B.S. Hood College 2002; Ph.D. UMBC 2008

The human adaptive immune system serves as the primary defense against pathogenic bacteria, viruses, and fungi. While the human adaptive immune response is powered by many components, the Major Histocompatibility Class II (MHCII) molecules lie at the heart of the machinery of the immune system. MHCII molecules bind to peptides derived from the invading species and display them on the surface of specialized cells of the immune system. The display of the MHCII-peptide complex is recognized by CD4+ T-cells, which then triggers a cascade of events leading to the immune response. This MHCII initiated cascade is essential for clearing the majority of bacterial, viral, and fungal infections. Lack of MHCII expression causes severe immunodeficiency disorders, whereas overproduction of these molecules can cause a variety of autoimmune diseases. Because these molecules play an essential role in generating immune response, regulating the production of MHCII molecules is critically important to balancing this response so that the immune system does not become aberrantly suppressed or hyperactive.      The production of MHCII molecules is regulated by a multi-protein complex known as the MHCII enhanceosome. The MHC II enhanceosome is comprised of four major components that assemble on the promoter of the MHCII genes. The key DNA-binding component of the MHCII enhanceosome is the Regulatory Factor X (RFX) protein complex. RFX is comprised of three subunits, RFX5, RFXAP, and RFXB. Mutations or deletions in any RFX subunit that prevents the formation of the MHCII enhanceosome abolishes MHCII expression. Because the RFX complex is specific for regulating MHCII gene expression, this complex is an excellent target for the rational design of therapeutic compounds that could disrupt complex formation, thus suppressing immune response. A better understanding of how the RFX complex assembles is required prior to designing such drugs. Our biophysical analysis of the RFX complex provides significant insight into how this complex assembles. We have specifically focused on domains of each subunit that we have identified as being directly involved in complex formation. We have shown that these domains are independently folded and can readily form a complex in the same manner as the full length subunits. Likewise, we have also determined the stoichiometric ratio in which the domains bind to one another in solution. Finally, we have initiated structural studies of each of these domains using NMR spectroscopy. These studies will provide the foundation for future work in solving the structure of the RFX complex.