Research
We study the initiation, progression, and spread of malignancy using biochemical and biophysical methods. We are especially interested in determining the structures of protein-DNA and protein-protein complexes involved in the initiation, maintenance, and spread of cancer.
Below are descriptions of ongoing projects, involving transcriptional regulation of the important oncogene c-myc, and the characterization of the prometastatic protein Autotaxin.
The disregulated expression of the c-myc oncogene is among the most frequent molecular abnormalities associated with cancer. Counterbalancing forces on TFIIH by two proteins – the Fuse Binding Protein (FBP) and the FBP Interacting Repressor (FIR) – govern c-myc transcription. FBP is a realtime regulator of c-myc, recognizing melted, single stranded DNA upstream of the P1 promoter called the Far Upstream Element (FUSE). FBP possesses a tyrosine-rich C terminal activation domain that drives transcription by interacting with TFIIH, and FIR possesses an amino terminal repression domain that opposes TFIIH activation by FBP, restoring c-myc transcription to basal levels. Significantly, mutations in TFIIH defective in FIR binding are seen in the hereditary neoplastic syndrome Xeroderma Pigmentosum, and a splice variant of FIR lacking the amino terminal repression domain, and therefore unable to repress c-myc, was recently identified in human colorectal cancers but not in adjacent normal tissue. We have determined the structures of FBP and FIR to their cognate DNA site FUSE, and have proposed a mechanism for transcriptional control of the oncogene based on the structures and associated biochemistry.
We are also interested in understanding the enzymatic and structural basis of the human extracellular lysophospholipase D (lyso-PLD) enzyme, autotoxin (ATX), and in evaluating the effects of small molecule modulators of autotoxin’s lyso-PLD activity on tumor cell migration and angiogenesis. ATX hydrolyzes lysophosphatidyl choline into choline and lysophosphatidic acid (LPA), an extracellular signaling phospholipid that activates G-protein-coupled cell surface receptors, triggers cell proliferation, migration, and survival, and ultimately mediates events central to organism fate such as wound healing, brain development and vascular remodeling. Generation of extracellular LPA by ATX promotes tumor invasion, metastasis, and neovascularization of ras-transformed cells. The potent mitogenic activity in human ovarian cancer ascitic fluid is mediated by LPA. In addition, ATX is markedly overexpressed in ovarian carcinoma, and the invasiveness of human breast cancer correlates directly with ATX expression.
Despite the important roles LPA and ATX play in cell signaling, development and oncogenesis, understanding the basis and regulation of extracellular LPA synthesis has been limited by a lack of information regarding the chemical and physical properties of ATX, the only identified enzyme responsible for generating extracellular LPA. To understand how ATX-mediated LPA production contributes to cancer development and progression, and to identify and develop small molecule inhibitors of LPA, we wish to determine the chemical mechanism and physiological roles of this important enzyme and the three-dimensional structure of the enzyme in complex with substrate, product, and inhibitors.
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People
Current members of the Braddock laboratory:
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Braddock, Demetrios, M.D., Ph.D. Assistant Professor of Pathology |
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Hsin-Hao Hsiao: 4th year graduate student, Molecular Biophysics and Biochemistry: Structural determinants of FBP/FIR/FUSE on c-myc regulation |
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William Chang: 3rd year gradute student, Molecular Biophysics and Biochemistry: Biophysical and biochemical characterization of Autotaxin.
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Chi-Yen Lin: Research Associate: TFIIH expression, purification, and crystallization. FBP purification and crystallization
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Amy Ouellette: Postdoctoral Associate: Biological studies related to Autotaxin and ATX inhibitors
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Po-Han Chen: 4th year undergraduate, Molecular, Cellular, and Developmental Biology, Yale University; Structural determinants of FBP/FIR/FUSE on c-myc regulation.
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Hongwen Zhou: Research Associate: Autotaxin expression, purification, and crystallization, TFIIH expression and purification. |
Former Staff
Isaac Young: Uniform Services University School of Medicine
Maxime Debrosse: Mount Sinai School of Medicine
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Publications
Selected Publications:
Crichlow, G. V., Zhou, H., Yang, Y., Hsiao, H-H, Frederick, K. B., Debrosse, M., Yang, Y., Folta-Stogniew, E. J., Chung., H-J, Fan, C., De La Cruz, E. M., Levens, D.L., Lolis E., and Braddock, D.T. “Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition.” In press EMBO (2007)
Huth, J.R., Liping, Y., Collins, I., Mack, Mendoza, R., Isaac, B., Braddock, D.T., Muchmore, S.W., Comess, K.M., Fesik, S.W., Clore, G.M., Levens, D., Hajduk, P.J. “Benzoylanthranilic Acid Inhibitors of FBP Binding to the c-myc Promoter” (2004) J. Med. Chem., 47, 4851-4857
Braddock, D.T., Baber, J. L., Levens, D., and Clore, G. M., “The Solution Structure of a Complex between the KH3 domain of hnRNP K and Single Stranded DNA.” (2002) EMBO, 21 3476-3485
Braddock, D.T., Louis, J. M., Baber, J. L., Levens, D., and Clore, G. M., “Solution Structure and Dynamics of a Specific Complex Between the KH3 and KH4 Domains of FBP and the Single Stranded Far Upstream Element of C-Myc.” (2002) Nature, 415 1051-1056
Braddock,D.T., Cai, M., Baber, J. L., Huang, Y., and Clore, G. M., “Rapid Identification of Medium to Large Scale Interdomain Motion in Modular Proteins using Dipolar Couplings” (2001) J. Am. Chem. Soc, 123 8634-8635
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