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

TSG-6是一种多功能的炎症诱导蛋白质，在炎症期间保护/稳定组织，在哺乳动物排卵/受精中也起着关键作用;例如，它可以保护软骨和骨骼免受破坏，我们正在研究它是否可用于治疗关节炎和骨质疏松症。TSG-6通过与广泛的蛋白质和多糖配体相互作用介导其多种功能（例如，我们在过去5年中发现了>10种新的结合伴侣，包括7种BMP、纤连蛋白、RANKL和2种血小板反应蛋白）[1，2]。虽然已经详细研究了其中一些相互作用（例如与糖胺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，包括鉴定作为关键节点的多分子复合物。这将为TSG-6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项目10/04-10/14）和排卵（MRC项目10/08-9/11）中的研究。该项目还将帮助开发可用于其他细胞外基质分子相互作用组研究的通用方法，包括多组分复合物的鉴定/表征以及它们如何进行构象调节。这些研究将通过使用双偏振干涉测量法（DPI）来促进，这是我们的工业合作伙伴Farfield开创的一项技术; Analight 4D已通过更新细胞矩阵研究欢迎信托中心获得资助，其中AJD/TAJ是其中的一部分。该仪器可用于真实的确定附着在表面上的分子的结构变化（>0.1 μ m），提供有关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物的组装并提供有关其结构/化学计量的信息。虽然DPI可用于从传感器表面的质量依赖性变化获得动力学信息，但从质量独立的构象变化获得动力学数据也是可能的，这是由Farfield开发的，即利用新的低容量反应池。学生将有助于这些发展，并比较DPI和表面等离子体共振（SPR）获得的动力学数据。单独的TSG-6-配体相互作用的相对亲和力/动力学将不会揭示结合的层次，特别是如果任何缔合涉及变构机制。TSG-6相互作用组的层次结构将使用配体捕捞技术（即固定的TSG-6蛋白和确定的蛋白质混合物）结合质谱分析进行研究;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复合物。DPI将结合其他生物物理技术，包括QCM-D（带耗散监测的石英晶体微天平）和SPR，研究它们的形成和化学计量。特别是，Farfield将帮助我们改进传感器表面蛋白质分子取向的方法，以允许在生物相关背景下组装复合物。1. Mahoney等人（2008）TSG-6通过抑制成骨细胞生成和破骨细胞活化调节骨重塑。J.Biol.Chem.283，25952-62 2. Kuznetsova等人（2008）TSG-6通过其CUB_C结构域结合纤连蛋白的细胞结合结构域并增加纤连蛋白基质组装。矩阵生物学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