Cells are capable of rapidly responding to changes in their environment, including the presence of pathogens or noxious conditions. Molecular signaling pathways that regulate these responses show highly dynamic patterns of activity. Indeed, the controls of half-life and protein degradation are hallmarks of many signaling pathways. In the case of the NF[kappa]B regulatory pathway, the key mediator of inflammatory responses, the inhibitor proteins I[kappa]B[alpha], -[beta], and -ε are known to be regulated by signal-responsive mechanisms freeing NF[kappa]B. Recent work also indicates that I[kappa]B[alpha] is synthesized in excess in resting cells to ensure that NF[kappa]B activity remains effectively inhibited. Rapid degradation of the excess I[kappa]B is critical so that NF[kappa]B activation can proceed rapidly when inflammatory responses are needed. Cellular protein degradation is catalyzed by the proteasome, a large molecular machine. Proteins are usually targeted for degradation by a specific post-translational modification, the covalent attachment of the small molecule ubiquitin. In this dissertation I report that I[kappa]B[alpha] does not require such modifications for its rapid degradation, but instead relies on a specific amino-acid sequence, termed a degron, that targets even heterologous proteins to the proteasome. Interestingly these amino acids are buried within the I[kappa]B[alpha]-NF[kappa]B complex, thus stabilizing I[kappa]B[alpha], and allowing it to effectively inhibit NF[kappa]B. This work describes a novel determinant of protein half-life control and shows that it is amenable to regulation by a post-translational mechanism to stabilize the target protein. In examining the other I[kappa]B family members, I show that while I[kappa]B[beta] may undergo similar control, I[kappa]B[epsilon] is in fact a stable protein that maybe less suited to ensure NF[kappa]B inhibition in resting cells

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an 'activator-induced inhibitor' regulatory motif. Within the NFκB signalling pathway, a key negative feedback regulator is IκBα. We show here that, contrary to current understanding, NFκB-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NFκB signalling to identify IκBα molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of IκBα and the IκBα-NFκB complex, as well as the free IκBα half-life, as key determinants of post-induction repression of NFκB and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required 'activator-induced inhibitor' relationship.

Whereas ubiquitin-dependent degrons have been characterized in some detail, how proteins may be targeted to ubiquitin-independent proteasomal degradation remains unclear. Here we show that IκBα contains an ubiquitin-independent degron whose activity is portable to heterologous proteins such as the globular protein GFP (green fluorescent protein) via a proteasome-dependent, ubiquitin-independent, non-lysosomal pathway. The ubiquitin-independent degradation signal resides in an 11-amino-acid sequence, which is not only sufficient but also required for IκBα's short half-life. Finally, we show that this degron's activity is regulated by the interaction with NFκB, which controls its solvent exposure, and we demonstrate that this regulation of the degron's activity is critical for IκBα's signaling functions.

Increasing incidence of inflammatory bowel disorders demands a better understanding of the molecular mechanisms underlying its multifactorial aetiology. Here we demonstrate that mice deficient for REGγ, a proteasome activator, show significantly attenuated intestinal inflammation and colitis-associated cancer in dextran sodium sulfate model. Bone marrow transplantation experiments suggest that REGγ's function in non-haematopoietic cells primarily contributes to the phenotype. Elevated expression of REGγ exacerbates local inflammation and promotes a reciprocal regulatory loop with NFκB involving ubiquitin-independent degradation of IκBɛ. Additional deletion of IκBɛ restored colitis phenotypes and inflammatory gene expression in REGγ-deficient mice. In sum, this study identifies REGγ-mediated control of IκBɛ as a molecular mechanism that contributes to NFκB activation and promotes bowel inflammation and associated tumour formation in response to chronic injury.