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:
- 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.
- 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.
- 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.
- 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.
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:
- 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.
- 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.
- 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.
- 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.
- Developments of assays: We will develop enzymatic assays for peroxidase-, catalase-, and cytochrome p450-type reactions.
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:
- We will use gel retardation assay and CD titration to characterize what DNA sequence recognized by the DNA-binding domain of ILF.
- We will determine the three dimensional structures and dynamic properties of DNA-binding domain of ILF and its DNA complex.
- We will determine the three dimensional structures and dynamic properties full length ILF and its DNA complex.
- 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.
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:
- 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)?
- 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.
- Three dimensional structure and dynamic properties of 28 kDa active SPE B with its inhibitor will be determined by NMR spectroscopy.
- 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.
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 (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.