2B1610 Destabilization of SOD1 facilitates abnormal scrambling of its disulfide bond in the familial form of amyotrophic lateral sclerosis(Proteins:Structure & Function II:Theory, Aggregation,Oral Presentation,The 50th Annual Meeting of the Biophysical Society of Japan)
2B1610 ジスルフィド結合の組換えによる不溶性SOD1オリゴマーの新たな形成メカニズム : 筋萎縮性側索硬化症における分子病理変化(蛋白質-構造機能相関II:理論,凝集,口頭発表,日本生物物理学会第50回年会(2012年度))

Keisuke Toichi, Yoshiaki Furukawa
2012 Seibutsu Butsuri  
TheBiophysicalSociety of Japan General IncorporatedAssociation cofiIin concentration), filaments at ca -7 kT protomer at ca 3.S kT. we estimated the binding energy of cofilin to actin and the energy required for twisting the neighboring 281510 U:,Ff-A"-rput7:-MFtrfiC7fF Amyloidogenic Peptides from Hen Lysozyme Hideki TachibanaT'], Masao Fajisawai'3, Ryohei Kono2'], Minoru Kato4 (islrh Bioi-Oriented Sci fech, Kinki Uhiv, 2}Vl kayama Mled Uhiv. 3High Press Prot Res Center, Kinki Uhiv, 4Cbll Pharm
more » ... Sci, Ritsumeikan Uhiv) Amyloid protofibri1-like assemb]y spontaneously forms from hen lysozyme disulfide-deficient variant protein termed OSS. Four peptide regions protected frorn the access of solvent in the OSS fibrillar state have been identified with NMR-detected hydrogenldeuterium exchange measurernents. These regions have high predicted beta-aggregation propensity, and rnost probably constltute the core ofOSS fibril. Here, the fibrillation of one of these peptides,Y52-R6], was investigated ln detail, When monitored with thioflavine T fluorescence the Y52-R6r peptide facile]y form amlyoid fibri]s under miidly acidic solution conditions, but not under neutral or alkaline conditions, which indicates that the presence of small amount of excess positive charge facititates amyloid-fibri11ation of this peptide. Atomic force microscopy observation showed that the YS2-R61 peptide fibrils were thin at the initial stage of fibri11ation when obtained from di[ute peptide solutions, and grew thick with prolonged incubation period by bundle formation. MMPBSA calcu]ation on two mo]ecules of the Y52-R61 peptides showed that an anti-parallel beta structure is more stable than a parallel one by about 40 kl, whjeh agrees with our previous finding withIRmeasurementthattheanti-paraltehbetastructurepredominantlyexistsin the OSS amyloid-like fibrils, 2B1522 Effects of raft components on the membrane-mediated aggregation of IAPP Kenji Sasahara {Kbbe Uitiversity) Human islet amyloid polypeptide (IAPP) is a 37-resudue peptide which is thc major constituent of amy]eid fibrils deposited in the pancreatic islets wjth type II diabetes metiitus (T2DM). IAPP is synthesized in pancreatic beta-celts and co-secreted wlth insulin. Numerous studies have shown that the interactions between IAPP and lipid membranes p]ay an impertant role in the pathogenesis of T2DM, in whjch the lipid membranes of beta-cel] are the target of IAPP toxicity. Despite reeent efforts toward biophysical characterization of the IAPPlipid interactions, the cffects of IAPP aggregation on the physiological propenies and organization of lipid membranes have not been welF characterized, which is of critical importanee in further understanding of the pathogenesis ofT2DM. In the present study, amyloid aggregation ofIAPP was induced on the supported lipid bilayers {DOPCIDOPStraft components) to investigate the effects of raft components on its aggregate formazion and the associated membrane damage. Microscopic observations indicated that IAPP aggregation is significant]y accelerated on the membrane inctuding the raft components, and that the aggregates are subsequently converted to the needlelike fibTilluT aggregates during the prolonged incubatien, which is promoted for the rafts-containing membranes, Furthermore, the conversion of IAPP to fibrillar forms was microscopieal]y found associated with the mernbTane darnage. envjronrnent of the cytoplasm, while the cytop]asm in SHuffleTM is more oxidizing by deleting cytoplasmic reductases keeping cysteines in their thiol state, In both ce]ls, fALS-mutant SOD1s were obtained as insoluble pellets but with distinct propenies; a disu]fide bond of SODI was either comp]etely reduced in BL21 or abnormah]y formed between SODI rnolecules in SHurneTM. Depending upon the intracellular redox environment where SODI resides, therefore, fALS-mutant SODI is considered to be misfolded into aggregates with distinct properties, which would be relevant in the patho]ogica] heterogeneity in fALS cases. 2Bls46 envvACm;x[[tueLzaLlsoDlrel ¢ itttttnvewpth=XA A copper chaperone-independent mechanism for activation of Cu, Zn-superoxide dismutase Yasuyuki Sakurai, YDshiaki Funikawa {Dept. qf Chem, Keio Univ.) Cu,Zn-superexide dismutase {SODI) is an antioxidant enzyrne that removes superoxide anion radica]s, and is widely eonserved among aerobic organisrns. In order for SODI to gain catalytic activity, two post-transtationa] proeesses are known to be necessary/ binding of a coppeT ion and formation of an intramolecular disu] fide bond. 1n eukaryetes, the copper chaperone CCS plays a pivota] role in the activatiDn of cytoplasmic SODI by delivering the cata]ytic eopper ion and introducing the intramolecular disulfide bond to SOD1. A ccsknockout meuse, however, exhibjts residua] SODI activity, implying that a CCS-independent pathway is also opeTative for SODI activation. To get insight into the mechanism of this CCS-independent pathway, we have focused upon the activation of Escherichia coli periplasmic SODI {SODC), which occurs independently of CCS, as the E. coli genome contains no CCS homologue, We have confirmed that an intramolecu]ar disu]fide bond as well as a bound copper ion is essential for cata]ytic activity of SODC and also found that the canonical binding ofcopperlzinc ions in SODC requires the formation of a disulfide bond. During the activation of SODC in E. coti cells, therefore, rnetal binding ls considered to be preceded by formation of a disulfide bond. This mechanism appears to fit we]1 with the oxidizing environment of the bacterial periplasm. We thus propose a regulatory role ofthe disulfide fonmation both in the CCS-dependent and independent activation ef SOD1 enzymes. 2Blsss swwfirantsutrvmaEnvml6 raseresu oppth=xh A mechanism controlling the morphoLogies of protein aggregates by "post-aggregation oiidation" Yasushi Mitomii, Takao Nomurai, Masaru Kurosawa2. Nobuyuki Nukina2, Yoshiaki Furukawai (LDept. qf Chem, Kbio Uhiv., 2RIKEN Brain Science Jnstitute)Misfolding of a protein molecule often leads to the formation of insoluble aggregates and is also characterized as a pathological haltmark in neurodegenerative diseases. One of the important factors regu]ating the aggregation of protejns is a post-translatienal modification; indeed, abnormal rnodi fications are eften identified in the aggregate-consisting proteins. Actuatty, however, it remains quite obscure when and how proteins are medified in the course of the aggregation. To reveal an unprecedented roTe ofmodifications in protein aggregation, we focus upon a protein, huntingtin (HTT). which has been known to form insoluble aggregates in a neurodegenerative disorder, Huntington disease (HD). By examining aggregation of recombinant HTT prote{ns, we have for the first time idcntified oxidation ofa Met residue in HTT during the aggregation in vitro. The Met residue has also becn found to oxidize in pathological HTT aggregates purified from HD-model mice. A trace amount of contaminated copper ions as well as a physiological concentration of hydrogen peroxide oxidized a Met residue in HTT, and more interesting]y, this Met oxidation occurred only in the aggregated HT'I' but not in the solub]e state. Furthemiore, we have found that the Met oxidation creates additiona] interactions among HTT aggregatcs and alters those overall morphologies. Protei" aggregates can thus be a target of oxidative modifications. and we propose that such a "post-aggregation" modification is a retevant factor to rcgulate properties of protein aggregates. 2B1534 Yoshiaki Fllrukavva (Dept. Redex control proteins, and which forrnation ofan its native structure. tr{ggers the formation ofinsolub]e aggregates of mutant SODI amyotrophie tatera] scl should be correctly intrace]]ular envlronment intracellular were expressed in E. e;,Jcofirf ± tsNvFptoxNmlctueLTscza6Mspterent lbl"=xAetq)fpase\coks Redox environment is an intracelluiar factor to operate distinct pathways for protein aggregation qfChemistry, Keio Uhiv.) of the thiol-disulfide status is critica] for functioning of many Cu,Zn-superoxide dismutase (SODI) is one of such proteins in intramolecu]ar disulfide bond is required for folding into Indeed, abnorma] reduction of a disu]fide bond in SODI in vitro. In addition, aggregation proteins is a pathological hallmark in a farnilial form of erosis(fALS);therefore,athiol-disulfidestatusofSOD1 regulated inside cel]s, Given that SODI exists in various organelles. however, it rernains obscure how their distinct redox affects misfolding pathways of SODI, To investigate effects of redox environment on SODI misfolding,
doi:10.2142/biophys.52.s41_6 fatcat:hwmr6dcrw5bwhbcmkteetm4bwq