office: CSL 325A
- B.S. Biochemistry, Brigham Young University, Provo, Utah, 1995
- Ph.D. Chemistry, University of California San Diego, La Jolla, California, 2001, Advisor: Dr. Gourisankar Ghosh
- Postdoc Structural Biology, University of California San Diego, La Jolla, California, 2005, Fellow of the University of California University-wide AIDS Research Program
- Assistant and Associate Professor, Department of Chemistry & Biochemistry, San Diego State University, 2005-present
- Assistant and Associate Professor, Molecular Biology Institute, San Diego State University, 2005-present
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-κB signal transduction pathway. NF-κB is a relatively small class of proteins that respond to diverse stimuli by activating the expression of numerous genes. NF-κB 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-κB function is integral to a cell's ability to fight off infection and respond to stress, too much of an NF-κB 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-κB cell survival program. Chronic inflammation due to hyperactive NF-κB has also been shown to contribute to sclerotic formation in arteries and heart disease.
The prototypical NF-κB functions as a heterodimer of p50 and p65 subunits. NF-κB is present in the cytoplasm of all cells as an inactive factor in complex with a member of the IκB inhibitor protein family. Diverse NF-κB-inducing stimuli lead to activation of the IκB 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-κB complex-associated IκB. Once phosphorylated, IκB is recognized by a specific E3 Ubiquitin-protein ligase complex leading to its poly-ubiquitinylation. The 26 S proteasome can then recognize and proteolyze IκB. Removal of IκB renders NF-κB 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 two NF-κB-related projects:
IKK structure and function.
IKK is a multisubunit kinase complex that specifically phosphorylates IκB. Purification of IKK from cytokine-induced HeLa cells revealed that it is composed of three subunits. These are referred to as IKKα (IKK1), IKKβ (IKK2), and IKKgamma (NEMO, FIP3). Although IKKα and IKKβ are highly conserved protein subunits, they differ significantly in their cellular function. For example, the IKKβ subunit has been shown to be responsible for activating NF-κB in response to inflammatory stimuli by catalyzing the attachment of two phosphates near the amino-terminus of the classical IκB proteins. Furthermore, IKKβ 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 IKKβ subunit. We are also interested in understanding the structure of multisubunit IKK complexes including the role of IKKgamma in regulating catalytic activity of the enzyme.
Nuclear IκB structure and function.
The classical NF-κB inhibitor proteins, IκBα, IκBβ, and IκBε, function primarily in the cell cytoplasm by masking NF-κB nuclear localization signals and blocking DNA binding. However, two additional classes of IκB proteins are also integral to NF-κB regulation. The proteins p105 and p100 play a dual roles as IκB proteins and precursors of the mature NF-κB p50 and p52 subunits, respectively. The identification of a third general class of IκB proteins that function exclusively in the nucleus has been made recently. The nuclear IκB proteins include Bcl-3, IκBζ (MAIL), and IκBNS. These proteins all show similar properties: their expression is regulated by NF-κB; they rapidly accumulate in the nucleus; and they have modulatory effects on NF-κB-dependent expression of specific target genes. We have shown that in contrast to classical IκB proteins, the nuclear IκBζ protein binds preferentially to the NF-κB p50 homodimer. We also found that formation of this protein-protein complex does not remove the NF-κB homodimer from binding to target DNA. We are currently interested in studying how assembly of an IκBζNF-κB p50:DNA complex in the nucleus activates the expression of specific NF-κB responsive genes such as the cytokine interleukin-6 (IL-6).
Other projects in which we are currently involved include:
Structure and function of a muscle assembly co-chaperone
In collaboration with the students in the laboratory of Dr. Sanford I. Bernstein in the Department of Biology at San Diego State University we are studying the structure and in vitro biochemistry of the factor UNC-45 that functions to help assemble skeletal muscles and to repair the misfolded heads of myosin motor proteins. We are also using a novel method for recombinant expression and purification of myosin isoforms from whole flies to study the structural and biophysical consequences of disease-causing mutations on motor proteins.
Structure and antigen binding of therapeutic antibodies
We collaborate with several local biotechnology and pharmaceutical companies to understand the structural mechanisms of clinically relevant antibodies. Current projects in this area include: selective antigen binding of anti-lipid antibodies, antibodies that employ metal ions as bridging factors in antigen binding, and novel bispecific antibody designs to specifically target difficult antigens.
- "High-affinity, pan-specific monoclonal antibodies that target cysteinyl leukotrienes and show efficacy in an acute model of colitis," King A.N., Fleming J.K., Knapik S.S., Visentin B., Wojciak J.M. & Huxford T. J. Lipid Res. 2017 (in press). doi: 10.1194/jlr.M075614
- "Effect of mutation and substrate binding on the stability of Cytochrome P450BM3 variants," Geronimo I., Denning C.A., Rogers W.E., Othman T., Huxford T., Heidary D.K., Glazer E.C. & Payne C.M. Biochemistry 55 3594-3606, 2016.
- "Probing kinase activation and substrate specificity with an engineered monomeric IKK2," Hauenstein A.V., Rogers W.E., Shaul J.D., Huang D.-B., Ghosh G. & Huxford T. Biochemistry 53 2064-2073, 2014.
- "A structural basis for IκB Kinase 2 activation via oligomerization-dependent trans auto-phosphorylation," Polley S., Huang D.-B., Hauenstein A.V., Fusco A.J., Zhong X., Vu D., Schröfelbauer B., Kim Y., Hoffmann A., Verma I.M., Ghosh G. & Huxford T., PLoS Biol. 11 e1001581, 2013.
- "Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system," Caldwell J.T., Melkani G.C., Huxford T. & Bernstein S.I. Methods 56 25-32, 2012.
- Ashlee King, M.S. Thesis 2013. Crystallization of nucleic acid complexes: NF-κB p50 homodimer bound to G-IRE sequences and Holliday Junctions bound to inhibitors of recombination.
- Norman Zhu, Ph.D. Dissertation 2013. Structure and biochemistry of IκBζ: A nuclear IκB protein.
- Arthur Hauensten, Ph.D. Dissertation 2013. The role of oligomerization in human IKK2 activation as revealed by structural and biophysical studies.
- James T. Caldwell, Ph.D. Dissertation 2013. First x-ray crystal structure of an insect muscle myosin.
- Scott W. Lashbrook, M.S. Thesis 2014. In vitro analysis of tyrosine auto-phosporylation by the IKK2 enzyme.
- Christy Milani, M.S. Thesis 2015. Ideal DNA binding site spacity by NF-κB p50 homodimer.
- Ellie Nguyen, M.S. Thesis 2016. Biochemical study of prolonged NF-κB responses regulated by IκBβ.
- Peggy Chatfield, M.S. Thesis 2017. Feline Infectious Peritonitis: Biochemistry of the fatal hyperimmune response to Feline Enteric Coronavirus.