个人简介

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

研究领域

Biochemistry

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. NFkB activation 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 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 (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. 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.

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Hauenstein A.V., Rogers W.E., Shaul J.D., Huang D.-B., Ghosh G. & Huxford T. (2014). Probing kinase activation and substrate specificity with an engineered monomeric IKK2. Biochemistry 53 2064-2073. PubMed Weerasinghe A.J., Amin S.A., Barker R.A., Othman T., Romano A.N., Parker Siburt C.J., Tisnado J., Lambert L.A., Huxford T., Carrano C.J. & Crumbliss A.L. (2013). Borate as a synergistic anion for Marinobacter algicola Ferric Binding Protein, FbpA: A role for boron in iron transprot of marine life. J. Amer. Chem. Soc. 135 14504-14507. PubMed 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. (2013). A structural basis for IkappaB Kinase 2 activation via oligomerization-dependent trans auto-phosphorylation. PLoS Biol. 11 e1001581. PubMed Caldwell J.T.., Melkani G.C., Huxford T. & Bernstein S.I. (2012). Transgenic expression and purification of myosin isoforms using the Drosophila melaogaster indirect flight muscle system. Methods 56 25-32. PubMed Fleming J.K., Wojciak J.M., Campbell, M.A. & Huxford T. (2011). Biochemical and structural characterization of lysophosphatidic acid binding by a humanized monoclonal antibody. J. Mol. Biol. 408 462-476. PubMed Lee C.F., Hauenstein A.V., Fleming J.K., Gasper W.C., Engelke V., Sankaran B., Bernstein S.I. & Huxford T. (2011). X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone from Drosophila melanogaster. Structure 19 397-408. PubMed Wojciak J.M., Zhu N., Schuerenberg K.T., Moreno K., Shestowsky W.S., Hiraiwa M., Sabbadini R. & Huxford T. (2009). The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen. Proc. Natl. Acad. Sci. USA 106 17717-17722. PubMed Shaul J.D., Farina, A. & Huxford T. (2008). The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity. Biochem. Biophys. Res. Commun. 374 592-597. PubMed Trinh D.V., Zhu N., Farhang G., Kim B.M. & Huxford T. (2008). The nuclear IkappaB protein IkappaBzeta specifically binds NF-kappaB p50 homodimers and forms a ternary complex on kappaB DNA. J. Mol. Biol. 379 122-135. PubMed Bergqvist S., Croy C.H., Kjaergaard M., Huxford T., Ghosh G. & Komives E.A. (2006). Thermodynamics reveal that helix four in the NLS of NF-kappaB p65 anchors IkappaBalpha, forming a very stable complex. J. Mol. Biol. 360 421-434. PubMed Hansen S.B., Sulzenbacher G., Huxford T., Marchot P., Taylor, P.W. & Bourne, Y. (2005). Structures of Aplysia AChBP complexes with agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24 3635-3646. PubMed Croy C.H., Bergqvist S., Huxford T., Ghosh G. & Komives E.A. (2004). Biophysical characterization of the free IkappaBalpha ankyrin repeat domain in solution. Protein Sci. 13 1767-1777. PubMed Malek S., Huang D.-B., Chen Y., Huxford T., Ghosh S. & Ghosh G. (2003). X-ray crystal structure of an IkappaBbeta/NF-kappaB p65 homodimer complex. J. Biol. Chem. 278 23094-23100. PubMed Huxford T., Mishler D., Phelps C.B., Chen Y., Sengchanthalangsy L.L., Reeves R., Hughes C.A., Komives E.A. & Ghosh G. (2002). Solvent exposed non-contacting amino acids play a critical role in NF-kappaB/IkappaBalpha complex formation. J. Mol. Biol. 324 587-597. PubMed Malek S., Chen Y., Huxford T. & Ghosh G. (2001). IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells. J. Biol. Chem. 276 45225-45235. PubMed Huxford T., Malek S., & Ghosh G. (2000). Preparation and crystallization of dynamic NF-kappaB/IkappaB complexes. J. Biol. Chem. 275 32800-32806. PubMed Phelps C., Sengchanthalangsy L.L., Huxford T. & Ghosh G. (2000). Mechanism of IkappaBalpha binding to NF-kappaB dimers. J. Biol. Chem. 275 29840-29846. PubMed Huxford T., Huang D.-B., Malek S. & Ghosh G. (1998). The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 95 759-770. PubMed Malek S., Huxford T. & Ghosh G. (1998). IkappaBalpha functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-kappaB. J. Biol. Chem. 273 25427-25435. PubMed Huang D.-B., Huxford T., Chen Y.-Q. & Ghosh G. (1997). The role of DNA in the mechanism of NF-kappaB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits. Structure 5 1427-1436. PubMed