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.
