11:10-12:00, Rm 103

Protein: modification and regulationR-18-01Gene engineering, purification, crystallization and preliminary X-raydiffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), theArabidopsis membrane proteinYoung Ha Kim¹, TaeWoo Kwon¹, Hee Jung Yang¹, Wanyeon Kim¹, Young Ju Yun¹,HyeSook Youn², Ji Young Lee¹and BuHyun Youn¹¹Department of Biological Sciences, College of Natural Sciences, Pusan National University,Busan 609-735, Korea, ²Department of Bioscience & Biotechnology/Institute of Bioscience, SejongUniversity, Seoul 143-747, KoreaCytochrome P450s are the most versatile biological catalysts in plants; however, thestructural basis for their broad substrate specificity has been limited. The activity of p-coumarate-3-hydroxylase is essential for the biosynthesis of lignin and otherphenylpropanoid products in plants; but, all attempts to express and purify C3H, havefailed. In this research, we have developed a bacterial expression and purification systemfor the active Arabidopsis C3H by combinational mutagenesis. The modified C3H couldbe purified in the absence of detergent, and crystallized in two forms (orthorhombic andtrigonal space group) under different conditions. X-ray diffraction was processed to a 4.0Å resolution (first type crystal) and a 3.8 Å resolution (second type crystal). Although thediffraction of C3Hmod crystals is still limited, the simplicity and rapidity of this technologyare drastic advantages in comparison with other methods, and may contribute tostructural analyses of other membrane proteins including P450s family. [Supported by theR&D Program through the NRF of Korea funded by the MEST (grant code: 2010-0021920 and 2010-0029553)]R-18-04Effect of thrombin on the expression of secretory group IIAphospholipase A2Wonwha Lee 1, 2 and Jong-Sup Bae 2¹Department of Biochemistry and Cell Biology, School of Medicine, ²College of Pharmacy,Kyungpook National University, Daegu, KoreaTNF-a up-regulate the expression of proinflammatory molecules in HUVECs. However,activated protein C (APC) down-regulates the expression of the same molecules. Theexpression level of sPLA2-IIA is known to be elevated in inflammatory disorders includingin sepsis. Here, we investigated the effects of APC and thrombin on the expression ofsPLA2-IIA and ERK in HUVECs. Thrombin had no effect on the expression of sPLA2-IIAin HUVECs, however, TNF-a potently induced its expression. The prior treatment of cellswith APC inhibited expression of sPLA2-IIA through the EPCR-dependent cleavage ofPAR-1. Further studies revealed that if HUVECs were pretreated with the zymogenprotein C to occupy EPCR, thrombin also inhibited the TNF-a-mediated expression ofsPLA2-IIA through the cleavage of PAR-1. The EPCR-dependent cleavage of PAR-1 byboth APC and thrombin increased the phosphorylation of ERK 1/2. Pretreatment of cellswith either LY294002 or MβCD abolished the inhibitory activity of both APC and thrombinagainst sPLA2-IIA expression, suggesting that the protein C occupancy of EPCR confersa PI3-kinase dependent protective activity for thrombin such that its cleavage of the lipidraftlocalized PAR-1 inhibits the TNF-α-mediated expression of sPLA2-IIA in HUVECs.R-18-02SUMO modification regulates the transcriptional activity of ataxin-1, theSCA1 gene productYuzhen Jiang and Seongman KangSchool of Life Sciences and Biotechnology, Korea University, Seoul 136-701, KoreaSpinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerativedisorder caused by the expansion of polyglutamine tract within ataxin-1, the SCA1 geneproduct.SCA1 is characterized by ataxia, progressive motor deterioration, and loss ofcerebellar Purkinje cells and brainstem neuron.Although the function of the ataxin-1protein is not clearly known, evidences showed that it is involved in transcriptionalregulation. It was reported that ataxin-1 is SUMO (small ubiquitin-like modifier) modified atmultiple sites. However, the function of this modification is still unknown. In this study, weconstructed SUMOylation-associating mutants of ataxin-1 and took out luciferase reportergene assays to investigate whether SUMOylation of ataxin-1 relates with itstranscriptional activity. Results showed that both repression and activation activities ofataxin-1 were affected by SUMO modification. In addition, the interaction between ataxin-1 and CtBP2, a transcriptional repressor, was also influenced. Therefore, we speculatethat SUMOylation of ataxin-1 may regulate its transcriptional activity by changing itsinteraction with transcription factors or co-factors.R-18-05NIR is regulated by post-translational modificationMyong Ho Jeong and Seung Jin HanSchool of Biological Sciences, Inje University, Gimhae 621-749, KoreaThe Novel INHAT Repressor (NIR), a histone acetylation inhibitor protein, interacts withtumor suppressor gene p53 and p63, and inhibits their transcriptional activity. In normalcell, majority of NIR exists in the nucleolus and small fraction of NIR is present in thenucleoplasm and cytosol. In this study, we examined the relationship between localizationof NIR and its stability. By checking the localization of three fragmented domains,INHAT1(N-term), ΔINHAT(Middle), INHAT2(C-term), we determined that thesefragments have NLS, NES and NoLS, respectively. The acetylated NIR was found only inthe nuclear not in the cytoplasm, and cannot be associated with p53 protein. The stabilityof NIR is regulated by its RNA binding property as the NIR disappeared by addition ofRNase on the cell. The degradation of NIR is associated with ubiquitination. Our datashow that the only cytoplasmic NIR was ubiquitinated. These results indicated thatlocalization of NIR is affected by acetylation and NIR is degradation by ubiquitination incytoplasm.R-18-03Regulation of Thrombin-mediated MMP-2 activation by HeparanSulfate proteoglycanHarim Jeon, Hee Doo Lee, Bikash Thapa, Yeon Hyang Kim, Bon-Hun Koo and Doo-Sik KimDepartment of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul,KoreaMMP-2 is synthesized as a zymogen that is activated through proteolysis of thepropeptide. Thrombin has been shown to be involved in MMP-2 activation via specificcleavages of the propeptide. Moreover, our previous study showed that thrombin alsodegraded MMP-2, but this degradation was reduced greatly under cell-associatedconditions with a concomitant increase in activation. However, the underlying molecularmechanisms remain to be elucidated. In the present work, we demonstrate that heparansulfate proteoglycan is essential for thrombin-mediated activation of pro-MMP-2. Bindingof heparan sulfate to thrombin is primarily responsible for this activation process,presumably through conformational changes at the active site. Furthermore, interaction ofMMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2degradation and inhibition of this interaction by heparan sulfate or hirudin fragment resultsin a decrease in MMP-2 degradation. Finally, we demonstrated interaction betweenexosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role ofhemopexin-like domain in MMP-2 degradation. Taken together, our experimental datasuggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activityby heparan sulfate proteoglycans.R-18-06Erythropoietin induces igf-1 from human scalp dermal papilla cellsYaeJi Jang, Mi Hee Kwack, Moon Kyu Kim, Jung Chul Kim, Young Kwan SungHair Research Center & Department of Immunology, School of Medicine, Kyungpook NationalUniversity, Daegu 700-422, KoreaThe mammalian hair follicle contains dermal papilla (DP) and dermal sheath derived fromthe mesenchyme. It also contains epithelial cells of the outer root sheath, inner rootsheath, matrix and hair shaft. The reciprocal interactions between the epithelium andmesenchyme are essential for postnatal hair growth and cycling of hair follicles. Recentstudies showed that erythropoietin (EPO)/ erythropoietin receptor (EPOR) signaling existin both human and mouse hair follicles. Very recently, we reported that EPO promoteshair shaft growth in cultured human hair follicles and EPOR is expressed in DP cells. Asfactors from the DP are believed to cause matrix cells to proliferate to elongate the hairshaft in cultured human hair follicles by EPO, we examined EPO-inducible paracrinefactors using three dimensional (3D) spheroid cultures of DP cells. We found that theexpression of EPOR is increased by spheroid cultures of DP cells and EPO inducesexpression of IGF-1, a well-known hair growth promoting factor from 3D DP cells. Ourdata strongly suggest that EPO-inducible IGF-1 from DP is a key factor responsible forhair shaft elongation by EPO in cultured human hair follicles.328 Korean Society for Biochemistry and Molecular Biology