BI1 The long-range thickening effect induced by cholesterol condensed in lipid bilayers.

J. Y. Lin (林建佑)1, J. W. Chen(陳俊文)1, J. H. Tsai(蔡景翰)1, J. H. Wang(王靖宏)1, W. C. Hung(洪偉清) 1

1Department of Physics, Chinese Military Academy,Fengshan, Kaohsiung, 83055 Taiwan

 

The condensing effect of cholesterol on phospholipid bilayers was systematically investigated for saturated and unsaturated chains, as a function of cholesterol concentration. X-ray lamellar diffraction was used to measure the phosphate-to-phosphate distances, PtP, across the bilayers. The measured PtP increases non-linearly with the cholesterol concentration until it reaches a maximum. With further increase of cholesterol concentration, the PtP remains at the maximum level until the cholesterol content reaches the solubility limit. The data in all cases can be quantitatively explained with a simple model that cholesterol forms complexes with phospholipids in the bilayers. The phospholipid molecules complexed with cholesterol are lengthened and this lengthening effect extends into the uncomplexed phospholipids surrounding the cholesterol. This long-range thickening effect is similar to the effect of gramicidin on the thickness of lipid bilayers due to hydrophobic matching.

 

 

BI2 Over-expression and purification of GST-MyD88

Shih-de Yang1、Cheng-Chin Kuo2、and Li-chu Tsai1

1 Department of Molecular Science and Engineering, National Taipei University of Technology
2 Agricultural Biotechnology Research Center , Academia Sinica, Taipei , Taiwan

 

Myeloid differentiation factor 88 (MyD88) is an adaptor protein which is involved in interleukin-1 recptor and Toll-like receptor induced activation of NF-κB. MyD88 is composed of a C-terminal TIR Domain and N-terminal death domain, which mediated the interaction of MyD88 with TL-1R/TLR-associated with other adaptor proteins of TL-1R/TLR pathway. In order to understand the protein-protein interaction and the function of MyD88, we plan to resolve the crystallographic structure of MyD88. Here, we describe how we purify GST-MyD88, containing the cloning, protein expression, and purification. MyD88 was constructed into pGEX4T-1 vector and transformed into E. coli BL21. The expression proteins were purified by an ion-exchange Q sepharose (anion exchange method) and a glutathione-S-transferase (GST)-affinity chromatography. Crystallization of the purified MyD88 protein is in progress.

 

 

BI3 Identification of functional domains for interactions between JAB1 and the hepatitis B virus surface antigen pre-S2 mutant

Yu-Jun Huang, Yi-Hsuan Hsieh, Wenya Huang

Department of Medical Laboratory Science and Biotechnology, National Cheng-Kung University, Tainan, Taiwan

 

Chronic hepatitis B virus (HBV) infection is the major cause for hepatocellular carcinoma (HCC) in Taiwan. In the late stages of chronic HBV infection, the pre-S mutant large surface antigen (LHBS) emerges naturally. The pre-S2 mutant LHBS, partially deleted in the pre-S2 region of the surface gene, induces oxidative stress, genomic instabilities, and nodular proliferation of hepatocytes, indicating that it regulates the tumorigenic processes. In our recent studies, we found that Jun activation domain-binding protein 1 (JAB1) directly interacts with pre-S2 mutant LHBS and results in p27 degradation, Rb phosphorylation, and S phase progression. In this study, we aim to map the functional domains that involve in the interactions between pre-S2 mutant LHBS and JAB1. The partially deleted pre-S2 mutant LHBS genes were cloned into the yeast two-hybrid vector pGBKT7, which contains the GAL4 DNA-binding domain. The JAB1 gene was cloned into pACT2, which contains the GAL4-activation domain. The interaction between JAB1 and each pre-S2 mutant LHBS construct was tested by yeast two-hybrid assays. We found that deletion of amino acids 61 to 119 of the pre-S2 mutant LHBS rendered it lost the activity to interact with JAB1. The other partially-deleted pre-S2 mutant LHBS constructs tested did not affect its interaction with JAB1. These findings indicate that the region of amino acids 61 to 119 of the pre-S2 mutant LHBS is the main domain to interact with JAB1. Further studies to identify the key residues for such interaction are in progress. Based on these results, the role of JAB1 as regard to the pre-S2 mutant LHBS-induced HCC can be further understood.

 

 

BI4 Novel photo-dynamic cancer therapy strategy development by nanodiamond-protein complex.

Pei-Hsin Chen 1, Chih-Yuan Cheng 2, Kuang-Ka Liu 3, An-Lun Lo 4, Chia-Liang Cheng 2, Jui-I Chao 3, Yen-Peng Ho 4, Chia-Ching Chang 1

1 Institute of Biochemical Engineering, National Chiao Tung University, Hsinchu, 300, Taiwan;2 Department of Physics, National Dong Hwu University, Hualien, 974, Taiwan;3 Institute of Pharmacology and Toxicology, Tzu-Chi University, Hualien, 970, Taiwan; 4 Department of Chemistry, National Dong Hwu University, Hualien, 974, Taiwan.

 

The proper strategy on the blockade of cancer cell proliferation is highly desirable in cancer therapy. In this study, we have developed a novel strategy to specific destroy the growth hormone receptors on the membrane of cancer cells following treatment with the recombinant growth hormone (rGH)-conjugated nanodiamond (ND) complex by triggering the explosion of ND with pulse laser. The cytotoxicity analysis indicated that the ND alone did not induce the cell death and altered the protein expression profiles in the A549 lung carcinoma cells. Moreover, the fish rGH increased the cell proliferation in the A549 cells. We have found that the rGHND complex was located on the surface of A549 cells. Interestingly, the cells contained the rGHND that were destroyed by exposing with the high energy of pulse laser from the ND explosion. In contrast, the cells without rGHND were not altered after the laser treatment. Accordingly, we designate this novel technique as nano-surgery, which may contribute to the novel therapeutic strategies in cancer therapy or other biomedical applications.

 

 

BI5 Protein folding and aggregation (rEaGH)

Izabela Zakowska 1, Chuan-Mei Tsai 2, Anna Svanidze 1,3, Pei-Hsin Chen 1, Fang-Hsing Chiang 1, Hsueh-Liang Chu 1, Chang-You Wu 1, Chia-Ching Chang 1,2,4*

1 Department of Biological Science and Technology, National Chiao Tung University, HsinChu, 30050, Taiwan;
2 National Nano Device Laboratories, HsinChu, 30078, Taiwan;
3 Institute of Chemistry, Academia Sinica, 128 Sec. 2, Academia Rd., Nankang, Taipei, 115, Taiwan;
4 Institute of Physics, Academia Sinica, 128 Sec. 2, Academia Rd., Nankang, Taipei, 115, Taiwan

 

Protein molecules in a solution undergo Brownian motion and collide with each other constantly. The collisions may lead to protein precipitation when the situation favors aggregation. The aim of this study was to study how fast proteins can stabilize themselves during the folding process. The refolding of recombinant fish growth hormone (rEaGH) has been examined by direct folding reactions (off path folding) in different temperatures. The mean collision times versus aggregation fraction were determined for different temperatures. The protein stabilization time parameter (τC) from the autocorrelation function was determined around 49.2 ㎲ in 281 K. It gives an estimate of the time limit before the protein aggregates. It is also a measure of time during which the protein folds into a stable state. Meanwhile, there were observed some fraction of the soluble protein and aggregated protein in the same time. The results suggest that spontaneously folding and aggregation are antagonistic reactions. The ability to control and repeal protein aggregation may be a key in the production and formation of the therapeutic proteins. This study was supported by grants of NSC 95-2811-M-009-009 and NSC 95-2112-M-009-019.

 

 

BI6 Real-time monitoring of plant AtRGS protein interaction with plant G-alpha protein, GPA1

Ching-Shin Huang 1, Yu-Chen Lin 1, Yi-Che Chen 1, Chii-Shen Yang 1

1 Institution of Microbiology and Biochemistry,
Department of Biochemical Science and Technology,
National Taiwan University

 

The G-protein system in Arabidopsis thaliana consists of one G-alpha, GPA1, one G-beta and two G-gamma proteins. Upon ligand activation, the G-protein coupled receptor (GCR1 or GCR2) could lead to a conformational change in GPA1 and trigger the release of GDP from the GPA1 and the binding of GTP. The GPA1-GTP, the activated form of GPA1, can then further initialize a series of signal cascading inside the cells. The intrinsic GTPase enzyme activity of GPA1 will hydrolyze the GTP into GDP and restore GPA1 to the ground state, GPA1-GDP. This GTPase activity of GPA1 can be accelerated by a G-protein signaling regulator (AtRGS) protein which interacts with GPA1 and turns off the GPA1 signaling state. Here, we report a real-time measurement of AtRGS interaction with GPA1 using a fluorescent probe, BODIPY-TR-GTP, which serves as a GTP analog and a decrease of fluorescence can be detected when it is hydrolyzed by the GPA1.

 

 

BI7 Theoretical Investigation for the Interaction of Adenine Adducts with Thymine

Prabhat K. Sahu, Chang –Wang Kuo, and Shyi –Long Lee*

National Chung Cheng University, Chia -Yi, TAIWAN

 

The existence of DNA adducts bring the danger of carcinogenesis because of mispairing with normal DNA bases. 1, N6-ethenoadenine adducts (εA) and 1,N6-ethanoadenine adducts (EA) have been considered as DNA adducts to study the interaction with thymine, as DNA base. Several different stable conformers for each type of adenine adduct with thymine, have been considered with regards to their interactions. The differences in its geometrical structure, energetic properties and hydrogen bonding strengths have also been compared with Watson-Crick adenine-thymine base pair (A-T). The aim of this research is to provide fundamental understanding of adenine adduct and thymine interaction at molecular level and to aid in future experimental studies towards finding the possible cause of DNA damage.