋白质和多糖配体的相互作用(例如，我们在过去5年中发现了10个新的结合伙伴，包括7个BMP、纤维连接蛋白、RANKL和2个凝血酶反应蛋白)[1，2]来调节其不同的功能。虽然其中一些相互作用已经被详细研究(例如与糖胺多聚糖、透明质酸和肝素)，但在大多数情况下，我们几乎没有生物物理数据。该项目的目的是对TSG-6-配体相互作用进行系统分析，以获得其动力学信息，并建立TSG-6相互作用组的层次结构，包括识别作为关键结点的多分子复合体。这将为TSG-6的结构/功能相互关系及其作用机制提供重要的新见解，这将补充我们目前对TSG-6在关节炎(ARC方案10/04-10/14)和排卵(MRC Project 10/08-9/11)中的研究。该项目还将帮助开发可用于研究其他细胞外基质分子相互作用组的通用方法，包括识别/表征多组分复合体及其构象调控方式。这些研究将通过使用双偏振干涉法(DPI)来促进，这是我们的工业合作伙伴Farfield首创的一项技术；Analight 4D已通过更新欢迎信托细胞矩阵研究中心获得资金，AJD/Taj是其中的一部分。该仪器可用于实时确定附着在表面的分子的结构变化(&gt；0.1？)，提供关于单个相互作用的亲和力和动力学的信息，但它也可以监测多分子复合体的组装并提供关于其结构/化学计量的信息。虽然DPI可以用来从传感器表面与质量相关的变化中获得动力学信息，但从与质量无关的构象变化中获得动力学数据也是可能的&这是由Farfield开发的，即利用一种新的低体积反应池。学生将对这些发展做出贡献，并比较DPI和表面等离子体共振(SPR)获得的动力学数据。在孤立的情况下，单个TSG-6配体相互作用的相对亲和力/动力学不会揭示结合的层次，特别是如果任何一个结合涉及变构机制。将使用配体钓鱼技术(即固定的TSG-6蛋白质和定义的蛋白质混合物)结合质谱分析来研究TSG-6相互作用组的层次结构；将探索pH和糖胺聚糖的影响(以模拟特定的微环境，例如炎症环境)，这将为TSG-6的调控提供重要的见解。这些研究还将确定TSG-6与其配体之间形成的关键络合物。它们的形成和化学计量将由DPI结合其他生物物理技术进行研究，包括QCM-D(具有耗散监测的石英晶体微天平)和SPR。特别是，Farfield将帮助我们改进传感器表面蛋白质分子定向的方法，以便在生物相关的背景下组装复合体。1.马奥尼等人。(2008)TSG-6通过抑制成骨细胞生成和破骨细胞激活来调节骨重建。J.Biol.化学。25952-62.库兹涅佐娃等人。(2008)TSG-6通过其CUB_C结构域与纤维连接蛋白的细胞结合域结合，增加纤维连接蛋白的基质组装。基质生物学27,201-10

TSG-6 is a multifunctional, inflammation-induced, protein that protects/stabilises tissues during inflammation & that also has a critical role in mammalian ovulation/fertilisation; e.g. it can protect cartilage & bone from breakdown & we have on-going investigations into whether it has utility for treatment of arthritis & osteoporosis. TSG-6 mediates its diverse functions via interactions with a wide-range of protein & polysaccharide ligands (eg we have discovered >10 new binding partners in the last 5 years, including 7 BMPs, fibronectin, RANKL & 2 thrombospondins) [1,2]. While a few of these interactions have been studied in detail (eg with the glycosaminoglycans hyaluronan & heparin), in most cases we have little biophysical data. The aim of this project is to undertake a systematic analysis of TSG-6-ligand interactions to obtain information on their kinetics & establish the hierarchy of the TSG-6 interactome, including the identification of multimolecular complexes that serve as key nodal points. This will provide important new insights into TSG-6 structure/function inter-relationships and its mechanism of action, which will complement our current research on TSG-6 in arthritis (ARC Programmes 10/04-10/14) & ovulation (MRC Project 10/08-9/11). The project will also aid the development of generic methodologies that can be utilised for the investigation of the interactome of other extracellular matrix molecules, including the identification/characterisation of multi-component complexes & how they are conformationally regulated. These studies will be facilitated by the use of dual polarisation interferometry (DPI), a technique that has been pioneered by our industrial partner Farfield; an Analight 4D has been funded through the renewal of the Welcome Trust Centre for Cell-Matrix Research of which AJD/TAJ are part. This instrument can be used to determine structural changes (>0.1 Å) of molecules attached to a surface in real time, providing information on affinities & kinetics of individual interactions, but it can also monitor the assembly of multi-molecular complexes & provide information on their structure/stoichiometry. While DPI can be used to obtain kinetic information from mass-dependent changes at the sensor surface, obtaining kinetic data from mass independent conformational changes is also possible & this is being developed by Farfield, ie utilising a new low volume reaction cell. The student will contribute to these developments and compare kinetic data obtained by DPI and surface plasmon resonance (SPR). In isolation the relative affinities/kinetics of the individual TSG-6-ligand interactions will not reveal the hierarchy of binding, particularly if any of the associations involve allosteric mechanisms. The hierarchy of the TSG-6 interactome will be investigated using ligand-fishing techniques (ie with immobilised TSG-6 protein and defined mixtures of proteins) combined with mass spectrometric analyses; the effect of pH & glycosaminoglycans will be explored (to mimic particular microenvironments, for example an inflammatory milieu) and this will provide important insights into TSG-6 regulation. These studies will also identify key complexes formed between TSG-6 & its ligands. Their formation & stoichiometry will be investigated by DPI in conjunction with other biophysical techniques including QCM-D (quartz-crystal microbalance with dissipation monitoring) and SPR. In particular, Farfield will help us improve the methodology for orientation of protein molecules on the sensor surface to allow the assembly of complexes in a biologically relevant context. 1. Mahoney et al. (2008) TSG-6 regulates bone remodeling through inhibition of osteoblastogenesis and osteoclast activation. J. Biol. Chem. 283, 25952-62 2. Kuznetsova et al. (2008) TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly. Matrix Biology 27, 201-10