Biophysical analysis of the RFX complex: An essential regulator of the adaptive immune system.

摘要

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. This display triggers a cascade of events leading to the immune response. The 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. The production of MHCII molecules is regulated by a multi-protein complex known as the MHCII enhanceosome. This regulatory unit 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, which 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. A better understanding of how the RFX complex assembles would provide great insight into the rational design of novel immunosuppressive therapeutic agents. Our biophysical analysis of the RFX complex elucidates this complex formation, in efforts to move towards this therapeutic goal. 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.