NMR studies of vaccinia virus interferon binding protein: interactions with GAGs

描述(申请人提供)：糖胺多聚糖(GAG)参与以高等生物的细胞外基质(ECM)为中心的一系列调控过程。GAG非同寻常的结构多样性使它们能够与多种生物分子相互作用来调节过程，包括免疫反应和细胞生长调节。GAG-病原体的相互作用也影响着微生物致病的大部分关键步骤，包括宿主细胞附着、入侵、细胞-细胞传递、全身传播和逃避宿主防御机制。此外，在将病原体转化为治疗药物方面取得了很大进展，特别是在癌症治疗中使用溶瘤病毒方面。显然，人们有兴趣改进针对癌症中上调的表面受体的靶向，包括GAG。痘病毒的几个特点使它们非常适合用作溶瘤病毒疗法。特别是，痘病毒天生具有广泛的肿瘤组织趋向性。虽然它们不使用GAG来附着或入侵，但它们确实会产生用于其他目的的GAG结合蛋白。这项研究的特定目标VACV B18是由痘苗病毒(痘病毒家族的成员)分泌的I型干扰素(干扰素)结合蛋白，它通过与干扰素结合来抑制免疫反应。这种蛋白质与细胞表面的GAG相互作用，以便将这种抑制效应的范围扩大到多个细胞表面。特别是，B18已被证明与肝素(HP)和硫酸肝素(HS)都有很强的结合；然而，这种识别和结合的结构基础仍然不清楚。这项提案将开发生化和溶液核磁共振方法，以解决B18-GAG结合中涉及的结构因素和分子间相互作用。了解这些特定的相互作用可以为为治疗目的操纵Gag-蛋白质相互作用奠定基础。为了确定GAG-B18结合的结构基础，将结合细胞培养、分离、消化和分离技术分离一系列HS低聚体，以制备用于结构研究的同位素标记低聚体。有了这些，现有的溶液核磁共振方法将与新的稀疏标记和长程顺磁微扰方法相结合，以确定野生型蛋白质的结构模型，无论是在其自由、非结合状态，还是在与特定HS寡聚体的络合物中。 对自由和结合结构的比较将阐明先前被认为重要的残基的结构作用，并导致鉴定对B18结合重要的HS片段特征。这些进展将提供适用于其他GAG结合蛋白的方法。该项目的成功完成不仅将产生新的方法学，而且将在分子水平上加深对痘苗病毒B18-HS分子间相互作用的了解，并为未来设计能够抑制B18免疫抑制作用的HS类似物提供分子基础。

DESCRIPTION (provided by applicant): Glycosaminoglycans (GAGs) are involved in a host of regulatory processes centered in the extracellular matrix (ECM) of higher organisms. The extraordinary structural diversity of GAGs enables them to interact with a wide variety of biological molecules to modulate processes, including immune response and regulation of cell growth. GAG-pathogen interactions also affect most, if not all, of the key steps of microbial pathogenesis, including host cell attachment, invasion, cell-cell transmission, systemic dissemination, and evasion of host defense mechanisms. Additionally, much progress has been made in turning pathogens to therapeutics, specifically in the use of oncolytic viruses in cancer treatment. There is obvious interest in improving targeting to surface receptors up-regulated in cancer, including GAGs. Several characteristics of poxviruses make them well-suited for use as oncolytic virus therapeutics. In particular, poxviruses inherently have broad tumor tissue tropism. While they do not use GAGs for attachment or invasion, they do produce GAG-binding proteins for other purposes. The particular target of this study, VACV B18, is the type I interferon (IFN) binding protein (IFN¿/¿BP) secreted by the vaccinia virus (a member of the poxvirus family), that suppresses immune response through binding to IFN. This protein interacts with cell surface GAGs in order to extend the range of this suppressive effect over multiple cell surfaces. In particular, B18 has been shown to bind strongly to both heparin (HP) and heparan sulfate (HS); however, the structural basis for this recognition and binding remains unknown. This proposal will develop biochemical and solution NMR methods to address the structural factors and intermolecular interactions involved in B18-GAG binding. Understanding these specific interactions can lay the basis for manipulating GAG-protein interactions for therapeutic purposes. In order to determine the structural basis of GAG-B18 binding, a series of HS oligomers will be isolated using a combination of cell culture, isolation, digestion, and separation technology that allow preparation of isotopically labeled oligomers for structural study. With these in hand, existing solution NMR methods will be combined with novel sparse labeling and long range paramagnetic perturbation methods to determine structural models of the wild-type protein, both in it's free, unbound state, and in complex with specific HS oligomers. A comparison of the free and bound structures will illuminate the structural role of residues previously identified as important, and lead to the identification of HS fragment characteristics important for B18 binding. These advances will provide methods which will be applicable to other GAG binding proteins. Successful completion of this project will not only generate novel methodology, but also enhance molecular-level understanding of intermolecular B18-HS interactions in vaccinia virus and provide a molecular basis for the future design of HS analogs that can inhibit the immune suppressive action of B18.