简介：Venomousanimalsontheearthhavebeenfoundtobevaluableresourcesforthedevelopmentoftherapeutics.Enzymaticandnon-enzymaticproteinsandpeptidesarethemajorcomponentsofanimalvenoms,manyofwhichcantargetvariousionchannels,receptors,andmembranetransporters.Comparedtotraditionalsmallmoleculedrugs,naturalproteinsandpeptidesexhibithigherspecificityandpotencytotheirtargets.Inthisreview,wesummarizethevarietiesandcharacteristicsoftoxinsfromafewrepresentativevenomousanimals,anddescribethecomponentsandapplicationsofanimaltoxinsaspotentialdrugcandidatesinthetreatmentofhumandiseases,includingcancer,neurodegenerativediseases,cardiovasculardiseases,neuropathicpain,aswellasautoimmunediseases.Inthemeantime,therearemanyobstaclestotranslatenewtoxindiscoverytotheirclinicalapplications.Thechallenges,strategies,andperspectivesinthedevelopmentoftheproteintoxin-baseddrugsarediscussedaswell.

简介：Thereisaccumulatingevidencethatcysteinesulfenation（cys-SOH）inproteinsplaysanimportantroleincellularresponsetooxidativestress.Thepurposeofthepresentstudywastoidentifymitochondrialproteinsthatundergochangesincys-SOHduringaging.Studieswereconductedinratswhentheywere5or30monthsofage.FollowingblockingoffreeproteinthiolswithN-ethylmaleimide,proteinsulfenicacidswerereducedbyarsenitetofreethiolgroupsthatweresubsequentlylabeledwithbiotin-maleimide.Sampleswerethencomparativelyanalyzedbytwo-dimensionalWesternblots,andproteinsshowingchangesinsulfenationwereselectivelyidentifiedbymassspectrometrypeptidesequencing.Asaresult,fiveproteinswereidentified.Proteinsshowinganage-relateddecreaseinsulfenationincludepyruvatecarboxylaseandpyruvatedehydrogenase;whilethoseshowinganage-relatedincreaseinsulfenationincludeaconitase,mitofilin,andtubulin（α-1）.Resultsofthepresentstudyprovideageneralpictureofmitochondrialproteinsulfenationinbrainoxidativestressandimplicatetheinvolvementofproteinsulfenationinoveralldeclineofmitochondrialfunctionduringbrainaging.

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简介：Micro-electrondiffraction(MicroED)isanemergingtechniquetousecryo-electronmicroscopetostudythecrystalstructuresofmacromoleculefromitsmicro-/nano-crystals,whicharenotsuitableforconventionalX-raycrystallography.However,thistechniquehasbeenpreventedforitswideapplicationbythelimitedavailabilityofproducinggoodmicro-/nano-crystalsandtheinappropriatetransferofcrystals.Here,wedevelopedacompleteworkflowtopreparesuitablecrystalsefficientlyforMicroEDexperiment.Thisworkflowincludesinsituon-gridcrystallization,single-sideblotting,cryo-focusionbeam(cryo-FIB)fabrication,andcryo-electrondiffractionofcrystalcryo-lamella.ThisworkflowenablesustoapplyMicroEDtostudymanysmallmacromolecularcrystalswiththesizeof2-10μm,whichistoolargeforMicroEDbutquitesmallforconventionalX-raycrystallography.Wehaveappliedthismethodtosolve2.5Acrystalstructureoflysozymefromitsmicro-crystalwithinthesizeof10×10×10μm^3.OurworkwillgreatlyexpandtheavailabilityspaceofcrystalssuitableforMicroEDandfillupthegapbetweenMicroEDandX-raycrystallography.

简介：Amolecularmodelofpancreaticzymogengranule（ZG）iscriticalforunderstandingitsfunctions.WehaveextensivelycharacterizedthecompositionandmembranetopologyofratZGproteins.Inthisstudy,wereportthedevelopmentoftargetedproteomicsapproachestoquantifyrepresentativemouseandhumanZGproteinsusingLC-SRMandheavyisotope-labeledsyntheticpeptides.TheabsolutequantitiesofmouseRab3DandVAMP8weredeterminedas1242±218and2039±151（mean±SEM）copiesperZG.ThesizedistributionandtheaverageddiameterofZGs750±23nm（mean±SEM）weredeterminedbyatomicforcemicroscopy.TheabsolutequantificationofRab3Dwasthenvalidatedusingsemi-quantitativeWesternblottingwithpurifiedGST-Rab3Dproteinsasaninternalstandard.Toextendourproteomicsanalysistohumanpancreas,ZGswerepurifiedusinghumanaciniobtainedfrompancreaticislettrans-plantationcenter.OnehundredandeightyhumanZGproteinswereidentifiedforthefirsttimeincludingboththemembraneandthecontentproteins.Furthermore,thecopynumberperZGofhumanRab3DandVAMP8weredeterminedtobe1182±45and485±15（mean±SEM）.ThecomprehensiveproteomicanalysesofmouseandhumanpancreaticZGshavethepotentialtoidentifyspecies-specificZGproteins.ThedeterminationofproteincopynumbersonpancreaticZGsrepresentsasignificantadvancetowardsbuildingaquantitativemolecularmodelofaprototypicalsecretoryvesicleusingtargetedproteomicsapproaches.TheidentificationofhumanZGproteinslaysafoundationforsubsequentstudiesofalteredZGcompositionsandsecretioninpancreaticdiseases.

简介：精子不是成熟的直到他们运输他们获得使肥沃的活动性和能力的epididymis通过顺序的修正的一颗卵。epididymis有三功能的区域，头，语料库，和尾，和epididymis的钠蛋白质在上述修正起重要作用。然而，有在头和尾之间的微分丰富的蛋白质大部分仍然是未知的。在精子成熟期间揭示头和尾的函数，从鼠标的头和尾的钠蛋白质被等压的标签为相对、绝对的quantitation(iTRAQ)分析。总的来说，128差别充实蛋白质被发现，哪个46是充实的头，82是充实的尾。生物信息的分析证明那类脂化合物新陈代谢在头是活跃的；当阴离子绑定和阳离子绑定活动和磷和organophosphate新陈代谢在尾是活跃的时。新epididymal钠蛋白质，包含1的充实头的PDZ领域(Pdzk1)，也把Na+/H+交换称为规章的余因子3(NHERF3)，它在胆固醇新陈代谢和肉毒碱运输起一个关键作用，在类脂化合物新陈代谢被发现。西方的弄污和immunofluorescence分析证明Pdzk1在epididymis然而并非在睾丸被表示，并且在精子尾巴的中间的片局部性。Pdzk1蛋白质水平也在normozoospermic人与那相比在asthenozoospermic病人的情况下在精子被减少，建议Pdzk1可以参予精子成熟规定并且可以与男不孕被联系。这些结果可以提供新卓见进精子成熟和男不孕的机制。

简介：Insulinsecretorygranules(ISGs),agroupofdistinguishingorganellesinpancreaticβcells,areresponsibleforthestorageandsecretionofinsulintomaintainbloodglucosehomeostasis.ThemolecularmechanismsofISGbiogenesis,maturation,transportation,andexocytosisarestilllargelyunknownbecausetheproteinsinvolvedinthesedistinctstepshavenotbeenfullyidentified.Subcellularfractionationbydensitygradientcentrifugationhasbeensuccessfullyemployedtoanalyzetheproteomesofnumerousorganelles.However,useofthismethodtoelucidatetheISGproteomeislimitedbyco-fractionatedcontaminantsbecause1SGsareverydynamicandhaveabundantexchangesorcontactswithotherorganelles,suchastheGolgiapparatus,lysosomes,andendosomes.Inthisstudy,wedevelopedanewstrategyforidentifyingISGproteinsbyproteincorrelationprofiling(PCP)-basedproteomics,whichincludedISGpurificationbyOptiPrepdensitygradientcentrifugation,label-freequantitativeproteome,andidentificationofISGproteinsbycorrelatingfractionationprofilesbetweencandidatesandknownISGmarkers.Usingthisapproach,wewereabletoidentify81ISGproteins.Amongthem,TM9SF3,anine-transmembraneprotein,wasconsideredahighconfidenceISGcandidateproteinhighlightedinthePCPnetwork.FurtherbiochemicalandimmunofluorescenceassaysindicatedthatTMgSF3localizedinISGs,suggestingthatitisapotentialnewISGmarker.

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简介：Disulfidebondsarevitalforproteinfunctions,butlocatingthelinkagesiteshasbeenachallengeinproteinchemistry,especiallywhenthequantityofasampleissmallorthecomplexityishigh.In2015,ourlaboratorydevelopedasensitiveandefficientmethodformappingproteindisulfidebondsfromsimpleorcomplexsamples（LuetaLinNatMethods12：329,2015）.Thismethodisbasedonliquidchromatography-massspectrometry（LC-MS）andapowerfuldataanalysissoftwaretoolnamedpLink.Tofacilitateapplicationofthismethod,wepresentstep-by-stepdisulfidemappingprotocolsforthreetypesofsamples--purifiedproteinsinsolution,proteinsinSDS-PAGEgels,andcomplexproteinmix-turesinsolution.Theminimumamountofproteinrequiredforthismethodcanbeaslowasseveralhundrednanogramsforpurifiedproteins,ortensofmicrogramsforamixtureofhundredsofproteins.Theentireworkflow--fromsamplepreparationtoLC-MSanddataanalysis--isdescribedingreatdetail.Webelievethatthisprotocolcanbeeasilyimplementedinanylaboratorywithaccesstoafast-scanning,high-resolution,andaccurate-massLC-MSsystem.

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简介：Fluorescentproteins(FPs)withemissionwavelengthsinthefar-redandinfraredregionsofthespectrumprovidepowerfultoolsfordeep-tissueandsuper-resolutionimaging.Thedevelopmentofred-shiftedFPshasevokedwidespreadinterestandcontinuousengineeringefforts.Inthisarticle,basedonacomputationaldesignandgeneticcodeexpansion,wereportarationalapproachtosignificantlyexpandandred-shiftthechromophoreofgreenfluorescentprotein(GFP).WeappliedcomputationalcalculationstopredicttheexcitationandemissionwavelengthsofaFPchromophoreharboringunnaturalaminoacids(UAA)andidentifyinsilicoanappropriateUAA,2-amino-3-(6-hydroxynaphthalen-2-yl)propanoicacid(naphthol-Ala).OurmethodologyallowedustoformulateaGFPvariant(cpsfGFP-66-Naphthol-Ala)withred-shiftedabsorbanceandemissionspectralmaximaexceeding60and130nm,respectively,comparedtothoseofGFP.TheGFPchromophoreisformedthroughautocatalyticpost-translationalmodificationtogenerateaplanar4-(p-hydroxybenzylidene)-5-imidazolinonechromophore.WesolvedthecrystalstructureofcpsfGFP-66-naphthol-Alaat1.3■resolutionanddemonstratedtheformationofamuchlargerconjugatedn-systemwhenthephenolgroupisreplacedbynaphthol.Theseresultsexplainthesignificantred-shiftingoftheexcitationandemissionspectraofcpsfGFP-66-naphthol-Ala.

简介：Thisstudywasconductedtoevaluatetheeffectsofplant/animal(P/A)proteinratios(viz.1∶4,1∶3,1∶2,1∶1,2∶1,3∶1,4∶1)ongrowthperformance,bodycomposition,apparentdigestibilityofdiets,andnonspecificimmunityofjuvenileseacucumber(Apostichopusjaponicus).Seacucumbersweredividedinto21plastictanks,andeachtankwasstockedwith15individuals(initialweight:about23.73g).Eachfeedwasallocatedtothreereplicatesofseacucumbers.Thefeedingexperimentlastedfor50days.Resultsindicatedthatweightgainrate(WGR)andbodywallweight(BWW)significantlyincreasedasdietaryratioofP/Aincreasedfrom1∶4to3∶1,andthendecreasedsignificantlywithfurtherincreaseofthisratio(P<0.05).Thebodywallcoefficient(BWC)showedasimilartendencytoWGRandBWW,butnosignificancewasdetectedamongdietarytreatments(P>0.05).Theapparentdigestibilityofdrymatter,proteinandlipidincreasedwithratioofP/Aincreasingfrom1∶14to2∶1(P<0.05),andthendecreasedwithfurtherincreaseofthisratio.Correspondingly,activitiesoftrypsinandamylaseweresignificantlyincreasedasP/Aincreasedfrom1∶4to2∶1(P<0.05).TheactivitiesofSODandCATshowedasimilartrendwithWGR,withthehighestvalueobservedintheratioof1∶2and1∶1,respectively.ResultsaboveshowedthatmoderateorrelativelyhigherratioofP/Aprotein(1∶1-3∶1)significantlyincreasedthegrowthperformance,apparentdigestibility,andnonspecificimmunityofseacucumber.Thiswillcontributetoimprovingthefeedformulationforjuvenilecucumbers.

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简介：ToinvestigatethechangesofthebiochemicalcompositionofAmericanshad(Alosasapidissima)eggsandlarvaeatembryonicandearlylarvalstages,sampleswerecollectedatdifferentdevelopmentstagesfromartificialfertilizationtotheendofyolkabsorptionincluding2h,12hand30hafterfertilizationandnewlyhatchedlarvaeincluding1and3daysafterhatching.Thecompositionoflipid,fattyacids,proteinandaminoacidswereanalyzed.Thecontentoftotalproteinexhibitedadecreasingtrendduringembryogenesisandlarvaldevelopment,andasignificantreductionwasdetectedafterhatching(P<0.05).Thetotallipidcontentremainedrelativestable.Asignificantreductionwasdetectedinalmostallaminoacidsafterhatchingexceptforglycine(P<0.05),whileasignificantdecreasewasfoundinthecontentofcysteine,proline,tyrosine,valine,isoleucine,leucineandphenylalanineduringtheyolk-sacphase(P<0.05).Ontheotherhand,allthegroupsoffattyacidsremainedstableduringtheperiodofembryogenesis.Butafterhatching,asignificantdecreasewasfoundinthecontentofC18:2n-6,C18:3n-6,SFAandratioofEPA/ARA(P<0.05),whileasignificantincreasewasfoundinthecontentofC18:3n-3,C20:4n-6,C22:6n-3andratioofn-3/n-6(P<0.05).Inconclusion,thecombineddatasuggestedthatAmericanshadutilizestheproteincontentaspreferentialenergysubstratesduringembryonicandearlylarvaldevelopmentswithsomespecificityintheconsumptionofdifferentaminoacids.