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Pharmocology and Molecular Sciences
725 N. Wolfe St.
Baltimore MD 21205
Chemical biology and molecular biology; use of small molecules as probes to various cellular processes and drug leads to treat diseases
We are interested in a molecular understanding of intracellular signal transduction pathways involved in T lymphocyte activation and apoptosis and regulation of endothelial cell proliferation. We employ a combination of techniques from protein biochemistry, molecular and cell biology and synthetic organic chemistry. Engagement of T cell receptor (TCR) with MHC-antigen complex can lead to opposite outcome, activation or death of T cells, depending on the strength or avidity of the signal. Transcriptional activation of the interleukin (IL)-2 gene is largely responsible for T cell proliferation while that of the Nur77 family of the orphan steroid receptors mediates T cell apoptosis. The second messenger calcium has been shown to be critical for both IL2 and Nur77 expression. We have been focusing on how calcium signal is transduced from the cytosol into the nucleus to activate IL2 and Nur77 genes. Two of the key mediators of calcium signaling for IL2 expression are the Ca2+, calmodulin-dependent protein phosphatase calcineurin and its substrate NFAT. In contrast, how calcium signal is transduced to activate the Nur77 gene has remained largely unknown. Unlike IL-2, the major calcium-responsive elements in the Nur77 promoter bind to a transcription factor known as myocyte enhancer factor 2 (MEF2). We discovered a novel calcineurin-binding protein named Cabin1. We have shown that Cabin1 plays important roles in both T cell activation and thymocyte apoptosis. Cabin1 serves as a negative regulator of calcineurin during T cell activation. Cabin1 also binds MEF2, repressing its transcriptional activity. TCR-induced calcium influx leads to the dissociation of MEF2 from Cabin1, as a result of competitive binding of activated calmodulin to Cabin1. The interplay between Cabin1, MEF2 and calmodulin represents a novel mechanism of calcium-dependent transcriptional regulation and defines a direct signaling pathway from calcium to MEF2. In addition to Cabin1, calcineurin is also required for full activation of MEF2, the mechanism of which remains unknown. We are currently investigating how calcineurin signal is integrated into MEF2 for Nur77 expression and how Cabin1 achieves transcriptional repression of MEF2. Angiogenesis, formation of new blood vessels, is necessary for tumor growth and metastasis as well as several other human diseases. Inhibition of angiogenesis is emerging as a promising strategy for treating cancer. Fumagillin and ovalicin are structurally-related natural products that exhibit potent anti-angiogenic and immunomodulatory activities. The inhibition of angiogenesis is mediated through the inhibition of cell cycle progression of endothelial cells, the central component of all blood vessels. An analog of fumagillin, TNP-470, is currently in clinical trials for several types of cancer. Using photoaffinity labeling and affinity chromatography in conjunction with mass spectrometry, we have purified and identified a molecular target for both TNP-470 and ovalicin as the type 2 methionine aminopeptidase (MetAP2), revealing a possible key role of MetAP2 in endothelial cell proliferation. Recently, we found that TNP-470 inhibits cell cycle progression of endothelial cells by activating p53 that induces p21, leading to the inhibition of CyclinE-Cdk2 activity. We are currently investigating how inhibition of MetAP2 leads to activation of p53 by identifying its substrates in endothelial cells. We are also investigating the molecular basis of the high-affinity and high-specificity binding of TNP-470 and ovalicin by MetAP2. Small ligand-receptor interactions underlie many fundamental biological processes and form the basis for pharmacological treatment of human diseases. As chemical diversity grows from combinatorial chemistry, there is an increasing demand for general, simple and sensitive methods for detecting interactions between ligands and proteins. To exploit the power of yeast genetics, we have developed a general method to detect ligand-protein receptor interactions in yeast by combining a hybrid ligand with the yeast two-hybrid system. By creating heterodimers of two small organic ligands and fusion proteins between the receptors for the individual ligands to a DNA binding domain and a transcription activation domain, respectively, we showed that hybrid ligands can activate reporter genes in yeast, leading to growth in selective medium or color change due to lacZ gene activation. The yeast three-hybrid system offers many exciting opportunities for future research including identification of new protein targets for “orphan” organic ligands, and selection of proteins possessing novel binding and catalytic properties.
Zhang L-H, and Liu JO. Sanglifehrin A, a novel cyclophilin-binding immunosuppressant, inhibits IL-2 dependent T cell proliferation at the G1 phase of the cell cycle. J. Immunol., 166:5611-5618, 2001.
Griffith EC, Licitra EJ and Liu JO. A yeast three-hybrid system for detecting ligand-receptor interactions. Meth. Enzymol., 328:89-103, 2000.
Zhang Y, Griffith EC, Sage J, Jacks T, and Liu JO. Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1/CIP1. Proc. Natl. Acad. Sci. USA, 97:6427-6432, 2000.
Youn H-D, Grozinger CM, and Liu JO. Calcium regulates transcriptional repression of myocyte enhancer factor 2 by histone deacetylase 4. J. Biol. Chem., 275:22563-22567, 2000.
Youn H-D. and Liu JO. Regulation of the MEF2 family of transcription factors by calcium-regulated recruitment of p300 and histone deacetylases. Immunity, 13:85-94, 2000.
Youn H-D, Chatila T, and Liu JO. NFAT mediates integration of calcineurin and MEF2 signals by the Coactivator p300. EMBO J., 19:4323-4331, 2000.
Youn H-D, Sun L, Prywes R and Liu JO. Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2. Science, 286:790-793, 1999.