生物大分子結構與功能第14章纖維蛋白課件

第十四章：纖維蛋白fibrousproteins

蛋白質(zhì)通?？煞譃閮煞N截然不同的功能類型，即由排列為長的蛋白纖維所構成的被動的結構材料和排列為小的致密結構域的多肽鏈所組成的細胞機理的活性成分。不管它們在結構和功能上的差異如何，這些類型的蛋白都含有由環(huán)區(qū)域所隔開的a螺旋和β折疊片。在大多數(shù)情況下，纖維蛋白含有特殊的重復氨基酸順序，這對于它們的特殊三維結構來說是必需的。 作為結構的材料纖維蛋白象其它的多聚物材料一樣在生物體內(nèi)它們都是長鏈分子，通過蛋白質(zhì)分子內(nèi)的各個分立的長鏈分子的交聯(lián)、內(nèi)部插入和纏繞的合適組合，形成具有許多不同功能性質(zhì)的蛋白質(zhì)纖維蛋白。根據(jù)組成纖維蛋白每個分子的二級結構，可分為三種不同的結構類型：角蛋白keratin和肌漿球蛋白myosin中的超卷曲a螺旋coiled-coil-helix，膠原蛋白collagen中的三螺旋，和淀粉樣纖維amyloidfibers和絲silk中的b折疊片。Thefundamentalstructuralunitisasimplerepeatingelementofsecondarystructure.Allfibrousproteinsareinsolubleinwater.Ahydrophobicaminoacidresiduesbothintheinterioroftheproteinandonthesurface.Fourmostrepresentativefibrousproteins:1,Keratins(角蛋白):

α-Keratininhair,fingernailsandanimalskin

β-Keratininfeathersandscales2,Collagen(膠原蛋白):inthematrixofbone,tendons,skin(upto1/3)3,Elastin(彈性蛋白):inligamentsandarterialbloodvessels(韌帶)(動脈血管)4,Fibroin(絲心蛋白):insilkmadebysilkwormsorspidersFibrousProteins:StructuralMaterialsofCellsandTissues主動脈腱Threemostabundantresiduesinfibrousproteins

存在于絲毛中的a螺旋纖維是有柔性的，它們可以伸展為近兩倍的長度，并且具有彈性以使得當張力被釋放時可以恢復到原始的長度。膠原蛋白纖維與之相比則更具有強度抗拉伸，因而相對具有剛性β折疊片纖維。既具有強度也有柔性最好的例子是蜘蛛網(wǎng)纖維，某些蜘蛛的網(wǎng)比相同粗細的鋼絲還要強，并且同時還具有柔性，以使得在蛛網(wǎng)上捕捉到昆蟲時不被弄破。纖維β折疊片在細胞內(nèi)也可由一些可溶性球蛋白的錯誤折疊產(chǎn)生因而會引起，像阿爾茨海默Alzheimer’s病和瘋牛病prion等疾病。 纖維蛋白常常形成原絲protofilaments或初纖維protofibrils，它們可以組裝為結構專一高度有序的絲蛋白或纖維蛋白。例如膠原蛋白、淀粉樣蛋白、中間體絲蛋白、管狀蛋白tubulin、肌漿球蛋白和纖維蛋白原fibrinogen

等。一般說來這些絲蛋白或纖維蛋白不能被結晶，因為它們僅在兩維方向上有序。但是有序的纖維能夠給出二維的衍射圖形，并可根據(jù)此二維圖形獲得它們大概的結構信息。但某些纖維蛋白或片段，例如纖維蛋白原原肌球蛋白tropomyosin、膠原蛋白、spectrin和肌漿球蛋白等已被結晶，并且獲得了它們的三維結構，這些詳細的三維結構信息對于解釋有序的絲蛋白和纖維蛋白的衍射圖形和電子顯微鏡照片是很有幫助的。在某些情況下纖維狀的蛋白組裝為一個高度有序的結構是一個動力學的過程，例如微管。它是由長的中空的球狀蛋白-管狀蛋白組成的多聚物，它可以連續(xù)地發(fā)生解聚和重聚化作用，并由此來控制細胞器organelles和細胞其它成分的定位。在成纖維細胞fibroblast中，單個微管的半衰期大約是10分鐘，而一個管狀蛋白分子的平均壽命要超過20小時，這樣每個管狀蛋白在它的壽命中將通過許多微管周期。

collagen這個字來源于西臘字，意思是漿糊。建立在不同家族成員的氨基酸順序測定的基礎上，現(xiàn)在的定義是collagen是一類蛋白質(zhì)。它們在細胞間的空間中組裝為纖維狀多分子的聚集，并且由具有大量的含有重復順序Gly-X-Y的三條肽鏈組成。X通常為脯氨酸，Y通常為羥基脯氨酸，羥基脯氨酸是由一種特殊的脯氨酰羥化酶prolylhydroxylase脯氨酸的后轉(zhuǎn)錄修飾所形成的。每一個膠原蛋白的多肽鏈含有大約1000個氨基酸殘基，一個完整的分子大約3000?長。 膠原蛋白鏈首先被合成為一個大的前體稱作原膠原蛋白procollagens。此原膠原蛋白在其頭尾兩端含有兩個約為200氨基酸殘基的原肽propeptides，這些原膠原蛋白的肽鏈在它們被組裝成三鏈分子之前被轉(zhuǎn)運到不光滑的內(nèi)質(zhì)網(wǎng)腔中，并在內(nèi)質(zhì)網(wǎng)腔中被羥基化和被進行其它的化學修飾，其末端的原肽對于合適的三鏈分子的形成是相當重要的。在它們形成三鏈結構之前首先形成鏈內(nèi)二硫鍵，這些二硫鍵可指導三條鏈的對齊，然后原肽被細胞間的酶所切開，并經(jīng)細胞排粒作用排出。兩端的原肽的刺激使得三鏈分子聚集為幾微米長50-200納米直徑的纖維蛋白，然后這些纖維蛋白邊邊平行相靠堆砌為平行束即膠原蛋白纖維，這樣的膠原蛋白纖維的強度要比相同尺度的鋼絲還要強。當成熟的膠原蛋白被變性的時候，由于沒有原肽的存在多肽鏈可以互相交聯(lián)在不同的部位上，而不能僅交聯(lián)在它們的末端上，因而形成隨機的三鏈，這些聚集作用只能形成膠明膠gelatin。一、膠原蛋白CollagenCollagencollagenhasevolvedtoprovidestrength.Itisfoundinconnectivetissuesuchastendons,cartilage軟骨,theorganicmatrixofbone,andthecornea角膜oftheeye.Thecollagenhelixisauniquesecondarystructurequitedistinctfromthe

helix.Itisleft-handedandhasthreeaminoacidresiduesperturnCollagenisalsoacoiledcoil,butonewithdistincttertiaryandquaternarystructures:threeseparatepolypeptides,called

chains(nottobeconfusedwith

helices),aresupertwistedabouteachother.Thesuperhelicaltwistingisright-handedincollagen,oppositeinsensetotheleft-handedhelixofthe

chains.Therearemanytypesofvertebratecollagen.Typically,theycontainabout35%Gly,11%Ala,and21%Proand4-Hyp(4-hydroxyproline,anuncommonaminoacid. Theunusualaminoacidcontentofcollagenisrelatedtostructuralconstraintsuniquetothecollagenhelix.Theaminoacidsequenceincollagenisgenerallyarepeatingtripeptideunit,Gly–X–Y,whereXisoftenPro,andYisoften4-Hyp.OnlyGlyresiduescanbeaccommodatedattheverytightjunctionsbetweentheindividual

chains.TheProand4-Hypresiduespermitthesharptwistingofthecollagenhelix.Theaminoacidsequenceandthesupertwistedquaternarystructureofcollagenallowaveryclosepackingofitsthreepolypeptides.4-Hydroxyprolinehasaspecialroleinthestructureofcollagen—andinhumanhistory. Thetightwrappingofthe

chainsinthecollagentriplehelixprovidestensilestrengthgreaterthanthatofasteelwireofequalcrosssection.Collagenfibrilsaresupramolecularassembliesconsistingoftriple-helicalcollagenmolecules(sometimesreferredtoastropocollagenmolecules)associatedinavarietyofwaystoprovidedifferentdegreesoftensilestrength.The

chainsofcollagenmoleculesandthecollagenmoleculesoffibrilsarecross-linkedbyunusualtypesofcovalentbondsinvolvingLys,HyLys(5-hydroxylysine);HisresiduesthatarepresentatafewoftheXandYpositionsincollagens.Theselinkscreateuncommonaminoacidresiduessuchasdehydrohydroxylysinonorleucine.Theincreasinglyrigidandbrittlecharacterofagingconnectivetissueresultsfromaccumulatedcovalentcross-linksincollagenfibrils.早期由P.Puuling,F.Crick和A.Rich等人對膠原蛋白纖維衍射研究的解釋指出，三條多肽鏈的每一條都被折疊為一個伸展的左手螺旋，在此螺旋中每螺旋圈有3.3個殘基，每個殘基沿螺旋軸上升的高度為2.9?。與之相比更致密的右手a螺旋每圈為3.6個殘基，每殘基的上升高度為1.5?，因此膠原蛋白螺旋中每圈的高度為9.6?，而右手a螺旋的每圈的高度為5.4?。膠原蛋白的這種伸展的肽鏈必須聚集形成穩(wěn)定的結構。圖14-1：每一條膠原蛋白分子的多肽鏈折疊為一種伸展的聚脯氨酸II型螺旋，其每圈沿螺旋軸上升9.6?，每圈3.3個殘基在膠原蛋白分子中三條這樣的多肽鏈繞著。一個公共的軸形成一個3000?長的桿狀超卷曲分子，其多肽鏈的氨基酸順序含有Gly-X-Y的重復序列一條多肽鏈兩圈的球棒模型一條膠原蛋白多肽鏈的模型，每個氨基酸殘基被表示為一個球膠原蛋白超螺旋的一小部分的結構。三條鏈分別被標以不同的顏色合成的脯氨酸或甘氨酸多聚物，也可以折疊為相似的伸展的左手螺旋，因此，此種螺旋的類型被稱作聚脯氨酸II型類型蛋白激酶的SH3結構域，可以識別其它蛋白中的短的富含脯氨酸的片段，當這些片段結合到SH3結構域上時，它們具有與膠原蛋白相似的螺旋構象。

膠原蛋白中的三條II型聚脯氨酸螺旋可通過繞中心軸的卷曲形成一個三體分子，此三體分子是一個具有大約100?重復距離的右手超螺旋圖，在此超螺旋中因為每條鏈中的第三個殘基的側(cè)鏈要緊挨著中心軸而在此位置上是沒有足夠空間容納側(cè)鏈的，因此它們只能是甘氨酸殘基，任何其它的殘基都將會破壞超螺旋結構。某些遺傳性的結締組織疾病就是由于編碼這些甘氨酸殘基的基因的突變造成的。此順序的要求是三螺旋類膠原蛋白結構域的一個標志，此標志被用來進行未知結構的蛋白的序列分析，三螺旋結構域已被發(fā)現(xiàn)存在于多種的超分子聚集的結構中。其范圍從腱tendons和軟骨cartilage的膠原蛋白纖維到基底膜的網(wǎng)絡形式和血互補成分C1q或糖結合collectin中的短的三螺旋平行串。Thestructureofcollagen(膠原蛋白)fibersTherepeatingtripeptidesequenceGly-X-ProorGly-pro-4Hyp)adoptsaleft-handedhelicalstructurewiththreeresiduesperturnb)space-fillingmodelofthesameαchainc)Threeoftheseheliceswraparoundoneanotherwitharight-handedtwistd)three-strandscollagensuperhelixshownfromoneend(Glyinred)

膠原蛋白三螺旋的詳細的結構細節(jié)已被人們進行了研究。H.Berman研究組測定了人工合成的類膠原蛋白肽段(Pro-Y-Gly)101.9?分辨率的晶體結構，此合成的類膠原蛋白多肽不能形成纖維但可結晶。為了研究正常的膠原蛋白三螺旋結構和甘氨酸位置被突變后的效應的細節(jié)，人們設計了特殊的氨基酸順序，在此結構的中間有一個甘氨酸被突變?yōu)楸彼帷＝Y構表明了直接的以及水介導的氫鍵在穩(wěn)定三螺旋結構的重要性。此外結構還表明丙氨酸的側(cè)鏈只要在螺旋的局部幾何學上作一些小的調(diào)整后，也可存在于三螺旋的內(nèi)部。圖14-2在肽鏈中部由甘氨酸突變?yōu)楸彼岬囊粋€類膠原蛋白的結構。模型每一條肽鏈都折疊為聚脯氨酸II型螺旋，并且三條多肽鏈形成一個類似于膠原蛋白分子一部分的超螺旋。丙氨酸殘基側(cè)鏈可適配在超螺旋的內(nèi)部，但引起了每條鏈扭曲的輕微變化，此調(diào)整可允許鏈間隙水分子參與鏈的連接，此種構象位移可幫助順序變化的適配

在正常的三螺旋膠原蛋白結構中，三條鏈是通過一條鏈的脯氨酸的C=O基團和另一條鏈的甘氨酸的NH基團形成直接的氫鍵緊密連接的。圖14-3膠原蛋白中三螺旋的鏈間氫鍵(a)正常膠原蛋白中三螺旋件的氫鍵(b)在肽鏈的中間部分由Gly-Ala替換結構中的水介導氫鍵由于丙氨酸側(cè)鏈的存在有四個水分子插入到三螺旋的內(nèi)部，在丙氨酸殘基周圍的區(qū)域三條多肽鏈被丙氨酸側(cè)鏈強行撐開，有四個水分子進入到鏈之間，直接的氫鍵被水介導的氫鍵所替換。

所有的側(cè)鏈以及甘氨酸的C=O基團在三條鏈中都位于三螺旋分子的外部以便與水分子接觸，這些水分子介導了羥基脯氨酸的羥基和每條鏈以及不同的鏈之間的C=O和NH基團之間的氫鍵。圖14-4類膠原蛋白多肽晶體結構中觀察到的鏈內(nèi)水橋的模型。三條肽鏈分別被標以綠色、紅色和藍色，水分子的氧原子被標以黃色，氫原子被標以白色(a)三個水分子與兩個C=O基團形成氫鍵并與其它水分子連接(b)長的水橋形成了圍繞三螺旋的網(wǎng)絡。這些水分子介導的氫鍵對于三螺旋的穩(wěn)定是主要的?？梢约俣ㄟ@可能也是羥基脯氨酸存在于膠原蛋白中的原因Collagenmolecules.Thechainsarecross-linkedbyunusualtypesofcovalentbondsinvolvingLys,HyLysorHisthatarepresentatafewoftheXandYpositionsincollagenStructureofcollagenfibrils.Collagen(Mr300,000)isarod-shapedmolecule,about3,000?longandonly15?thick.Itsthreehelicallyintertwined

chainsmayhavedifferentsequences,buteachhasabout1,000aminoacidresidues.Collagenfibrilsaremadeupofcollagenmoleculesalignedinastaggeredfashionandcrosslinkedforstrength.Thespecificalignmentanddegreeofcross-linkingvarywiththetissueandproducecharacteristiccross-striationsinanelectronmicrograph.Intheexampleshownhere,alignmentoftheheadgroupsofeveryfourthmoleculeproducesstriations640?apart.BiosynthesisandassemblyofcollagenSteps:1.Translationontheribosome(synthesizingtheprimarystructure);2.Hydroxylationofthelysineandprolinesidechains(stillontheribosome);3.Releasefromtheribosomeandadditionofsugarstolysinestocreateprocollagen;4.Assemblyofcentralregionsintotriplehelices/foldingofN-terminalandC-terminalendsintoglobularstructures;5.Exportofprocollagencomplexesfromthecytosoltotheextracellularspace;6.CleavageofN-terminalandC-terminalregions,yieldingthetropocollagentriplehelix;7.Oxidationofsomelysineresiduestoaldehyde乙醛derivatives,whichcross-linkwithlysineresiduesoroneanotheronotherstrandstosolidifythefiber成骨不全 Atypicalmammalhasmorethan30structuralvariantsofcollagen,particulartocertaintissuesandeachsomewhatdifferentinsequenceandfunction. Somehumangeneticdefectsincollagenstructureillustratethecloserelationshipbetweenaminoacidsequenceandthree-dimensionalstructureinthisprotein.Osteogenesisimperfectischaracterizedbyabnormalboneformationinbabies;Ehlers-Danlossyndromeischaracterizedbyloosejoints.Bothconditionscanbelethal,andbothresultfromthesubstitutionofanaminoacidresiduewithalargerRgroup(suchasCysorSer)forasingleGlyresidueineachα

chain(adifferentGlyresidueineachdisorder).Thesesingle-residuesubstitutionshaveacatastrophiceffectoncollagenfunctionbecausetheydisrupttheGly–X–Yrepeatthatgivescollagenitsuniquehelicalstructure.GivenitsroleinthecollagentriplehelixGlycannotbereplacedbyanotheraminoacidresiduewithoutsubstantialdeleteriouseffectsoncollagenstructure.先天性結締組織發(fā)育不全綜合征二、超卷曲通常形成纖維蛋白寡聚體和球狀蛋白

a螺旋超卷曲的基本特征是氨基酸順序是由七重(a-g)重復所識別的。位置a和d通常是疏水氨基酸，亮氨酸拉鏈DNA結合蛋白也是利用超卷曲形成均二聚體和異二聚體球蛋白的。各種纖維蛋白也有七重重復順序，它們也可利用超卷曲形成寡聚體，主要是二聚體和三聚體。包括肌漿球蛋白、纖維蛋白、原肌動蛋白、交聯(lián)蛋白像spectin和dystrophin以及間絲蛋白(desmin)、角蛋白(keratin)，vimentin和神經(jīng)絲蛋白等。

角蛋白是皮膚毛發(fā)和皮革等的主要結構蛋白，它屬于間絲蛋白intermediatefilaments超家族的成員。在人的基因組中由大約60種不同大小的基因組成。該家族的所有成員都含有一個同源的約有300個氨基酸殘基的中央?yún)^(qū)域，此中央?yún)^(qū)域具有典型的能夠形成超卷曲的七重重復順序。圖14-5：間絲蛋白單體的結構域和大多數(shù)的間絲蛋白具有一個很相似的通常大約由310個氨基酸殘基組成的并形成伸展的a螺旋的棒狀結構域，它們的氨基端和羧基端結構域是非a螺旋，并且大小和順序的變化很大。假定這些中央?yún)^(qū)域形成一個雙股超卷曲的均二聚體棒狀結構域和兩端的兩個球狀結構域，棒狀結構域相互作用形成絲的核心，而大小不同的球狀結構域凸出在絲的表面上。此多肽鏈組裝為間絲蛋白的假說圖示于圖14-6。圖14-6間絲蛋白構造的模型。(a)單體。(b)兩個單體配對形成超卷曲二體。(c)兩個二體形成反平行四體。(d)每個四體內(nèi)二體之間產(chǎn)生搖晃以利于與另外一個四體的作用。(e)四體沿螺旋方向包裹最后形成10nm的索狀間絲蛋白α-KeratinTheα-keratinshaveevolvedforstrength.Foundinmammals,theseproteinsconstitutealmosttheentiredryweightofhair,wool,nails,claws,quills,horns,hooves,andmuchoftheouterlayerofskin.Theα-keratinsarepartofabroaderfamilyofproteinscalledintermediatefilament(IF)proteins.OtherIFproteinsarefoundinthecytoskeletonsofanimalcells.AllIFproteinshaveastructuralfunctionandsharestructuralfeaturesexemplifiedbytheα-keratins.Proposedstructureforkeratin-typeintermediatefilamentsa)twoleft-handedsensehelicesb)Two-chaincoiled-coilsc)Protofilamentsd)Protofibrilsandfourprotofibrilsformaninterme-diatefilementAnindividualpolypeptideinthe-keratincoiledcoilhasarelativelysimpletertiarystructure,dominatedbyan-helicalsecondarystructurewithitshelicalaxistwistedinaleft-handedsuperhelix.Theintertwiningofthetwo-helicalpolypeptidesisanexampleofquaternarystructure.Coiledcoilsofthistypearecommonstructuralelementsinfilamentousproteinsandinthemuscleproteinmyosin.Thequaternarystructureofα-keratincanbequitecomplex.Manycoiledcoilscanbeassembledintolargesupermolecularcomplexes,suchasthearrangementofα-keratintoformtheintermediatefilamentofhair.ahairisanarrayofmanyα-keratinfilamentsStructureofhair:Hair-keratinisanelongated

helixwithsomewhatthickerelementsneartheaminoandcarboxyltermini.Pairsofthesehelicesareinterwoundinaleft-handedsensetoformtwo-chaincoiledcoils.Thesethencombineinhigher-orderstructurescalledprotofilamentsandprotofibrils.Aboutfourprotofibrils—32strandsof-keratinaltogether—combinetoformanintermediatefilament.Theindividualtwo-chaincoiledcoilsinthevarioussubstructuresalsoappeartobeinterwound,butthehandednessoftheinterwindingandotherstructuraldetailsareunknown.三、連續(xù)的β折疊片構成淀粉樣纖維

淀粉樣變性amyloidosis是因為正常的球狀和可溶的蛋白沉積在組織，例如大腦和眼睛的細胞間空間中，變?yōu)榉€(wěn)定的不可溶纖維的疾病。這種纖維的沉積是與幾種淀粉樣疾病相關的，包括傳染性海綿狀腦病(transmissablespongiformencephalopathies)和克羅伊茨費爾特-雅各布綜合癥(Creutzfeld-Jacob)、阿爾茨海默病Alzheimer’sdisease和II型糖尿病(typeIIdiabetes)。來自于不同來源的淀粉樣纖維(Amyloidfibrils)共屬于一個超結構，此超結構是一個大約直徑為為100?的長直無分支的棒狀體。人們已經(jīng)根據(jù)甲狀腺素運載蛋白transthyretin蛋白的淀粉樣纖維結構提出了纖維結構的一致性模型。C.Blake和L.Serpell利用同步輻射光源獲得了高分辨率的淀粉樣纖維衍射圖形，表明這種來自于病人的淀粉樣纖維是因家族的淀粉樣變性(familialamyloidoticpolyneuropathy)。這些病人的transthyretin蛋白發(fā)生了一個單點突變Val-Met，transthyretin蛋白的功能是把甲狀腺素(thyroxine)攜帶進血流中，這種突變引起了可溶的球狀transthyretin蛋白分子的聚合，并進而變?yōu)槌练e在心臟和眼睛上。C.Blake研究組早期已經(jīng)測定了transthyretin可溶性蛋白的晶體結構結構，表明它是一個均四聚體，每個亞基折疊為一個反平行的β結構,圖14-7transthyretin球狀形式的一個亞基的結構，β鏈分別被標以A到H，鏈C和D在淀粉樣纖維中被認為是非折疊的。圖14-8transthyretin蛋白纖維形成的β折疊片螺旋的模型。由24條β鏈組成的β螺旋的重復單位中，每條β鏈平均扭曲了15°，四個transthyretin多肽鏈以不同的顏色表示。由于在球狀蛋白中β鏈之間的平均距離大約為4.8?，沿纖維方向的重復單位相應于24條β鏈，每條β鏈扭曲15°。此值與球狀蛋白中的β折疊片有著很好的吻合。根據(jù)高分辨率的纖維衍射圖形，人們還建議每一個重復單位由四個transthyretin分子構成，每個transthyretin貢獻六條β鏈，形成β折疊片螺旋。此模型還認為在形成纖維時丟失了兩條β鏈，圖14-7中的C和D鏈，此推論已被部分的實驗所支持。transthyretin纖維的電子顯微鏡照片表明它的寬度大約為130?，是由四個直徑為50-60?的平行原絲構成的。Blake和Serpell提出每一個原絲是由四個連續(xù)扭曲的β折疊片所組成的，此扭曲的β折疊片以無限的長度沿纖維軸方向前進，β鏈垂直于纖維軸。每條β鏈含有大約10個氨氨基酸殘基，四個β折疊片配對排列，每對通過β折疊片之間的疏水內(nèi)核獲得穩(wěn)定。就像β

結構的球狀蛋白的情況一樣。兩對β折疊片之間也以互相配對方式形成在衍射圖上扭曲的β螺旋，構成了原絲的正常的內(nèi)核結構，且此內(nèi)核結構必須被其它物質(zhì)例如多肽鏈的環(huán)區(qū)域所包圍，而這些環(huán)區(qū)域則對纖維衍射沒有貢獻。此模型暗示著transthyretin蛋白從球狀向纖維狀重折疊，但是在其它蛋白中大量的重折疊事件的發(fā)生是主要為a螺旋的球狀亞基轉(zhuǎn)變形成淀粉樣β折疊片纖維。例如prions和a-乳清蛋白(a-lactalbumin)就是這種情況。即使此模型的細節(jié)可能是不正確的，但它不僅可解釋相應的纖維衍射，而且也能從球狀蛋白的β折疊片的結構研究獲得相應的知識。Theamyloidosescompriseaspectrumofdiseasescausedbythesystemicorlocaliseddepositionofcharacteristicfibrillarmaterial,termedamyloidfibrils.Thesedepositscanbefoundinvariousorgansandtissuesthroughoutthebody.Eachamyloidosisisclassifiedaccordingtothechemicalnatureoftheproteinthatformstheinitialamyloidfibrildeposit.Amyloidfibrilsareubiquitousstructuresthatarerichincrossβ-sheetsandtypicallyhaveafibrillarmorphology,whichcanvaryinlengthanddiameter.Amyloidfibrilsaredetected

invitro

and

invivo

usingspecific-bindingmolecules,namelyCongoRed,thiophenederivativesandThioflavin-SandT.ThemostcommonamyloidosesareAlzheimer’sandParkinson’sdiseaseandtype2diabetes,inwhichamyloidfibrilsarefounddepositedinthecentralnervoussystemandinbetacellsfromthepancreas胰腺,respectively.agroupofdiseasesthatroughlyaffectsapproximately8,000-10,000peopleworldwide.Theseamyloidosesarecausedbytheaggregationoftransthyretin（TTR）,anamyloidogenicproteinthatcangiverisetoamyloidfibrils.Transthyretin(TTR)wasfirstdiscoveredinthecerebrospinalfluid(CSF)腦脊髓in1942andthensequencedin1984,receivingthenameprealbumin前白蛋白becauseofitselectrophoreticmigrationpatterncomparedtoalbumin白蛋白.Afterwards,aimingtobetterdescribeitsfunctionality,itsnamewaschangedtotransthyretin-thetransporterofthyroxine甲狀腺素(T4)andretinol視黃醇、維生素A.TTRtransportsretinolthroughbindingtoretinol-bindingprotein(RBP)andT4duetotheformationofahydrophobicchannel,whichexistsonlywhenTTRistetrameric.Althoughitsfunctionmayvary,TTRishighlyconservedfromhumanstobacteria.TTRisa55kDahomotetramericproteinpredominantlysynthesisedbytheliverandchoroidplexus脈絡膜叢,whichislocatedinthebrain.AlthoughTTRisknownprimarilyasatransporter,emergingevidencehasdemonstratedthatTTRcanalsoactasaproteaseandaneuroprotectivemolecule.ThecrystallographicstructureofhumanTTR,whichwassolvedin1971,revealedthateachTTRmonomeriscomposedof127aminoacidresidues,forming8β-strandsnamedfromA-H,whicharearrangedinaβ-sandwichoftwofour-strandedβ-sheetsandonesmallα-helixfoundbetweenβ-strandsEandF.TTRmonomersinteractviahydrogenbondsbetweentheantiparallel,adjacentβ-strandsH-H’andF-F’toformadimericspecies.Thetwodimers(A-BandC-D)predominantlyformthetetramerthroughhydrophobiccontactsbetweentheresiduesoftheA-BandG-Hloops.Thetetramerformsacentralhydrophobicpocket(T4channel)withtwobindingsitesforhormones.EachTTRmonomercontainsonecysteineresidueatposition10andtwotryptophanresiduesatpositions41and79,whichcanbeusedasatoolformonitoringTTRunfolding.ManygroupshavestudiedthestructureofTTRanditsaggregationprocesstounderstandthetriggeringfactorsthatfavourTTRaggregation,whichoccursinanon-nucleatedmannerthatisknownasadownhillpolymerisationreactionbecausetetramerdissociationintomonomersistherate-limingstepoftheaggregationreaction

X-raystructureofTransthyretin.TTRisahomotetramericproteincomposedoffourmonomersof127aminoacids.Structurally,initsnativestate,TTRcontainseightstands(A-H)andasmallα-helix.ThecontactsbetweenthedimersformtwohydrophobicpocketswhereT4binds(T4channel).Asshowninred,eachmonomercontainsonesmallα-helixandeightβ-strands(CBEFandDAGH).Adaptedfromamodel;PDBcode1DVQ.X-raystructureofTransthyretin.TTRisahomotetramericproteincomposedoffourmonomersof127aminoacids.TTRtransportsretinol（視黃醇；維生素A）throughbindingtoretinol-bindingprotein(RBP)andT4duetotheformationofahydrophobicchannel,whichexistsonlywhenTTRistetramericAlthoughitsfunctionmayvary,TTRishighlyconservedfromhumanstobacteriaTheTTRgene,whichislocatedonchromosome18atposition12.1,presentsmanypolymorphisms,exceptforonedeletion,leadingtoover80aminoacidsubstitutionsintheTTRpolypeptidesequence.ManyTTRvariants,suchasV30MandL55P,areassociatedwithfamilialamyloidoticpolyneuropathy(FAP),alethalautosomaldominantdisorderinwhichmutantformsofTTRaggregatetoformamyloidfibrilsthatdepositintissues,especiallytheperipheralnervoussystem(PNS).TheseamyloiddepositsarepredominantlycomposedofintactorfragmentedTTRintheformoffibrillarspeciesthatprogressivelyaccumulate.Theinitialsymptomistypicallyasensoryperipheralneuropathyinthelowerlimbs,withpainandseverelyaffectedtemperaturesensationandlaterfollowedbymotorimpairments.MostpatientswithFAPhaveearlyandsevereimpairmentoftheautonomicnervoussystem,commonlymanifestedbydyshidrosis出汗障礙,sexualimpotence,alternatingdiarrhoea腹瀉andconstipation便秘,orthostatichypotension低血壓,andurinaryincontinence尿失禁.Thediseaseonsettypicallydependsonthemutationandtheethnic種族的backgroundofeachpatientandbeginsatapproximatelythethirdorfourthdecadeoflife,althoughtherearemanycasesinwhichthediseaseonsetoccurslaterinlife.NotallTTRvariantgenecarriersdevelopFAP,indicatingthatotherfactorsmustbeinvolvedinthedevelopmentofclinicalsymptoms.

TTR-relatedamyloidosesTherapiesforTTR-relatedamyloidoses:Whenandwheretoinhibitaggregation?ThetreatmentforTTR-relatedamyloidosesvariesaccordingtothesymptomspresentedbythepatient(cardiac,autonomicorcentral).Currently,FAPistheonlyformofTTR-relatedamyloidosesthathasatreatment,whichisorthotopiclivertransplantation(OLT).Althoughatreatmentexists,somepatientshavedisplayeddiseaseprogressionafterOLT,whichhasbeenshowntobeduetocontinuedamyloidformationfromwt-TTRandisespeciallytrueinpatientswithactiveFACsymptoms.Inaddition,afterOLT,somepatientsdevelopCNSAsymptomsduetotheproductionofmutantTTRbythechoroidplexus脈絡叢.AnotherproblemrelatedtoOLTtherapyisthatthisprocedureisalsoknownas“dominolivertransplantation”,inwhichtheliveroftheFAPpatientistransplantedintoanon-FAPpatient.However,thenon-FAPliverreceptorsdevelopFAPsymptomsinlessthan5yearsandnotlaterinlifeasexpected.ThesephenomenasuggestthatOLTtherapyispossiblebutthatitisnotafullyeffectiveandsafeprocedure.Inaddition,forFACorSSApatients,hearttransplantationshaveonlybeenperformedsuccessfullyworldwideinafewpatientspresentingcardiacamyloidosis.Insummary,theseclinicaldataemphasisetheimportanceofdevelopingnewtherapiesforTTR-relatedamyloidosis.AlthoughmanystudieshaveattemptedtoinfertheconditionsthataffectTTRstabilityandamyloidogenicity,whythediseasepenetrance,pathologyandclinicalcoursearesodifferentbetweenmutationsisstillnotcompletelyunderstood.Herein,wepresentsomeofthedataobtainedbydifferentgroupsthatprovidesevidenceonTTRstabilityandamyloidogenicity.ManygroupshavecollecteddataonTTRstabilitybystudyingthestructureofTTRanddesigningTTR-bindingcompounds.However,asfurtherquestionshavebeenraisedaboutTTRstability,moreevidencehasdemonstratedthatmostoftheanswerswithregardtoTTRstabilityliewithinitsthermodynamicpropertiesandrateoftetramerdissociation.WhyTTRaggregates?Althoughrestrictedtoasingleaminoacidsubstitution,TTRmutationsmaysignificantlyalterthekineticsofTTRaggregation.IntryingtounderstandwhichfeaturesofTTRmutantsareresponsibleforitsalteredamyloidogenicity,alargeamountofcrystallographicdatahasbeencollectedbydifferentgroupsworldwide.Unfortunately,theconclusionreachedwasthattherewasnosignificantdeviationinTTRstructure.Theonlyexceptiontothisconclusionwasthatthereweresignificantdeviationsforthepositionsatwhichtheaminoacidsubstitutionstookplace.AlthoughTTRmutationsdonotsignificantlyalterthetetramericstructurecomparedtowt-TTR,asingleaminoacidsubstitutionisenoughtoalterthethermodynamicstabilityandrateofdissociationofthetetramer

Transthyretin(TTR)amyloidcascade.TTRmaintainsatetramertomonomerdissociationrate,normallyformingfoldedmonomers.Thesemonomerscanpartiallyunfoldduetopointmutations,acidicconditions,metals,glycosaminoglycans(GAGs),anioniclipids,highhydrostaticpressure(HHP)andposttranslationalmodifications.Thisunfoldedmonomerispronetoaggregation,formingprefibrillarspeciessuchasoligomersand,consequently,matureamyloidfibrils.Inaddition,thealternativelyfoldedtetramersformedduringacompression-decompressioncycleofHHPcanaggregateeitherdirectlyorafterdissociatingintomonomers.TTR-stabilisers:AnewandpowerfulapproachtostopTTRaggregationTTRcanbindavarietyofmolecules,fromsmallhydrophobiccompoundstolargeglycosaminoglycans,withgreatflexibility.Asmentionedbefore,TTRiscomposedoffouridenticalmonomericsubunitsthatassemblearoundacentralchannel;therefore,thetetramerpossessesamolecularsymmetrywithtwohormone-bindingsitespertetramer(T4channel).Underphysiologicalconditions,onlyoneofthehormone-bindingsitesoftheT4channelisoccupiedbyT4.However,hormonebindingisgovernedbyaphenomenontermednegativecooperativity,inwhichthebindingofT4tothesecondbindingsitereducesthehormone-bindingaffinityofthefirsthormone-bindingsite.ThefactthatTTRisnottheonlyT4transporterinthehumanbodyandthatunboundT4concentrationsinbloodareverylow(lessthan0.1μM,34)agreewiththefactthatmostTTR

invivo

doesnothaveT4boundtoitstwobindingsites.ThisinformationhasmadeitpossibletodesignanoveltherapyusingsmallmoleculesthatbindtotheT4channelwithhighaffinityandreduceTTRaggregation.Miroyetal.haveshownthatthebindingofT4itselfstabilisestheTTRtetramerandconsequentlyinhibitsamyloidformation.Sincethen,manypharmacologicalagentsandnaturalproducts,suchasplantflavonoids,nonsteroidalanti-inflammatorydrugs(NSAIDs),andinotropicbipyridines,havebeentested,andtheseagentshavebeendemonstratedtobestrongcompetitorsforT4bindingtoTTRandevenpossesshigherbindingaffinitiesthanT4.Interestingly,manyofthesesmallmoleculessharestructuralsimilaritieswithT4,typicallypresentingoneormorearomaticrings.X-raycrystallographicstudieshavedemonstratedthatthesecompoundsbindtotheTTRhydrophobicchannel,similarlytoT4,duetothehydrophobicpropertiesofthecompounds.AsobservedforT4andevenRBP,asmentionedabove,thebindingofNSAIDstoTTRistypicallynegativelycooperative.However,dependingontheligand,thiscooperativitymaychange.ThecurrentlytestedNSAIDSincludediclofenac,diflunisalandflufenamicacid.Interestingly,thefirsttwocompoundsarecurrentlyapprovedbytheFoodandDrugAdministration(FDA)forthetreatmentofotherpathologicalconditions.StructuraldataforTTRcomplexedwithflufenamicacidanddiflunisaldemonstratethatthesecompoundsmediatehydrophobicandhydrogenbondinteractionsbetweenthesubunits,whichstabilisetetramericTTR.ThedetailedcomparisonsofseveralstructuresofapoTTR(nativeTTR)andTTRcomplexeswithsmallmoleculesprovideinsightsintothemechanismofligand-inducedconformationalchangesonligandbinding,

Althoughpromising,thechronicuseofNSAIDShasbeencorrelatedwithdyspepsiasymptoms,smallintestinebleedingandulcers.Recently,anewTTR-stabilisingdrug,Tafamidis,wasapprovedforuseinEurope.Thisclassofcompoundsfunctionsbypreventingtheformationofamyloidfibrils,retainingthenativetetramericstateofTTR.ThismodeofactionispossiblebecausethemostacceptedtheoryregardingamyloidfibrilformationisthatTTRtetramersneedtodissociatetoapartiallyfoldedmonomer,whichthenrapidlyself-assemblestogiverisetosolubleoligomerintermediatesbeforereachingtheamyloidstate.TherearemanystudiesintroducingtheuseofTTR-stabilisingdrugsorantisenseoligonucleotides(ASO)asnew,moreeffectivetherapiesagainstTTRamyloidosis.Thereiscurrentlyanon-goingtrialtotesttheefficacyofusingASOspecificallytotargetTTRinFAPpatientsanditssafetyinhealthysubjects[ISISpharmaceuticals,ISI-TTRrx,49].TherationalebehindASOtherapyandothersmallinterferingRNA(siRNA)therapiesisthattheseoligonucleotidesinterferewithTTRproductioni