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).
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). Although IKKalpha and IKKbeta are highly conserved protein subunits, they differ significantly in their cellular function. For example, the IKKbeta subunit has been shown to be responsible for activating NF-kappaB in response to inflammatory stimuli by catalyzing the attachment of two phosphates near the amino-terminus of the classical IkappaB proteins. Furthermore, IKKbeta itself is subject to phosphorylation-dependent regulation of its own catalytic activity. We are interested in understanding the detailed mechanisms of substrate specificity and phosphorylation-dependent regulation of the IKKbeta subunit.
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 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. We have shown that in contrast to classical IkappaB
proteins, the nuclear IkappaBzeta protein binds preferentially to the NF-kappaB
p50 homodimer. We also found that formation of this protein-protein complex
does not remove the NF-kappaB homodimer from binding to target DNA. We are
currently interested in studying how assembly of an IkappaBzeta/NF-kappaB
p50/DNA complex in the nucleus activates the expression of specific NF-kappaB
responsive genes such as the cytokine interleukin-6 (IL-6).
Other Projects in which we are currently involved include:
3) Structure and function of a muscle repair protein. In collaboration with the Drosophila Genetics laboratory of Dr. Sanford I. Bernstein in the Department of Biology at San Diego State University we are studying the structure of the factor UNC-45 that functions in muscles to repair the misfolded heads of myosin motor proteins.
4) The recognition and modification of sphingolipids. The hydrolytic products of membrane sphingolipids are potent signaling molecules. In collaboration with the local biotechnology company LPath, Inc. and the synthetic organic laboratory of Dr. Tom Cole, we are studying the structures of antibodies that have been raised to recognize specific sphingolipid hydrolysis products. We are also participating in the design and assay of small molecule inhibitors of sphingomyelinase enzymes from pathogenic bacteria and spider venom.
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