The research of my laboratory focuses on elucidating the molecular basis of cell communication. My current researches deal with the biology of bacterial adenylyl cyclase toxins, proteins that secreted by human bacterial pathogens. These toxins by themselves are not active till they enter into target cells and are associated with cellular proteins that serve as the activator. These adenylyl cyclase toxins become highly active and can then raise the intracellular cyclic AMP (cAMP) of its host cells to pathogenic levels. Cyclic AMP is a prototypic diffusible second messenger that controls diverse physiological responses. The unregulated increase of intracellular cAMP level can alter the functions of host cells to benefit the bacterial propagation.

      One of such adenylyl cyclase toxins is edema factor (EF) secreted by Bacillus anthracis, the etiologic agent for anthrax. The other is CyaA secreted Bordetella pertussis that causes whooping cough. Both EF and CyaA bind the cellular calcium sensor, calmodulin, with high affinity. We have solved the x-ray structures of EF and CyaA as well as applied biochemical and biophysical analyses to address how calmodulin binds and activates EF and CyaA. We will continue these approaches to elucidate the principles in how protein-protein interaction leads to catalytic activation as well as how two proteins from two different organisms evolve to gain the desired biological activities. Many bacterial toxins, such as Botulinum toxin (BoTox) and Cholera toxin, have been developed as the experimental and therapeutic tools. We are currently exploring the therapeutic potential of adenylyl cyclase toxin in cancer treatment.

      Adenylyl cyclase toxins have also been identified biochemically from Pseudomonas aeruginosa which is one of hospital-acquired pathogens that threatens the health of the immuno-compromised patients such as those with AIDS or cystic fibrosis (ExoY). Genomic sequences of Yersinia pestis (plague), Yersinia pseudotuberculosis (gastrointestinal syndromes), Vibrio Cholerae (massive diarrhea) reveal two novel members of adenylyl cyclase toxins. This suggests that adenylyl cyclase toxin may be used broadly by pathogenic bacteria to alter the host defense. We will apply biochemical, structural, and pharmacologic approaches to analyze the roles of these adenylyl cyclase toxins in bacterial pathogenesis.

      The incident of bioterrorism-related anthrax in 2001 has moved the challenge against anthrax from an obscure agricultural problem to the center of biodefense. Given the ease of making antibiotic-resistant anthrax strains and unknown enemies, the best defense against anthrax is to build up a battery of possible antidotes against anthrax. We have developed several small molecular anti-anthrax toxin leads that can potently inhibit the action of anthrax toxins, EF and lethal factor. We will continue to discover and improve anti-anthrax toxin leads, which could then be further developed as the adjunct therapeutic against anthrax infection.

References

1. Tang, W.-J. and Gilman, A. G. (1991) Type-specific regulation of adenylyl cyclase by G protein bg subunits. Science 254:1500-1503. 2. Tang, W.-J., Krupinski, J. and Gilman, A.G. (1991)  Expression and characterization of calmodulin activated (type 1) adenylyl cyclase. J.Biol.Chem..266:8595-8603. 3. Tang, W.-J. and Gilman, A.G. (1995) Forskolin and Gsa sensitive soluble adenylyl cyclase.  Science 268:1769-1772. 4. Yan, S.-Z., Hahn, D., Huang, Z.-H., and Tang, W.-J. (1996) Two cytoplasmic domains of mammalian adenylyl cyclase form a Gsa and forskolin-activated enzyme in vitro. J. Biol. Chem.. 271:10941-10945. 5. Yan, S.-Z., Huang, Z.-H., Shaw, R.S., and Tang,W.-J. (1997) The conserved asparagine and arginine are crucial for catalysis of mammalian adenylyl cyclase.  J. Biol. Chem.. 272:12342-12349. 6. Yan, S.-Z., Huang, Z.-H., Rao, V.D., Hurley, J.H., and Tang, W.-J. (1997) Three discrete regions of mammalian adenylyl cyclase form a site for Gsa activation.  J. Biol. Chem. 272:18849-18854. 7. Yan,S.-Z., Huang, Z.-H., Andrews R.K., and Tang,W.-J. (1998) Conversion of forskolin insensitive to sensitive (mouse type IX) adenylyl cyclase. Mol. Pharm. 53:182-187. 8. Drum, C.L., Yan, S.-Z., Sarac, R., Mabuchi, Y., Beckingham, K., Bohm, A., Grabarek, Z., and Tang, W.-J. (2000) An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.  J. Biol. Chem. 275:36334-36340. 9. Yan, S.-Z. Beeler, J. Chen, Y., Shelton, R.K., and Tang, W.-J. (2001) The regulation of human type 7 adenylyl cyclase by C1b and E. coli peptidyl prolyl isomerase, slyD. J. Biol. Chem 276: 8500-8506. 10. Drum, C.L., Yan, S.-Z.,  Bard, J., Shen, Y.-Q., Lu, D., Soelaiman, S., Grabarek, Z., Bohm, A., and Tang, W.-J. (2002) Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin. Nature 415:396-402. 11. Shen, Y.-Q., Lee, Y.-S., Soelaiman, S., Bergson, P., Lu, D., Chen, A., Beckingham, K., Grabarek, Z., Mrksich, M., Tang, W.-J. (2002) Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins. EMBO J. 21: 6721-6732. 12. Soelaiman, S., Wei, B., Bergson, P., Lee, Y.-S., Shen, Y., Mrksich, M., Shoichet, B., Tang, W.-J. (2003) Structure-based inhibitor discovery against anthrax adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough. J. Biol. Chem. 278:25990-25997. 13. Shen, Y.-Q., Zhukovskaya, N.L., Zimmer, M.I., Soelaiman., S., Wang, C.R., Gibbs, C.S., and Tang, W.-J. (2004) Selective inhibition of anthrax edema factory by adefovir, a drug for chronic hepatitis B virus infection. Proc. Natl. Acad., Sci., U.S.A. 101:3242-3247. 14.  Guo, Z., Shen, Y.-Q., Zhukovskaya, N.L. Florian, J., and Tang, W.-J. Structural and kinectic analyses of the interaction of anthrax adenylyl cyclase toxin with reaction products, cAMP and pyrophosphate. J. Biol Chem. 279:29427-29435. 15.  Lee, Y.-S., Bergson, P., He, W.-S., Mrksich., M., and Tang., W.-J. (2004) Discovery of a small molecule that inhibits the interaction of anthrax edema factor with its cellular activator, calmodulin. Chem & Biol. 11:1139-1146. 16.  Beeler, J.A., Yan, S.-Z., Bykov, S., Murza, A., Asher, S., and Tang, W.-J. (2004) A stable C1b soluble protein and its regulation of soluble type 7 adenylyl cyclase:  a prototype for soluble C1b model. Biochemistry. 43(49):15463-15471. 17.  Shen, Y., Zhukovskaya, N.L., Guo, Q., Florian, J., and Tang, W.-J.  (2005)  Calcium-independent calmodulin binding and two-metal-ion catalytic mechanism of anthrax edema factor.  EMBO J. 24:929-941. 18.  Guo, Z., Shen, Y., Lee., Y.-S., Gibbs, C.S., Mrksich, M., and Tang., W.-J. (2005) Structural basis for the interaction of adenylyl cyclase toxin of Bordetella pertussis with calmodulin.  EMBO J. 24:3190-3201.