Stabilization, Assembly, and Toxicity of Trimers Derived from Aβ.docx

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1、ThisisanopenaccessarticlepublishedunderanACSAuthorChoiceLicense,whichpermitscopyingandredistributionofthearticleoranyadaptationsfornon-commercialpurposes.ArticleTIAICISJOURNALOFTHEAMERICANCHEMICALSOCIETYpubs.acs.org/JACSStabilization,Assembly,andToxicityofTrimersDerivedfromApAdamG.Kreutzer,StanYoo,R

2、yanK.Spencer,andJamesS.Nowick*GDepartmentofChemistry,UniversityofCalifornia,Irvine,Irvine,California92697-2025,UnitedStates*SupportingInformationABSTRACT:OligomersoftheP-amyloidpeptideA(3haveemergedasimportantcontributorstoncurodcgcncrationinAlzheimersdisease.MountingevidencesuggeststhatAptrimersand

3、higher-orderoligomersderivedfromtrimershavespecialsignificanceintheearlystagesofAlzheimersdisease.Elucidatingthestructuresofthesetrimersandhigher-orderoligomersisparamountforunderstandingtheirroleinneurodegeneration.Thispaperdescribesthedesign,synthesis,X-raycrystallographicstructures,andbiophysical

4、andbiologicalpropertiesoftwostabilizedtrimersderivedfromthecentralandC-terminalregionsofAp.Thesetriangulartrimersarestabilizedthroughthreedisulfidecross-linksbetweenthemonomersubunits,TheX-raycrystallographicstructuresrevealthatthestabilizedtrimersassemblehierarchicallytoR)rmhexamers,dodccamcrs,anda

5、nnularporclikcstructures.Solution-phasebiophysicalstudiesrevealthatthestabilizedtrimersassembleinsolutiontoformoligomersthatrecapitulatesomeofthehigher-orderassembliesobservedcrystallographically.Thestabilizedtrimerssharemanyofthebiologicalcharacteristicsofoligomersoffull-lengthAp,includingtoxicityt

6、owardaneuronallyderivedhumancellline,activationofcaspase-3mediatedapoptosis,andreactivitywiththeoligomer-specificantibodyAll.ThesestudiessupportthebiologicalsignificanceofthetriangulartrimerassemblyoTTPP-hairpinsandmayofferadeeperunderstandingofthessonJBPOL-S 二 qndoJeqs A3EE 三号 oi MOL- uo suo 二 do s

7、a.op388u-Bqs/8JOsoBsqnd/_sd=q UssuHe9N9ZN 一急 ZZOZ Nz 工 co com. w 一二wrlPDPBO?二 0(】回INTRODUCTIONmolecularbasisofAlzheimersdisease.InAlzheimer/sdisease,thep-amyloidpeptideApassemblestoformamultitudeofsolubleoligomersaswellasinsolublefibrils.1,2TheApoligomershaveemergedasneurotoxicagentsthatleadtoneurod

8、egenerationinAlzheimersdisease.TheheterogeneityandmetastabilityoftheApoligomerspresentsatremendouschallengeinunderstandingthemolecularbasisofAlzheimersdisease.Specifically,thelackofhomogeneousoligomersprecludesdetailedconelationofthebiologicalpropertiesofA0oligomerswiththeirstructuralandbiophysicalp

9、roperties.ToreducetheheterogeneilyamongassembliesoftheAPpeptide,researchershavepreparedandstudiedApoligomersthatconsistofA0monomerslinkedbychemicalcross-links.39ThesestudieshavehelpeddeterminetheimportanceofdifferentresiduesinA(3oligomerizationandhavedemonstratedthatAp4()andAp42formdifferenttypesofo

10、ligomers.Crosslinkedoligomershavebeenfoundtobetoxictowardratpheochromocytoma(PC12)cellsandtoinhibitlong-termpotentiationinrats,providingevidencefortheroleofA0oligomersinneurodegenerationinAlzheimersdisease.Althoughcross-linkingA0decreasestheheterogeneityofApoligomers,cross-linkinghasnotyetproducedst

11、ructurallyhomogeneousoligomers.Thehigh-resolutionstructuresofthecross-linkedoligomersthathavebeengeneratedthusfanthrougheitherasingledisulfidebondorthroughphotoinducedcross-linkingofunmodifiedproteins(PICUP),remainunknown.Inthepastcoupleofyears,ourlaboratoryhasidentifiedandelucidatedhithertoundiscov

12、eredmodesofsupramolecularACSPublications2016AmericanChemicalSocietyassemblyofmacrocyclic0-sheetpeptidesderivedfromamyloidogenicpeptidesandproteins.111-13WepreviouslyreportedtheX-raycrystallographicstructuresoftwohomologoustrimersformedbytwomacrocyclicp-sheetpeptidesderivedfromA(317-36.10,14Thesepept

13、idescontainAp17-23andAB30-36p-strandscovalentlylinkedbytwo6-linkedornithine(sOrn)turnmimicsandaredesignedtomimicanAP17-36(3-15hairpin.Figure1illustratesthesepeptides,1and2,andshowstheirrelationshiptoanApi7-36p-hairpin.The6OrnthatconnectsresiduesD23andA30replacestheAP24-29loop;the6Ornthatconnectsresi

14、duesL17andV36reinforces(3-sheetstructure.16Wcincorporatedornithine(a-linkcd)asahydrophilicisostereofmethionineatposition35toimprovethesolubilityofthepeptides.14Peptides1and2bothcontainanN-methylgrouptoblockuncontrolledaggregation:peptide1containsanN-methylgrouponG33；peptide2containsanNmethylgrouponF

15、20.X-raycrystallographyrevealedthatpeptides1and2foldtoformp-hairpinsthatassembletoformoligomers.IntheX-Journal of the American Chemical SocietvArticle968 DOI: 13.1021/jacs.6bll748J. Am. Chem. Soc. 2017,139, 966-975raycrystallographicstructuresofpeptides1and2,threep-hairpinsassembleinatriangularfashiontoformtrimers,whicharestabilizedbyhydrogenbondingandhydrophobicinteractionsbetweenmonomers(Figure2).Althethreecornersofeachtrimer,themainchainofresidueV)sononemacrocyclicpshccthydrogenbondswiththemainchainofresidueE22ontheadjacentmacrocyclicp