The transcription factor NFkB is a master regulator of many important cellular processes, including inflammatory responses to invading pathogens. Patients with defects in NFkB signaling are susceptible to life threatening infections and have defects in skin development, known as the syndrome of ectodermal dysplasia with immune deficiency. We have identified a patient with ectodermal dysplasia with immune deficiency with a novel heterozgous mutation in IkappaBalpha (IkBa) a regulator of NFkB activity. This mutation results in the deletion of the first twelve amino acids of IkBa, termed lkBaDelta1-12. lkBaDelta1-12 cannot be normally phosphorylated and degraded following inflammatory stimuli, and, therefore impairs NFkB activation. This novel mutation in IkaBa identifies a previously unappreciated subdomain that is essential to its function and the regulation of NFkB function. The analysis of this IkaBa mutation will be performed by Douglas McDonald, M.D., Ph.D. in the Division of Immunology at Children's Hospital, Boston. Dr. McDonald's goals are to enhance understanding of the roles of IkBa and NFkB in human immunity. These studies will also greatly enrich Dr. McDonald's scientific and clinical skills as a physician scientist. The goals of the studies proposed are to characterize the role of the N-terminal 12 amino acids of IkBa in IkBa processing. We will study the importance of the N-terminal 12 amino acids of IkBa in protein-protein interactions with IKKbeta, the protein kinase that phosphorylates IkBa, leading to IkBa degradation and NFkB activation. We will examine the mechanisms by which lkBaDelta1-12 impairs NFkB function by use of electrophorectic mobility shift assays and the use of NFkB-Luciferase reporter constructs to quantitate NFkB activity. We will further study the role of NFkB in the development and function of the immune system by generating an lkBaDelta1-12 knock-in mouse model of impaired NFkB function. This mouse model will have the advantage of being non-lethal, compared to previous NFkB knock-out mouse models, which have lethal phenotypes. The lkBaDelta1-12 knock-in model will recapitulate the condition found in the patient who is the inspiration behind these studies. The mouse knock-in model will allow for analysis of the importance of IkBa and NFkB in immune system development and function and provide better insights into how the immune system protects against infectious diseases.
