Human Interferon Alphas: Structure and Function

人干扰素α：结构和功能

• 批准号: 7313430
• 负责人: Kathryn C. Zoon
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7313430
• 关键词: Human; Interferon; Alphas; Structure; Function

Our studies are directed toward understanding the structure of the members of the human interferon (IFN)-alpha family and how they elicit their biological (antiviral, antiproliferative and immunomodulatory) activities. Previously, it was shown that specific regions of the IFN-alphas are associated with specific types of biological functions using IFN alpha hybrid and mutant molecules. To determine the domains of the IFN-alphas that are important for antiviral and antiproliferative activities we genetically engineered, expressed and purified 14 hybrid IFN-alpha species derived from human IFN-alpha 21a and IFN-alpha 2c.The secondary/tertiary structures of the human IFN hybrids were examined using a broad range of monoclonal antibodies (mAbs). Results have shown structural differences among our constructs as well as the parent molecules, IFN alphas 2c and 21a. Based on these data, we extended our studies on the interactions of the IFN hybrids with the IFN receptor subunit IFNAR2-ECD (Interferon alpha receptor 2-extracellular domain). ELISA results showed that helix E (138-154) is the most critical region of the interferon molecule for interaction with the IFNAR2-ECD. Our data suggests that the epitope 112-132 (helix D), which is recognized by several C-terminal anti-IFN-alpha mAbs, is not involved in the interaction of our constructs with IFNAR2-ECD. We also deduced that two antigenically distinct IFNs may bind to IFNAR2EC simultaneously in an independent manner. In addition, we are examining the interaction between IFN-alpha 2 subvariants (2a, 2b and 2c) and the extracellular domain of human IFN receptor (IFNAR2-EC) using a sandwich ELISA and anti-IFN monoclonal antibodies. The amino acid residue at position 34 seems to be crucial for the structure/function of human IFN-alphas and for IFN/IFN receptor interactions.We also studied the structure/function differences between 6-histidine-tagged and untagged IFNs. In an effort to better understand the mechanisms of action and signaling pathways of the IFN alphas using Daudi (Burkitt's Lymphoma) cells, we have initiated gene expression microarray and proteomics analyses. It is anticipated that these two technologies will provide insight into the different levels of antiproliferative and antiviral activities observed with the various IFN-alpha hybrids and mutants. Oligonucleotide microarray analysis was used to evaluate gene expression profiles of the IFN-alphas. Data showed that there are distinct expression patterns corresponding to the IFN alphas (parental and hybrids). These diversities in gene regulation may contribute to different biological activities. Comparative analysis of cell lysates produced from IFN-alpha treated and untreated Daudi cells printed on protein microarrays and interrogated with antibodies against major forms of signaling proteins has been performed. In addition, we examined the relative abundance of proteins observed after treatment of Daudi cells with different interferon-alphas (IFN-alpha 2c and IFN-alpha 21a) using Isotope-Coded Affinity Tags (ICAT) technology. Using pathway analysis software, we are studying the up-regulation and down-regulation of specific proteins in a variety of signal transduction pathways following IFN treatment. Finally we have studied the antiviral properties of our human interferon alpha constructs against the SARS virus in Vero cells. Preliminary results suggest that three constructs have very high antiviral activity.

我们的研究旨在理解人干扰素（IFN） - 阿尔法家族的成员的结构，以及它们如何引起其生物学（抗病毒，抗逆变和免疫调节）活性。以前，已经表明，使用IFN Alpha杂交和突变分子的特定区域与特定类型的生物学功能有关。为了确定对抗病毒和抗增殖活性很重要的领域，我们在基因工程，表达和纯化的14种杂种IFN-alpha物种中得出了人类IFN-Alpha 21a和Ifn-Alpha 2c。结果显示了我们的构建体以及父分子之间的结构差异，即ifn alpha 2c和21a。基于这些数据，我们扩展了有关IFN杂交与IFN受体亚基IFNAR2-ECD（干扰素α受体2-纤维化结构域）相互作用的研究。 ELISA结果表明，Helix E（138-154）是与IFNAR2-ECD相互作用的干扰素分子中最关键的区域。我们的数据表明，几个C末端抗IFN-Alpha mAb识别的表位112-132（Helix D）与我们的构建体与IFNAR2-ECD的相互作用不参与。我们还推断出两个抗原不同的IFN可能以独立的方式同时与IFNAR2EC结合。此外，我们还使用三明治ELISA和抗IFN单克隆抗体研究了IFN-Alpha 2亚变量（2A，2B和2C）与人IFN受体（IFNAR2-EC）的细胞外域之间的相互作用。位置34处的氨基酸残基似乎对于人IFN-ALPHASS和IFN/IFN受体相互作用的结构/功能至关重要。我们还研究了6-Histidine标记的IFN和未删除的IFN之间的结构/功能差异。 为了更好地了解使用Daudi（Burkitt的淋巴瘤）细胞的IFN Alpha的作用和信号传导途径的机制，我们启动了基因表达微阵列和蛋白质组学分析。可以预料，这两种技术将提供有关与各种IFN-Alpha杂种和突变体观察到的不同水平的抗增殖和抗病毒活性的见解。寡核苷酸微阵列分析用于评估IFN-ALLASE的基因表达谱。数据表明，存在与IFNALPHAS（父母和杂种）相对应的不同表达模式。基因调节中的这些多样性可能有助于不同的生物学活动。对蛋白微阵列上印有经过处理和未经处理的Daudi细胞产生的细胞裂解物的比较分析，并用针对主要信号蛋白的抗体受到询问。此外，我们使用同位素编码的亲和力标签（ICAT）技术检查了具有不同干扰素 - 阿尔帕2C和IFN-Alpha 21a的Daudi细胞后观察到的蛋白质的相对丰度。使用途径分析软件，我们正在研究IFN处理后各种信号转导途径中特定蛋白的上调和下调。最后，我们研究了人类干扰素α构建体针对Vero细胞中SARS病毒的抗病毒特性。初步结果表明，三个构建体具有很高的抗病毒活性。