Woei-Jer Chuang

Chair Professor

Professor Woei-Jer Chuang


 
[Disintegrin] [Microperoxidase] [ILF] [SPE B] [Shark Cartilage] [Cordyceps Sinensis]

Disintegrin/Integrin

Structural and Functional Studies of Rhdostomin and its Integrin Complex

Our aim in this project is to study the structure and function relationships of rhodostomin (Rho), peptides derived from Integrin a and b subunits, and their complexes. We have made progress in the following studies:

  1. Expression: We have expressed Rho, and its D51E and R49K mutants in yeast expression system (Pichia pastoris), and the yield is more than 8 mg per liter. Based on functional and structural studies, they have correct folding.
  2. Function: The IC50 for the inhibition of platelet aggregation by wild-type, R49K, and D51E mutants are 70 nM, 215 nM, and 50 mM, respectively. R49K and D51E mutants are 3.1-fold and 714-fold weaker than wild-type Rho. These results indicate that negative charge residue, D51, plays the most important role in the binding between disintegrin and integrin.
  3. NMR assignment and structure: We have successfully labeled Rho and its D51E mutant with 15N. Therefore, we are able to resolve the ambiguities in 2D NOESY and to obtain high resolution structure. Based on our CD and NMR analyses, the secondary structures of Rho and its D51E mutant at pH 2.2 are almost the same and their tertiary structures are similar except that slight change in NH and 15N chemical shifts of D51 (E51) and M52. The determinations of the differences between tertiary structures of Rho and its D51E mutant are on going.
  4. pH dependence on structure: CD analysis show that the secondary structures of Rho and its R49K and D51E produced in Pichia are pH independent; however, we found that NH and 15N chemical shifts of C6, S7, S8, A17, T18, I47, D63, and Y67 are pH sensitive(DNH > 0.5 ppm and D15N > 2ppm). Determination of the solution structures of Rho and its D51E mutant at pH (2, 4, 6 and 8) are on going.

 

Microperoxidase

EFFECTS OF AXIAL LIGANDS AND METAL IONS ON THE STRUCTURE AND FUNCTION RELATIONSHIPS OF MICROPEROXIDASES

Our aims of this project are to study the structure and function relationships and mechanisms of heme enzymes including peroxidases, catalase, cytochrome p450, cytochrome c, and cytochrome b5. A number of novel microperoxidases are prepared, different enzymatic assays are used, and the solution structures and dynamic properties of these microperoxidases also will be studied by NMR methods. In order to accomplish our aims, we propose the following experiments:

  1. Effect of Fifth Ligand: We will use genetic, enzymatic, and chemical approaches to prepare His-Ac-MP-8, Tyr-Ac-MP-8, and Cys-Ac-MP-8 which will be models for peroxidase, catalase, and cytochrome P450, respectively. The effects of proximal ligands on the peroxidase, catalase, and monooxygenase activities of these heme models will be determined.
  2. Effect of Sixth Ligand: We will prepare His-Ac-MP-8-His, and His-Ac-MP-8-Met as models for cytochrome b5, and cytochrome c, respectively.
  3. Effect of Distal Residues: We will covalently couple the propanoic acids on heme groups His-Ac-MP-8 with the amine group of peptides or amino acid analog. The effects of distal residues on the activities of peroxidase, catalase, and monooxygnase will be determined.
  4. Effect of Metal Center: We will prepare Fe3+-, Cu2+-, and Mn3+-Ac-MP-8 to study the role of metal ions and their oxidation states in activities of peroxidases.
  5. Developments of assays: We will develop enzymatic assays for peroxidase-, catalase-, and cytochrome p450-type reactions.

 

DNA-binding domain of interleukin binding factor

Functional and Structural Studies of Interleukin Enhancer Factor

The long-term goals of this project are to study the structure and function relationships and dynamic properties of interleukin enhancer factor (ILF) and its DNA complex also will be studied. Since ILF can inhibit the replication of HIV-1, we eventually will design the transciptional drugs for targeting LTR of HIV-1. Specific aims are listed as follows:

  1. We will use gel retardation assay and CD titration to characterize what DNA sequence recognized by the DNA-binding domain of ILF.
  2. We will determine the three dimensional structures and dynamic properties of DNA-binding domain of ILF and its DNA complex.
  3. We will determine the three dimensional structures and dynamic properties full length ILF and its DNA complex.
  4. Design of the transciptional drugs for targeting LTR of HIV-1 by using the solution structure of the complex of DNA-binding domain of ILF and HIV-1 LTR.

 

Streptococcal pyrogenic exotoxin B

Title: Functional and Structural Studies of SPE B.

The long-term goals of this project are to study the structure and function relationships of SPE B and to design its inhibitor, a structure-based drug. SPE B or its zymogen (ProSPE B) is a GAS virulence factor in some patients. However, little is known including activation mechanism, downstream substrates, and the activity-structure relationships of SPE B. In order to provide the molecular aspect of SPE B in this PPG, we have successfully cloned ProSPE B in E. Coli and purify it to be homogenous. Here, we propose to use the recombinant ProSPE B to study the following questions and experiments:

  1. Activation mechanism of SPE B? The activation of SPE B occurs via autocatalytic cleavage or via the proteolytic digestion by plasmin, matirx metalloproteases (MMPs), or elastase? Does the activation of SPE B involve either in a single activation step or in a consecutive series (cascade)?
  2. Downstream substrates of SPE B? Recent reports showed that some extracellular matrices, urokinase receptor (uPAR), MMP-2, and cytokines are the downstream substrates of SPE B. We propose to study the other potential substrates in detail including: Extracellular Matrices: fibronectin, vitronectin , laminin, collagens, and elastin. Zymogens: MMPs, plasminogen, urokinase (u-PA), and tissue-type plasminogen activator (t-PA). Receptors: uPAR and integrins.
  3. Three dimensional structure and dynamic properties of 28 kDa active SPE B with its inhibitor will be determined by NMR spectroscopy.
  4. Rational drug design of SPE B. The central theme in this PPG is to study the role of SPE B in pathogenesis of GAS infection. This study provides the clinical, cellular, and molecular aspects, and animal model of SPE B. Particularly, we are interested in the molecular aspects of SPE B and in design of the inhibitors for clinical treatment. Based on our functional and structural studies, we will identify the role of SPE B as virulence factor, and the 3D structure of active SPE B will be used for rational drug design. The resulting drugs will be applied for the clinical treatment of virulent GAS infection.

 

Shark cartilage

Title: Purification of angiogenesis inhibitors from shark cartilage for cancer therapy

Shark cartilage has been reported to have strong antiangiogenic activity and to inhibit tumor neovascularizationtion (Lee & Langer, 1983). Cartilage is an acidic glycosaminoglycan/protein complex including chondroitin sulfates A, B, and C. Many cartilage factors contribute the inhibition of tumor angiogenesis, and characterizations of them are limited. There are only two cartilage factors including: a 27.65 kDa protein derived form cartilage was purified that inhibits angiogensis in vivo and capillary endothelial cell proliferation and migration in vitro and it is also an inhibitor of mammalian collagenase (Moses et al., 1990); a family of 3.5 kDa of proteins from shark cartilage also have analgeic and antiinflammatory effects and act as scavengers for reactive oxygen species (Fontenele et al., 1997). However, little is known about their mechanisms, and the contributions of other cartilage factors to the inhibition of tumor angiogenesis are unclear. Recently Gwo Chyang Pharmaceutical Co. has successfully extracted the effective compounds (U-995) from shark cartilage and applied to the inhibitions of sarcoma-180 tumor and the metastasis of lung tumor. To identify the role of anti-angiogenesis factors from shark cartilage in cancer therapy, we here propose to collaborate with Gwo Chyang Pharmaceutical Co. and to purify the angiogenesis inhibitors from shark cartilage.

 

Cordyceps sinensis

Cordyceps sinensis (Berk.) Sacc. is a time-honored tonic food and herbal medicine in China. Many of its traditional uses has been viewed from the basis of pharmacological activities. The ongoing exploration of C. sinensis in its wild form and cultured, fermented mycelial products derived from it, are reviewed from English and Chinese literature. The reviews of C. sinensis in preclinical in vitro and in vivo studies, and open-label and double-blinded clinical trials indicate that C. sinensis has effect on the treatments of respiratory, renal, hepatic, cardiovascular, immunologic, and nervous systems, cancer, glucose metabolism, inflammatory conditions, and toxicological conditions.

The long-term goals of this project are:
  • to extarct and purify the effective compounds from cordycpes sinensis;
  • to develop the bioassays; and
  • to estabish the animal models.

Prof. Woei-Jer Chuang's own Home Page

Selected publications:

  1. Lin CP, Kang KH, Lin TH, Wu MY, Liou HC, Chuang WJ, Sun WZ, Fu WM. Role of Spinal CXCL1 (GROα) in Opioid Tolerance: A Human-to-rodent Translational Study. Anesthesiology. 2014 [Abstract]

  2. Wang CC, Lai WC, Chuang WJ. Type I and II β-turns prediction using NMR chemical shifts. J Biomol NMR. 2014 [Abstract]

  3. Zheng PX, Chiang-Ni C, Wang SY, Tsai PJ, Kuo CF, Chuang WJ, Lin YS, Liu CC, Wu JJ. Arrangement and number of clustered regularly interspaced short palindromic repeat spacers are associated with erythromycin susceptibility in emm12, emm75 and emm92 of group A streptococcus. Clin Microbiol Infect. 2014 [Abstract]

  4. Lin CS, Chao SY, Hammel M, Nix JC, Tseng HL, Tsou CC, Fei CH, Chiou HS, Jeng US, Lin YS, Chuang WJ, Wu JJ, Wang S. Distinct structural features of the peroxide response regulator from group a streptococcus drive DNA binding. PLoS One. 2014 [Abstract]

  5. Lu SL, Tsai CY, Luo YH, Kuo CF, Lin WC, Chang YT, Wu JJ, Chuang WJ, Liu CC, Chao L, Chao J, Lin YS. Kallistatin modulates immune cells and confers anti-inflammatory response to protect mice from group A streptococcal infection. Antimicrob Agents Chemother. 2013 [Abstract]

  6. Wang CH, Chiang-Ni C, Kuo HT, Zheng PX, Tsou CC, Wang S, Tsai PJ, Chuang WJ, Lin YS, Liu CC, Wu JJ. Peroxide responsive regulator PerR of group A Streptococcus is required for the expression of phage-associated DNase Sda1 under oxidative stress. PLoS One. 2013 [Abstract]

  7. Chen YC, Cheng CH, Shiu JH, Chang YT, Chang YS, Huang CH, Lee JC, Chuang WJ. Expression in Pichia pastoris and characterization of echistatin, an RGD-containing short disintegrin. Toxicon. 2012 [Abstract]

  8. Cheng CH, Chen YC, Shiu JH, Chang YT, Chang YS, Huang CH, Chen CY, Chuang WJ. Dynamics and functional differences between dendroaspin and rhodostomin: insights into protein scaffolds in integrin recognition. Protein Sci. 2012 [Abstract]

  9. Chiang-Ni C, Zheng PX, Tsai PJ, Chuang WJ, Lin YS, Liu CC, Wu JJ. Environmental pH changes, but not the LuxS signalling pathway, regulate SpeB expression in M1 group A streptococci. J Med Microbiol. 2012 [Abstract]

  10. Shiu JH, Chen CY, Chen YC, Chang YT, Chang YS, Huang CH, Chuang WJ. Effect of P to A mutation of the N-terminal residue adjacent to the Rgd motif on rhodostomin: importance of dynamics in integrin recognition. PLoS One. 2012 [Abstract]

  11. Chiang-Ni C, Wu AB, Liu CC, Chen KT, Lin YS, Chuang WJ, Fang HY, Wu JJ. Emergence of uncommon emm types of Streptococcus pyogenes among adult patients in southern Taiwan. J Microbiol Immunol Infect. 2011 [Abstract]

  12. Su YF, Chuang WJ, Wang SM, Chen WY, Chiang-Ni C, Lin YS, Wu JJ, Liu CC. The deficient cleavage of M protein-bound IgG by IdeS: insight into the escape of Streptococcus pyogenes from antibody-mediated immunity. Mol Immunol. 2011 [Abstract]

  13. Dai YC, Chuang WJ, Chua KY, Shieh CC, Wang JY. Epitope mapping and structural analysis of the anti-Der p 1 monoclonal antibody: insight into therapeutic potential. J Mol Med (Berl). 2011 [Abstract]

  14. Chu YP, Chang CH, Shiu JH, Chang YT, Chen CY, Chuang WJ. Solution structure and backbone dynamics of the DNA-binding domain of FOXP1: insight into its domain swapping and DNA binding. Protein Sci. 2011 [Abstract]

  15. Anangi R, Chen CC, Lin YW, Cheng YR, Cheng CH, Chen YC, Chu YP, Chuang WJ. Expression in Pichia pastoris and characterization of APETx2, a specific inhibitor of acid sensing ion channel 3. Toxicon. 2010 [Abstract]

  16. Luo YH, Chuang WJ, Wu JJ, Lin MT, Liu CC, Lin PY, Roan JN, Wong TW, Chen YL, Lin YS. Molecular mimicry between streptococcal pyrogenic exotoxin B and endothelial cells. Lab Invest. 2010 [Abstract]

  17. Tsou CC, Chiang-Ni C, Lin YS, Chuang WJ, Lin MT, Liu CC, Wu JJ. Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes. Int J Med Microbiol. 2010 [Abstract]

  18. Chang CW, Tsai WH, Chuang WJ, Lin YS, Wu JJ, Liu CC, Tsai PJ, Lin MT. Procaspase 8 and Bax are up-regulated by distinct pathways in Streptococcal pyrogenic exotoxin B-induced apoptosis. J Biol Chem. 2009 [Abstract]

  19. Chang CW, Wu SY, Chuang WJ, Lin YS, Wu JJ, Liu CC, Tsai PJ, Lin MT. The IL-8 production by Streptococcal pyrogenic exotoxin B. Exp Biol Med (Maywood). 2009 [Abstract]

  20. Chiang-Ni C, Zheng PX, Ho YR, Wu HM, Chuang WJ, Lin YS, Lin MT, Liu CC, Wu JJ. emm1/sequence type 28 strains of group A streptococci that express covR at early stationary phase are associated with increased growth and earlier SpeB secretion. J Clin Microbiol. 2009 [Abstract]

  21. Chen CY, Shiu JH, Hsieh YH, Liu YC, Chen YC, Chen YC, Jeng WY, Tang MJ, Lo SJ, Chuang WJ. Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: importance of the interactions between the D residue and integrin. Proteins. 2009 [Abstract]

  22. Su YF, Wang SM, Lin YL, Chuang WJ, Lin YS, Wu JJ, Lin MT, Liu CC. Changing epidemiology of Streptococcus pyogenes emm types and associated invasive and noninvasive infections in Southern Taiwan. J Clin Microbiol. 2009 [Abstract]

  23. Wang CC, Houng HC, Chen CL, Wang PJ, Kuo CF, Lin YS, Wu JJ, Lin MT, Liu CC, Huang W, Chuang WJ. Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity. J Biol Chem. 2009 [Abstract]

  24. Chiang-Ni C, Tsou CC, Lin YS, Chuang WJ, Lin MT, Liu CC, Wu JJ. The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes. Gene. 2008 [Abstract]

  25. Tsou CC, Chiang-Ni C, Lin YS, Chuang WJ, Lin MT, Liu CC, Wu JJ. An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses. Infect Immun. 2008 [Abstract]

  26. Hsu JF, Chuang WJ, Shiesh SC, Lin YS, Liu CC, Wang CC, Fu TF, Tsai JH, Tsai WL, Huang YJ, Hsieh YH, Wu JJ, Lin MT, Huang W. Streptococcal pyrogenic exotoxin B cleaves human S-adenosylhomocysteine hydrolase and induces hypermethioninemia. J Infect Dis. 2008 [Abstract]

  27. Wang SM, Lu IH, Lin YL, Lin YS, Wu JJ, Chuang WJ, Lin MT, Liu CC. The severity of Streptococcus pyogenes infections in children is significantly associated with plasma levels of inflammatory cytokines. Diagn Microbiol Infect Dis. 2008 [Abstract]

  28. Su CC, Lin YP, Cheng YJ, Huang JY, Chuang WJ, Shan YS, Yang BC. Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol. 2007 [Abstract]

  29. Luo YH, Kuo CF, Huang KJ, Wu JJ, Lei HY, Lin MT, Chuang WJ, Liu CC, Lin CF, Lin YS. Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis. Kidney Int. 2007 [Abstract]

  30. Chang CW, Tsai WH, Chuang WJ, Lin YS, Wu JJ, Liu CC, Tsai PJ, Lin MT. The fate of SPE B after internalization and its implication in SPEB-induced apoptosis. J Biomed Sci. 2007 [Abstract]

  31. Wang CC, Chen JH, Lai WC, Chuang WJ. 2DCSi: identification of protein secondary structure and redox state using 2D cluster analysis of NMR chemical shifts. J Biomol NMR. 2007 [Abstract]

  32. Lu TJ, Lai WY, Huang CY, Hsieh WJ, Yu JS, Hsieh YJ, Chang WT, Leu TH, Chang WC, Chuang WJ, Tang MJ, Chen TY, Lu TL, Lai MD. Inhibition of cell migration by autophosphorylated mammalian sterile 20-like kinase 3 (MST3) involves paxillin and protein-tyrosine phosphatase-PEST. J Biol Chem. 2006 [Abstract]

  33. Chiang-Ni C, Wang CH, Tsai PJ, Chuang WJ, Lin YS, Lin MT, Liu CC, Wu JJ. Streptococcal pyrogenic exotoxin B causes mitochondria damage to polymorphonuclear cells preventing phagocytosis of group A streptococcus. Med Microbiol Immunol. 2006 [Abstract]

  34. Wang CC, Chen JH, Yin SH, Chuang WJ. Predicting the redox state and secondary structure of cysteine residues in proteins using NMR chemical shifts. Proteins. 2006 [Abstract]

  35. Chen CY, Cheng CH, Chen YC, Lee JC, Chou SH, Huang W, Chuang WJ. Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris. Proteins. 2006 [Abstract]

  36. Hsieh HC, Hsieh YH, Huang YH, Shen FC, Tsai HN, Tsai JH, Lai YT, Wang YT, Chuang WJ, Huang W. HHR23A, a human homolog of Saccharomyces cerevisiae Rad23, regulates xeroderma pigmentosum C protein and is required for nucleotide excision repair. Biochem Biophys Res Commun. 2005 [Abstract]

  37. Wang CH, Lin CY, Luo YH, Tsai PJ, Lin YS, Lin MT, Chuang WJ, Liu CC, Wu JJ. Effects of oligopeptide permease in group a streptococcal infection. Infect Immun. 2005 [Abstract]

  38. Tsai WH, Chang CW, Chuang WJ, Lin YS, Wu JJ, Liu CC, Chang WT, Lin MT. Streptococcal pyrogenic exotoxin B-induced apoptosis in a549 cells is mediated by a receptor- and mitochondrion-dependent pathway. Infect Immun. 2004 [Abstract]

  39. Shiu JH, Chen CY, Chang LS, Chen YC, Chen YC, Lo YH, Liu YC, Chuang WJ. Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding. Proteins. 2004 [Abstract]

  40. Jeng WY, Tsai YH, Chuang WJ. The catalase activity of Nalpha-acetyl-microperoxidase-8. J Pept Res. 2004 [Abstract]

  41. Yan JJ, Liu CC, Ko WC, Hsu SY, Wu HM, Lin YS, Lin MT, Chuang WJ, Wu JJ. Molecular analysis of group A streptococcal isolates associated with scarlet fever in southern Taiwan between 1993 and 2002. J Clin Microbiol. 2003 [Abstract]

  42. Chen CY, Luo SC, Kuo CF, Lin YS, Wu JJ, Lin MT, Liu CC, Jeng WY, Chuang WJ. Maturation processing and characterization of streptopain. J Biol Chem. 2003 [Abstract]

  43. Chuang WJ, Wu CH, Huang HN, Chen SH, Hsiao G, Lin CH, Sheu JR. Comparison of the binding character of triflavin on resting and activated alpha(IIb)beta(3) integrin in human platelets by electron microscopy. Thromb Res. 2003 [Abstract]

  44. Jeng WY, Chen CY, Chang HC, Chuang WJ. Expression and characterization of recombinant human cytochrome c in E. coli. J Bioenerg Biomembr. 2002 [Abstract]

  45. Liu PP, Chen YC, Li C, Hsieh YH, Chen SW, Chen SH, Jeng WY, Chuang WJ. Solution structure of the DNA-binding domain of interleukin enhancer binding factor 1 (FOXK1a). Proteins. 2002 [Abstract]

  46. Huang BM, Ju SY, Wu CS, Chuang WJ, Sheu CC, Leu SF. Cordyceps sinensis and its fractions stimulate MA-10 mouse Leydig tumor cell steroidogenesis. J Androl. 2001 [Abstract]

  47. Jiang ST, Chiu SJ, Chen HC, Chuang WJ, Tang MJ. Role of alpha(3)beta(1) integrin in tubulogenesis of Madin-Darby canine kidney cells. Kidney Int. 2001 [Abstract]

  48. Jiang ST, Chuang WJ, Tang MJ. Role of fibronectin deposition in branching morphogenesis of Madin-Darby canine kidney cells. Kidney Int. 2000 [Abstract]

  49. Jiang ST, Chiang HC, Cheng MH, Yang TP, Chuang WJ, Tang MJ. Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells. Kidney Int. 1999 [Abstract]

  50. Shieh B, Su IJ, Chuang WJ, Li C. Dye-induced denaturation of DNA dissolved in water. Biotechniques. 1998 [Abstract]

  51. Chen H, Wu YI, Hsieh YL, Shi GY, Jiang MJ, Chang WC, Chuang WJ, Kan WM, Tang MJ, Jen CJ. Perturbation of platelet adhesion to endothelial cells by plasminogen activation in vitro. Thromb Haemost. 1997 [Abstract]

  52. Young KC, Shi GY, Chang YF, Chang BI, Chang LC, Lai MD, Chuang WJ, Wu HL. Interaction of streptokinase and plasminogen. Studied with truncated streptokinase peptides. J Biol Chem. 1995 [Abstract]

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Updated: 2014.11.19