In this laboratory we use our knowledge and experience in the area of protein structure and function to determine the chemical mechanisms employed by interesting biological factors. The major focus of the laboratory is in understanding regulation in the transcription factor NF-kappaB signal transduction pathway. NF-kappaB is a relatively small class of proteins that respond to diverse stimuli by activating the expression of numerous genes. NF-kappaB responsive genes include many of the key components of the cellular survival program including inflammatory cytokines, mediators and effectors of both innate and adaptive immunity, and inhibitors of apoptosis. Although proper NF-kappaB function is integral to a cell's ability to fight off infection and respond to stress, too much of an NF-kappaB response can contribute to states of chronic inflammation such as arthritis, asthma, multiple sclerosis, and colitis. Recently, it has been shown that chronically inflamed tissues can serve as hotbeds for tumor formation. Cellular processes that recognize and kill tumors in healthy tissues fail to function effectively under the influence of the NF-kappaB cell survival program. Chronic inflammation due to hyperactive NF-kappaB has also been shown to contribute to sclerotic formation in arteries and heart disease.

The prototypical NF-kappaB functions as a heterodimer of p50 and p65 subunits. NF-kappaB is present in the cytoplasm of all cells as an inactive factor in complex with a member of the IkappaB inhibitor protein family. Diverse NF-kappaB-inducing stimuli lead to activation of the IkappaB kinase complex (IKK). IKK is a large multisubunit complex that specifically phosphorylates a pair of serine amino acid side chains in the amino-terminal region of NF-kappaB complex-associated IkappaB. Once phosphorylated, IkappaB is recognized by a specific E3 Ubiquitin-protein ligase complex leading to its poly-ubiquitinylation. The 26 S proteasome can then recognize and proteolyze IkappaB. Removal of IkappaB renders NF-kappaB active. It rapidly translocates from the cytoplasm to the nucleus where it binds specifically to DNA elements within the promoter regions of target genes and activates their transcription (Figure 1).

We are currently working on the following NF-kappaB-related projects:

1) IKK structure and function. IKK is a multisubunit kinase complex that specifically phosphorylates IkappaB. Purification of IKK from cytokine-induced HeLa cells revealed that it is composed of three subunits. These are referred to as IKKalpha (IKK1), IKKbeta (IKK2), and IKKgamma (NEMO, FIP3). Both IKKalpha and IKKbeta contain similar domain organizations with an amino-terminal kinase domain followed by helical regions. We are interested in determining the structure of IKK including stoichiometry and identification of domains as well as correlating this with function in order to determine the source of specificity and mechanisms of regulation.

2) Nuclear IkappaB structure and function. The classical NF-kappaB inhibitor proteins, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, function primarily in the cell cytoplasm by masking NF-kappaB nuclear localization signals and blocking DNA binding. However, two additional classes of IkappaB proteins are also integral to NF-kappaB regulation.
The proteins p105 and p100 play a dual roles as IkappaB proteins and precursors of the mature NF-kappaB p50 and p52 subunits, respectively. The identification of a third general class of IkappaB proteins that function exclusively in the nucleus has been made recently. The nuclear IkappaB proteins include Bcl-3, IkappaBzeta (MAIL), and IkappaBNS. These proteins all show similar properties: their expression is regulated by NF-kappaB; they rapidly accumulate in the nucleus; and they have modulatory effects on NF-kappaB-dependent expression of specific target genes. In particular, we are interested in studying how IkappaBzeta (MAIL) functions in the nucleus to activate cytokine interleukin-6 (IL-6) expression in an NF-kappaB-dependent manner.

3) Natural inhibitors of NF-kappaB. Several natural cellular or pathogenic products have been shown to affect NF-kappaB regulation. We are interested in determining the mechanisms employed by these factors. In one case, the small protein Murr1 (COMMD1) has been shown to interfere with regular NF-kappaB activation. As a consequence, NF-kappaB-mediated replication of the HIV-1 virus is inhibited by treatment of Murr1. We are currently working to determine the mechanism of Murr1 inhibition of NF-kappaB activation.

A second area of focus in the lab is structure and ligand binding of the nicitinicoid family of ligand-gated ion channels. This class of transmembrane channel proteins functions to mediate fast synaptic transmission in the central nervous system and includes the receptors for acetylcholine, 5-hydroxytryptamine (5-HT; serotonin), gamma-amino-butyric acid (GABA), and glycine. In collaboration with researchers in the neurobiology laboratory of Dr. Palmer Taylor at the University of California San Diego School of Medicine, we recently determined the structure of a protein derived from the glial cells of the saltwater snail Aplysia californica that shows significant sequence homology and similarity in ligand binding specificity with the human nicotinic acetylcholine receptor ligand binding domain. We plan to expand this area of research by conducting structural characterization of ligand-bound complexes as well as working on mammalian receptors.