NF-kappa-B has been shown to play a role in tumorgenesis by inducing cell proliferation, anti-apoptosis, and angiogenesis. We have recently observed that a small pool of NF-kappa-B inhibitor molecule, I-kappa-B-alpha, remains free. This pool of free I-kappa-B-alpha, which is maintained through its continuous synthesis and degradation, is necessary for the proper inhibition of NF-kappa-B in resting cells.

We have further shown that free I-kappa-B-alpha degradation does not require its phosphorylation or ubiquitination, suggesting an alternative degradation pathway. Given that the 26S proteasome is known to carry out ubiquitin (Ub) dependent degradation, it is possible that a different form of the proteasome is involved in the Ub- independent degradation of free I-kappa-B-alpha. Similarly, the continuous processing of p105, an I-kappa-B-like precursor of NF-kappa-B p50 subunit, requires the proteasome but does not require phosphorylation and ubiquitination of p105. In in vitro degradation assays, 20S proteasome, which is the catalytic core of the proteasome, can degrade recombinant I-kappa-B-alpha, and generates p50 from p105.

We hypothesize that a pool of 20S proteasome can degrade proteins in an Ub-independent manner. Its activity could be regulated either by post-translational modifications and/or different subunit composition. We also hypothesize that the 20S proteasome must recognize a signal in the non- ubiquitinated protein that acts as a degradation signal for the protein. Thus, the project goals are to: 1) characterize the form of 20S proteasome involved in Ub- independent degradation; and 2) identify the proteasome recognition signal on the substrate for its Ub- independent degradation.

Jessica has shown that "active" and "inactive" 20S can be obtained by performing the purification in the absence and presence of reducing agent, respectively. She has further shown that the 20S proteasome isolated from cells treated with oxidizing agents rapidly degrades both recombinant and endogenous IkappaBalpha, compared to untreated cells. Her next goal is to identify subunit composition and modification using mass spectroscopy.

It is reasonable to consider then that oxidative modification on the substrate also affects its ability to be degraded. It is hypothesized that the degradation signal for these oxidized substrates are hydrophobic residues, which are exposed upon unfolding of the protein. Jessica will use a mouse embryonic fibroblast cell line to identify the substrate recognition signal in I-kappa-B-alpha using mutational and functional analysis.

The regulation of protein stability by the proteasome system is a central molecular event in cancer biology, since it regulates the degradation of proteins involved in apoptosis, angiogenesis, cell cycle regulation, and transcription. This is why proteasome inhibitors are being considered as drugs for breast cancer treatment.

PUBLICATIONS

Moorthy, A., Savinova, O., Ho, J.Q., Wang, V., Vu, D., Ghosh, G. The 20S proteasome processes NF-kappa-B1 p105 into p50 in a translation-independent manner. The EMBO Journal (2006) 25:1945-1956.