- 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
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
We are currently working on the following two NF-kappaB-related projects:
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.
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. We have
shown that in contrast to classical IkappaB proteins, the nuclear IkappaBzeta
protein binds preferentially to the NF-kB 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-kB responsive genes such as the cytokine interleukin-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
Structure and selectivity of anti-lipid antibodies
The hydrolytic products of membrane sphingolipids are potent signaling
molecules. In collaboration with the San Diego-based biotechnology company
LPath, Inc. we are studying the structures of antibodies that have been raised
to recognize specific biologically active lipids.
Phosphate recognition by STS-1
STS-1 is an enigmatic factor that influences signal transduction in T
lymphocytes in response to engagement of their T-cell receptor. In
collaboration with the laboratory of Dr. Peter van deer Geer in the Department
of Chemistry & Biochemistry at San Diego State University we are working to
understand the mechanism by which phosphorylation of specific signaling
proteins influences the function of STS-1.
- "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.
56 25-32, 2012.
- "Biochemical and structural characterization of lysophosphatidic acid binding
by a humanized monoclonal antibody," Fleming J.K., Wojciak J.M., Campbell M.A.
& Huxford T. J. Mol. Biol. 408 462-476, 2011.
- "X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone
from Drosophila melanogaster," Lee C.F., Hauenstein A.V., Fleming J.K., Gasper
W.C., Engelke V., Sankaran B., Bernstein S.I. & Huxford T. Structure 19
- "Understanding the logic of IkappaB:NF-kappaB regulation in structural
terms," Huxford T., Hoffmann A. & Ghosh G. Curr. Top. Microbiol. Immunol. 349
- "A structural guide to proteins of the NF-kappa B signaling module," Huxford
T. & Ghosh G. Cold Spring Harbor Perspectives in Biology 1 a000075, 2009.
- "The crystal structure of sphingosine-1-phosphate in complex with a Fab
fragment reveals metal bridging of an antibody and its antigen," Wojciak J. M.,
Zhu N., Schuerenberg K.T., Moreno K., Shestowsky W.S., Hiraiwa M., Sabbadini
R., & Huxford T., Proc. Natl. Acad. Sci. USA 106 17717-17722, 2009.
- "The human IKKbeta subunit kinase domain displays CK2-like phosphorylation
specificity," Shaul J.D., Farina, A. & Huxford T., Biochem. Biophys. Res.
Commun. 374 592-597, 2008.
- "The nuclear IkappaB protein IkappaBzeta specifically binds NF-kappaB p50
homodimers and forms a ternary complex on kappaB DNA," Trinh D.V., Zhu N.,
Farhang G., Kim B.J. & Huxford T., J. Mol. Biol. 379 122-135, 2008.
MS Theses and Ph.D. Dissertations
- Dan V. Trinh, M.S. Thesis 2007.
Biochemical characterization of IkappaBzeta interactions with NF-kappaB/DNA complexes.
- Jacob D. Shaul, M.S. Thesis 2008.
IKKbeta phosphorylates IkappaBalpha with CK2-like specificity upon removal of C-terminal elements
- Ghazal Farhang, M.S. Thesis 2008.
Assaying transcriptional activation by NF-kappaB p50 Homodimer/IkappaBzeta complexes on kappaB DNA promoters
- Michaela S. Norrbom, M.S. Thesis 2008.
Expression, purification, and in vitro characterization of the Drosophila melanogaster IkappaB Kinase beta subunit
- Jon K. Fleming, M.S. Thesis 2010.
Crystallization and x-ray structure determination of the antigen binding portion of a humanized monoclonal anti-lysophosphatidic acid antibody
- Jesal C. Patel, M.S. Thesis
Biochemical characterization of a phosphate-binding domain in STS-1
- Mark Villaluz, M.S. Thesis 2011.
Purification and Kinetic Characterization of Soluble Sphingomyelinases C and D.
- Danielle DiTirro, M.S. Thesis 2011.
Large-scale expression and characterization of a human multi-subuit IKK complex.
- Frank Erasmus, M.S. Thesis 2012. Characterization of metal binding by the anti-sphingosine-1-phosphate metalloantibody LT1002.
- Deepthi Anipindi, M.S. Thesis 2012. X-ray crystal structures of two thermostable metal binding variants of Streptococcal Protein Gβ1 domain.