Synergistic Inhibition of the Complement Alternative Pathway by FHR Family Proteins - PROJECT SUMMARY/ABSTRACT The complement system is a network of cell surface-associated and soluble plasma proteins comprising a proteolytic cascade with essential functions in innate immune surveillance and inflammation. While there are three canonical complement pathways, the alternative pathway (AP) is the primary means of complement amplification and innate immune effector generation in vivo. Because of this, AP activity is tightly controlled via the functions of the Factor H (FH) family of regulatory proteins, all of whom act on C3b or its complexes. The FH-Related Proteins (FHRs) share many features with FH, but have distinct regulatory properties that are not yet fully understood. Rare genetic variants in all FHRs are associated with developing complement-driven pathologies, indicating that FHRs play vital roles in controlling complement AP activity in various contexts in vivo. Despite the existence of dedicated regulatory proteins, excessive complement AP activity is a driver of many autoimmune, inflammatory and degenerative diseases. Among these are rare diseases like atypical hemolytic uremic syndrome and paroxysmal nocturnal hemoglobinuria, but also far more common conditions like dry age-related macular degeneration – a disease affecting nearly 20 million adults in the USA. Recent FDA approvals of several complement AP inhibitory drugs have diversified the therapeutic landscape, but the high cost of these drugs is a major burden for both patients and healthcare systems. To develop more affordable therapies, there remains a need to study the basic mechanisms underlying AP inhibition and regulation. We previously characterized a potent AP inhibitory protein called Efb-C. Through structure/function studies, we found that Efb-C inhibits complement by stabilizing an open conformation of C3b that forms the AP Convertase enzyme inefficiently. Further consequences of Efb-C binding to C3b have not been systematically investigated. However, we recently found that Efb-C binding to C3b leads to recruitment of the endogenous complement regulator, FHR2, resulting in a strong, synergistic inhibition of the AP. Aside from FHR2, there are four additional FHRs (ie, FHR1, FHR3, FHR4, and FHR5). Whereas FHRs 1, 2, and 5 inhibit complement activity, FHR4 seems to promote complement function. Whether and how Efb-C influences these FHRs is unclear. Here, we will use X-ray crystallography to determine how Efb-C binding to C3b promotes the interaction with FHR2, leading to synergistic inhibition of the AP. We will also examine whether Efb-C binding to C3b alters the function of the remaining FHR family members. Finally, we will test the hypothesis that other molecules which share the Efb-C binding site on C3b will exert similar effects on FHRs and thereby inhibit the complement AP. To do so, we will complete a site-selective phage display screen to identify such peptides and characterize their effects on FHRs and complement function. Completing this work will provide insights into C3b structure and FHR function, and will enable development of more cost-effective therapies for complement-driven diseases.
