Human Interferons: Structure and Function

人类干扰素：结构和功能

• 批准号: 7592300
• 负责人: Kathryn C. Zoon
• 金额: $ 228.75万
• 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/7592300
• 关键词: Affect; Aftercare; Amino Acids; Antibodies; Antiviral Agents; Apoptosis; Autophagocytosis; Binding; Biological; Biological Assay; Biological Process; Burkitt Lymphoma; Cancer cell line; Cell Line; Cells; Cessation of life; Complex; Computer software; Data; Down-Regulation; Electrophoresis; Engineering; Extracellular Domain; Family; Gene Expression; Gene Expression Regulation; Gene Proteins; Genes; Growth; Human; Human Activities; Hybrids; IFNAR2 gene; Insecta; Interferon Alfa-2c; Interferon Receptor; Interferon Type II; Interferon-alpha; Interferons; Isotopically-Coded Affinity Tagging; Methods; Microarray Analysis; Molecular Cloning; Molecular Profiling; Monoclonal Antibodies; Mutation; Numbers; Oligonucleotide Microarrays; Parents; Pathway Analysis; Pattern; Printing; Process; Property; Protein Family; Protein Microchips; Proteins; Proteome; Proteomics; RNA Interference; Range; Relative (related person); Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Protein; Structure; Structure-Activity Relationship; Technology; Title; Transcriptional Activation; Up-Regulation; base; cell growth; comparative; insight; interferon alpha receptor; member; mutant; protein purification

Project Title: Human Interferon alphas: Structure and 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 21b 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 21b. 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). IFN-alpha2c and the IFN hybrid CM3 were selected for this study based on their cell binding and biological properties. Using a number of biological, immunological and physiochemical methods we show evidence that each of the described IFN-alpha subtypes affected the binding of the other IFN-alpha to IFNAR2-EC by affecting the stability of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-alpha2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In an effort to better understand the mechanisms of action and signaling pathways of the IFN alphas using Daudi (Burkitts 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. We examined the relative abundance of proteins observed after treatment of Daudi cells with different interferon-alphas (IFN-alpha 2c and IFN-alpha 21b) 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. 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 as well as Western analyses have been performed to validate the ICAT data. Proteome analysis requires fast and reliable methods of protein purification. We have made and expressed several IFN-alpha molecular clones containing specific mutations in both E.coli and insect cells. We are examining Daudi cell growth inhibition as it relates to growth arrest, apoptosis and autophagy as gene expression microarray data suggests that IFN-alpha 2c treatment of Daudi cells upregulates genes/proteins involved in these processes. In addition, preliminary assays have shown the possible existence of different signaling mechanisms in different cell lines which have been treated with IFN-gamma. We are pursuing these findings and intend to use proteomic and gene expression microarray analyses to elucidate them. To further investigate the mechanisms of antiproliferative and antiviral activities of IFN-alpha and -gamma, RNAi was transfected into several cancer cell lines. We focused on genes related to the Jak-Stat and death signaling pathways. After inhibition of some these genes, the antiproliferative and antiviral activities of IFN-alpha and gamma were partially or fully abrogated. The results suggest that these genes have important roles in these activities.

项目标题：人干扰素alpha：结构和功能 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，我们在基因设计，表达和纯化的14种杂种IFN-alpha物种中得出了人类IFN-Alpha 21b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IFNALPHAS 2C和21B。 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。 根据其细胞结合和生物学特性选择了IFN-Alpha2c和IFN杂化CM3。使用许多生物学，免疫学和生理化学方法，我们表明了证据表明，每个描述的IFN-Alpha亚型都会通过影响复合物的稳定性来影响其他IFN-Alpha与IFNAR2-EC的结合。此外，具有不同IFNAR2-EC突变体的天然电泳表明，IFN-Alpha2c和CM3在IFNAR2-EC的结合结构域中利用不同的氨基酸。 为了更好地了解使用Daudi（Burkitts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。我们检查了使用同位素编码的亲和力标签（ICAT）技术对Daudi细胞处理后观察到的蛋白质相对丰度。 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。 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主​​要形式的抗体以及西方分析进行了质疑以验证ICAT数据。 蛋白质组分析需要快速可靠的蛋白质纯化方法。 我们制作并表达了几个IFN-α分子克隆，这些克隆都包含大肠杆菌和昆虫细胞中的特定突变。 我们正在研究Daudi细胞生长的抑制作用，因为它与生长停滞，凋亡和自噬有关，因为基因表达微阵列数据表明，Daudi细胞的IFN-Alpha 2C处理可以上调参与这些过程的基因/蛋白质。此外，初步测定表明，在已通过IFN-GAMMA处理的不同细胞系中可能存在不同的信号传导机制。 我们正在追求这些发现，并打算使用蛋白质组学和基因表达微阵列分析来阐明它们。为了进一步研究IFN -Alpha和-gamma的抗增生性和抗病毒活性的机制，RNAi被转染到几种癌细胞系中。我们专注于与JAK-STAT和死亡信号通路有关的基因。抑制某些基因后，IFN-Alpha和Gamma的抗增殖和抗病毒活性被部分或完全消除。 结果表明这些基因在这些活动中具有重要作用。