11:10-12:00, Rm 103

Protein: modification and regulationR-18-13Effect of lysine-containing cationic domains to thermal transition ofelastin-like proteins and their sensitivity to salt and pHWon Bae JeonLaboratory of Biochemistry and Cellular Engineering, Daegu Gyeongbuk Institute of Science andTechnology, Daegu 711-873, KoreaElastin-like proteins (ELPs) that are modeled on the VPGXG pentapeptide repeat oftropoelastin, in which X is any amino acid except proline, display a soluble-insolublephase transition in response to changes in temperature, salt, or pH, and thus haveemerged as promising smart proteins for biomedical applications. The temperature atwhich the soluble-insoluble conformational change occurs, known as the inversetransition temperature (Tt), has been used to characterize the responsiveness of ELPs toa change in stimulating factors. A series of elastin-like proteins, SKGPG[V(VKG)3VKVPG]n-(ELP1-90)WP (n = 1, 2, 3, and 4), were biosynthesized based on thehydrophobic and lysine linkage domains of tropoelastin. The formation of self-assembledhydrophobic aggregates was monitored in order to determine the influence of lysinecontainingcationic domains on phase transition properties as well as the sensitivity tochanges in salt and pH. The thermal transition profiles of the proteins fused with only oneor two cationic blocks (n = 1 or 2) were similar to that of the counterpart ELP1-90. Incontrast, diblock proteins that contain 3 and 4 cationic blocks displayed a triphasic profileand no transition, respectively. Upon increasing the salt concentration and pH, a stimulusinducedphase transition from a soluble conformation to an insoluble aggregate wasobserved. The effects of cationic segments on the stimuli sensitivity of cationic bimodalELPs were interpreted in terms of their structural and molecular characteristics.R-18-16Glutathione enhances the functional efficiency of a traffickingchaperone involved in intracellular B12 metabolismJinju Jeong, Yeonju Choi and Jihoe Kim*School of Biotechnology, Yeungnam University, KoreaThe most common inherited disorder of intracellular B12 metabolism belongs to cblCgenetic complementation group. The defective gene product CblC is a traffickingchaperone for escorted delivery of B12 in the cytoplasm. The protein binds andprocesses vitamin B12 in enzyme cofactor assimilation, but the binding affinity waspreviously reported to be relatively low (Kd in a micromolar range) considering submicromolarB12 concentrations in cells. Using a bovine homologue protein bCblC, wefound that the binding affinity for vitamin B12 is increased up to 95-fold (Kd = 0.34 ±0.06µM) in the presence of reduced glutathione (GSH), which is abundant (1 ~ 10 mM) incells. The increased binding affinity appeared to be due to stabilization of ligand-boundbCblC upon binding of GSH, preventing the release of the ligand. These resultsdemonstrate that bCblC is more efficient B12 trafficking chaperone in vivo than previouslyreported. In addition, these findings suggest that vitamin B12 together with glutathioneshould be considered for the treatment of patients with a CblC defect.R-18-14Potential role of sumoylation in the activation and nuclear translocationof STAT6Seol-Hee Kim, Tran Thi Minh Tam and Choong-Eun LeeLaboratory of Immunology, Department of Biological Science, Sungkyunkwan University, Suwon440-746, KoreaSTATs, signal transducers and activators of transcription, undergo various posttranslationalmodifications (PTM) including phosphorylation, acetylation, methylation, andsumoylation which may affect STAT activation process and cytokine response. Recentlywe have reported the antagonistic regulation by IL-4 and IFN-alpha signaling mediatedthrough cytosolic retention of novel phospho-STAT(pY-STAT6:pY-STAT2:p48) complex.To elucidate molecular basis of the cytosolic localization, we have investigated the role ofPTMs in the nuclear translocation of STATs. The perturbation of methylation oracetylation did not affect the translocation of phospho-STAT1 or phospho-STAT6 inducedby IFN-alpha and/or IL-4. Interestingly, however, our data suggest the role of sumoylationfor STAT6 translocation. The sumo-plot prediction program revealed potentialsumoylation motifs in STAT6 at K131, K307, K618 and K636. In addition, the overexpressionof sumo-GFP and STAT6 resulted in an extensive cytoplasmic co-retention ofsumo-STAT6 and pY-STAT6 analyzed by confocal microscopy and immunoblot. Theresults indicate an involvement of sumoylation in the pY-STAT6-containing complexformation and inhibition of its nuclear translocation as implicated in the PIAS/STAT1interaction.R-18-18Characerization of cross-linked immobilized acyl-CoA thioesterase1precursor from the Listeria innocus CLIP 11262 and amyloid fibrils asnano-scaffoldSeul-Gi Kim, Song-Yi Bae, Seung-Joon Kim and T. Doo-Hun Kim*Department of Molecular Science and Technology, Ajou University, Suwon 443-749, KoreaCross-linked enzyme aggregates (CLEAs) were appeared as interesting enzyme designfor immobilization. In this approach used the LI22, which an enzyme that Acyl-CoAthioesterase1 precursor from L. innocus CLIP 11262. Efficient cross-linking was observedwhen using 80%-saturation ammonium sulfate followed by cross-linking for 12h-14h with0.5%(v/v) glutaraldehyde. The residual stability of the CLEA with respect to paranitrophenylactate was evaluated. The CLEA product was much more resistant todenature, heat than the soluble enzyme. As compared to its soluble enzyme form, solubleenzyme retained 20% of its activity after 1 h of incubation at 80 C and 75% of LI22-CLEA’s activity still observed after 2h of incubation at 80℃. During 15day of storage at 4℃, the LI22-CLEA retained more than 85% of its initial activity. Bovine insulin amyloidfiber were made on several conditions at pH2.0. We report successful functionalization ofamyloid fibrils by conjugation to LI22 using glutaraldehyde. We are currently investigatingbiochemical and biophysical properties of LI22-CLEA using several biochemical andbiophysical approaches including scanning electron microscopy (SEM). And we expectseveral roles of functionalized bovine insulin fibrils as nano-scaffold.R-18-15Tissue-specific and age-dependent expression of protein argininemethyltransferases (PRMTs) in rat tissuesEunyoung Hong, Eunil Lee, Yongchul Lim, Minyoung Oh, Daeho Kwon¹Department of Preventive Medicine & Medical Research Center for Environmental Toxico-Genomics and Proteomics, Korea University, Seoul, Korea, ²Department of Public Health, Schoolof Medicine, Graduate School of Medicine, Korea University, Seoul, Korea, ³Cellular andDevelopmental Biology, Division of Brain Korea 21 Program for Biomedical Sciences, KoreaUniversity, College of Medicine, Seoul 136-705, Korea, ⁴Department of Microbiology College ofMedicine Kwandong University 522 Naegok-dong, Gangneung-si, Kwangwon-do 210-701, KoreaProtein arginine methyltransferases generate asymmetric and symmetric dimethylargininesby catalyzing the transfer of methyl groups to the arginines in proteins. Wepreviously observed the expression and activities of PRMTs were significantly reduced inreplicatively senescent fibroblasts compared with young fibroblasts. In the present study,we investigated the expression of four PRMT members (PRMT 1, 4, 5, 6), symmetric andasymmetric arginine dimethylated proteins in age-dependent rat tissues. When theexpression of PRMTs and the methylation of their substrates were examined, asignificant reduction was found between old and young rat tissues. Although differenceswas distributed by a tissue-specific manner, most PRMTs were significantly downregulatedin several aged rat tissues compared with young rat tissues. Furthermore,when enzyme activity was determined with tissue extract, PRMT’s activity from aged rattissues was reduced. It was not routinely correlated with the expressed PRMTs, however,the data indicated that not only was the expressed PRMTs decreased in aged rat tissues,but also the enzymatic activities were reduced. These results suggest that the tissuespecificexpression patterns of PRMTs and their activities might affect the process of rattissue aging.330 Korean Society for Biochemistry and Molecular BiologyR-18-19The expression levels of Mfn1 in oxidative stress induced mitochondrialhyper-fusion is controlled by mitochondrial E3 ligase, MARCH5Yong-Yea Park and Hyeseong ChoDepartment of Biochemistry, Ajou University School of Medicine, Graduate School of MolecularScience and Technology, Ajou University, Wonchon-dong, Yeongtong-gu, Suwon 443-721, KoreaA mitochondrial E3 ubiquitin ligase, MARCH5, regulates mitochondrial morphology bybinding and controlling mitochondrial dynamics proteins. We previously reported thatMARCH5 lacking cells showed mitochondrial elongation which promotes cellularsenescence through Drp1 and accumulation of Mfn1. In this study, we proposed that thestress-induced mitochondrial hyperfusion (SIMH) induced by elevation of Mfn1 can beresolved by MARCH5. In response to Antimycin A (AMA) treatment, a mitochondrialcomplex III inhibitor, cells displayed SIMH and elevation of Mfn1 level was accompanied.Depletion of Mfn1 levels abrogates SIMH, indicating that Mfn1 plays an important role inSIMH. Notably, in AMA-treated cells, the interaction between MARCH5 and Mfn1 wasgreatly enhanced and ubiquitylation of accumulated Mfn1 was accelerated. Thus, inSIMH abnormally elevated Mfn1 levels in cells may trigger the activation of MARCH5,which returns the Mfn1 level to the basal. Together, we propose a novel mechanism thatthe balance of Mfn1 partly mediated through MARCH5 is important in maintaining of cellintegrity against cellular stress.