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

痘苗病毒干扰素结合蛋白的 NMR 研究：与 GAG 的相互作用

• 批准号: 8649447
• 负责人: Kari Joanne Pederson
• 金额: $ 5.51万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649447
• 关键词: Address; Affect; Affinity; Amino Acids; Antiviral Agents; Antiviral Response; Area; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Blood; Carbohydrates; Carrier Proteins; Cell Culture Techniques; Cell surface; Cell-Matrix Junction; Cells; Characteristics; Coagulation Process; Code; Complex; Development; Digestion; Escherichia coli; Escherichia coli Proteins; Extracellular Matrix; Family; Future; Glycobiology; Glycoproteins; Glycosaminoglycans; Goals; Hand; Heparin; Heparitin Sulfate; Host Defense Mechanism; Human; Immune; Immune response; Infection; Inorganic Sulfates; Interferon Type I; Interferons; Label; Lanthanoid Series Elements; Lead; Learning; Length; Malignant Neoplasms; Mammalian Cell; Membrane Proteins; Metabolic; Methodology; Methods; Molecular; Molecular Conformation; Monkeypox; N-terminal; Oligosaccharides; Oncolytic; Oncolytic viruses; Organism; Pathogenesis; Pattern; Peptides; Polysaccharides; Poxviridae; Preparation; Process; Production; Property; Proteins; Proteoglycan; Relaxation; Research Personnel; Role; Series; Smallpox; Solid; Solutions; Stream; Structural Models; Structure; Surface; Techniques; Technology; Therapeutic; Tumor Tissue; Unspecified or Sulfate Ion Sulfates; Vaccines; Vaccinia Virus Study; Vaccinia virus; Virulence Factors; Virus; analog; base; cancer therapy; cell growth regulation; design; experience; flexibility; immunoregulation; improved; inhibitor/antagonist; insight; interest; intermolecular interaction; member; microbial; novel; pathogen; protein expression; public health relevance; receptor; research study; skills; success; sulfation; tissue tropism; transmission process

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.

描述（由申请人提供）：糖胺聚糖（GAG）参与以高等生物细胞外基质（ECM）为中心的许多调节过程。糖胺聚糖的非凡结构多样性使它们能够与各种生物分子相互作用以调节过程，包括免疫应答和细胞生长调节。GAG-病原体相互作用还影响微生物致病的大多数（如果不是全部的话）关键步骤，包括宿主细胞附着、侵入、细胞-细胞传播、系统传播和逃避宿主防御机制。此外，在将病原体转化为治疗剂，特别是在癌症治疗中使用溶瘤病毒方面取得了很大进展。人们对改善针对癌症中上调的表面受体（包括糖皮质激素）的靶向有明显的兴趣。痘病毒的几个特征使其非常适合用作溶瘤病毒治疗剂。特别地，痘病毒固有地具有广泛的肿瘤组织嗜性。虽然它们不使用GAG进行附着或入侵，但它们确实产生用于其他目的的GAG结合蛋白。本研究的特定靶点VACV B18是由牛痘病毒（痘病毒家族的成员）分泌的I型干扰素（IFN）结合蛋白（IFN <$/<$BP），其通过与IFN结合来抑制免疫应答。这种蛋白质与细胞表面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）和硫酸乙酰肝素（HS）强烈结合;然而，这种识别和结合的结构基础仍然未知。该提案将开发生物化学和溶液NMR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，将使用细胞培养、分离、消化和分离技术的组合分离一系列HS寡聚体，这些技术允许制备同位素标记的寡聚体用于结构研究。有了这些，现有的溶液核磁共振方法将与新型稀疏标记和远程顺磁扰动方法相结合，以确定野生型蛋白质的结构模型，无论是在自由、未结合状态还是与特定HS低聚物的复合体中。 的自由和绑定结构的比较将阐明以前确定为重要的残基的结构作用，并导致识别的HS片段的特性重要的B18结合。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，而且还将增强对牛痘病毒中分子间B18-HS相互作用的分子水平理解，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。