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

描述（由申请人提供）：糖胺聚糖（GAGs）参与以高等生物细胞外基质（ECM）为中心的一系列调节过程。特异的gag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的调节。gag -病原体相互作用也影响微生物发病的大部分关键步骤，包括宿主细胞附着、入侵、细胞-细胞传播、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剂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。在癌症中，包括gag在内的表面受体上调的靶向性有明显的兴趣。痘病毒的一些特性使它们非常适合用作溶瘤病毒治疗药物。特别是痘病毒本身具有广泛的肿瘤组织亲和性。虽然它们不使用gag进行附着或入侵，但它们确实会产生用于其他目的的gag结合蛋白。本研究的特定靶点VACV B18是由痘苗病毒（痘病毒家族成员）分泌的I型干扰素（IFN）结合蛋白（IFN¿/¿BP），它通过与IFN结合来抑制免疫反应。该蛋白与细胞表面gag相互作用，以扩大这种抑制作用在多个细胞表面的范围。特别是，B18已被证明与肝素（HP）和硫酸肝素（HS）结合强烈；然而，这种识别和结合的结构基础仍然未知。本提案将发展生化和溶液核磁共振方法来解决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操纵gag -蛋白相互作用以达到治疗目的奠定基础。为了确定GAG-B18结合的结构基础，我们将采用细胞培养、分离、消化和分离技术相结合的方法分离一系列HS低聚物，从而制备同位素标记的低聚物进行结构研究。有了这些，现有的溶液核磁共振方法将与新的稀疏标记和远程顺磁摄动方法相结合，以确定野生型蛋白的结构模型，无论是在其自由状态，非结合状态，以及与特定HS低聚物的复合物。

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.