UC San Diego

In Drosophila, the Immune Deficiency (IMD) pathway is indispensable for proper innate immune responses. In this pathway, infection by gram-negative bacteria elicits a signaling cascade culminating in the rapid induction of antimicrobial peptide gene expression by the NF-κB transcription factor Relish. Signal-dependent activation of Relish is an essential component of the IMD pathway and is regulated by the Drosophila melanogaster IκB Kinase (DmIKK) complex. DmIKK is composed of two subunits: the catalytic subunit DmIKKβ (homologous to mammalian IKKβ) and non-catalytic subunit DmIKKγ (homologous to mammalian NEMO/IKKγ). Since the discovery of the IMD pathway in 1996, much of the molecular components of this pathway have been identified. However, the molecular details of regulation have remained equivocal. This dissertation investigates the biochemistry, substrate specificity and activation mechanism of this kinase complex using recombinant DmIKKβ:γ complex co-expressed using the Baculovirus system. In chapter III, phylogenetic analysis of extant metazoan taxa reveals that DmIKK has diverged significantly in sequence. Primary sequence comparison with mammalian IKK subunits and other metazoan taxa identified conserved as well as unique regions that may be important for its regulation and/or activity. Chapter IV is an in vitro study on the nature and assembly of the complex in solution using biophysical methods such as analytical scale size-exclusion chromatography (SEC), multi-angle laser light scattering (MALLS) and analytical ultracentrifugation (AUC). We show that our recombinant DmIKKβ:γ complex can non-covalently interact with poly-ubiquitin via its DmIKKγ subunit. This interaction may be a component of its signal-dependent regulatory mechanism. In chapter V, structural studies using negative stain transmission electron microscopy (TEM) examine the three-dimensional structure of this critical enzyme complex.