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by: Ashley Kunze


Ashley Kunze
GPA 3.61


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This 17 page Class Notes was uploaded by Ashley Kunze on Thursday October 22, 2015. The Class Notes belongs to CHEM 279 at University of California Santa Barbara taught by Staff in Fall. Since its upload, it has received 32 views. For similar materials see /class/226954/chem-279-university-of-california-santa-barbara in Chemistry and Biochemistry at University of California Santa Barbara.




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Date Created: 10/22/15
INSIGHT Stability and flexibility of epigenetic gene regulation in mammalian development Wolf Reik1 During development cells start in a pluripotent state from which they can differentiate into many cell types and progressively develop a narrower potential Theirgeneexpression programmes become more defined restricted and potentially locked in39 Pluripotent stem cells express genes that encode a set of core transcription factors while genes that are required later in development are repressed by histone marks which confer shortterm and therefore flexible epigenetic silencing By contrast the methylation of DNA confers longterm epigenetic silencing of particular sequences transposons imprinted genes and pluripotencyassociated genes in somatic cells Longterm silencing can be reprogrammed by demethylation of DNA and this process might involve DNA repair It is not known whether any of the epigenetic marks has a primary role in determining cell and lineage commitment during development Development is by definition epigenetic Differences in the prof grammes of gene expression that result in the development of different organs and tissues occur without changes to the sequence of our DNA with one or two exceptions There is nothing mysterious in this con cept subsets of the 30000 genes in our genome are active in different tissues and organs depending on their regulation by different sets or combinations of transcription factors This implies that if we were to take all of the transcription factors that activate genes in a liver cell and transfer them to a brain cell while inactivating all brainespecific trane scription factors then the brain cell would turn into a liver cell A recent study provides tantalizing insight into this concept of epigenetic control of development Takahashi and Yamanaka identie fied four transcriptional regulators that when expressed in broblasts resulted in L L 39 1 J to L L 39 ESelike cells Extending this concept a little further in somaticecell nuclear transfer the nucleus of a somatic cell from an adult individual is transplanted into an oocyte from which the nucleus has been removed re ultingin 1 39 theadult nnrlen and f I development of the cloned animal loning however is inefficient because most if not all cloned animals have epigenetic defects particularly in DNA methylation Therefore our lack of understanding of how epigenetic marks are reproe rammed is a key obstacle to cloningz Similarly the reprogramming of broblasts to become ESJike cells is a rare event in vitro and epigenetic defects uch J eC 411 promoter f 39 I 39 fthe enrorlerl factor have been noted in these ESJike cells These observations highlight that in addition to transcription face tors changes in 39 uring J r A 39 J orcausedby pig i 39 2397 L fDNA at CpG sequences in vertebrates4 5 modification of histone tails6 and the presence of nonenucleosomal chromatineassociated proteins7 Therefore 11 Iain vv 11v 391quot z r 1 1 late epigenetic marks that differ froin those of plurip otent cells and dif ferentiated cells of different lineages also accumulate different marks epigenetic marks which can be removed before a cell divides or within few cell divisions with the longeterm stability and heritability of other marks which can be maintained for many divisions Fig 1 During the early stages of development genes that are required later in 1 1r 391 1 MLquot modi cations which are highly exible and easily reversed when expression of these genes is needed During differentiation genes that are crucial for pluripotency are silenced by histone modifications as well as by DNA methylation Some of these genes are also silent in mature germ cells meaning that epigenetic marks probably need to be reversed rapidly after fertilization to allow reeexpression of pluripotencyeassociated genes in Held in a renre ed to be stably maintained from the gametes into the early embryo and the adult organism Methylation of imprinted genes can only be erased in primordial germ cells PGCs the cells that ultimately give rise to the germ line Probably because there is a requirement for both removing epigenetic marks and retaining epigenetic marks between generations Pigeneticv v u L v L w J 1 3911 erations In this review I addresshow the fascinating interplay between transcription factors and epigenetic factors is beginning to provide an explanation for how plurip otency and development are regulated y I39m 39 39 In this section three issues are k m in an 39 rlcn addressed First are differentiation 1 A 39 quot ilenced manner in pluripotent cell types in order to be activated later And is the removal of epigenetic marks from these genes needed for their activation Second are rmlr I q M V l l lVl l l 1 cell types This inactivation could in principle be irreversible because somatic cell types are not required to give rise to pluripotent cells One exception is the germ line where reactivation of pluripotencyeassociated genes is needed at the initial stages of development however later the silencing of these genes is essential for the differentiation of mature germ cells And therefore third is the removal of permanent silencing marks 39 V t V I In this review I focus on the role of epigenetic regulation in devele from L iei LillLdLlUu crucial opment particularly comparing the shorteterm exibility of certain genes such a plur39l 7 W A Irene earlyin I lLaborator Ul TL mh M R T UK 02007 Nuuu Puhllnhlng Group INSIGHT There is recent evidence forthe first type of epigenetic regulation that isthe temporary inactivation of differentiationispecific genes in pluripotent cell types Fig 2a Genes that are A 39 d J 39 J A ment and differentiation 7 for example those in the homeobox Hox distaliless homeobox Dlx paired box Pax and sineioculisirelated homeobox Six gene families 7 are held repressed in pluripotent ES cells by the n p PcGH r tein A t in mice and humans This system marks the histories associatedwith these genes by inducing methylation of the lysine residue at position 27 of histone H3 H3K27Bquot ES cells that lack EED embryonic ecto derm develop 7 merit a component of the Pporotein repressive complex PRC have partly derepressed J t A J m v v 1 W lntprp nch r within bivalent chromatin regions which contain both inactivating 1 I 1 1H J V V 1 I I 911 dentified demethylase 12 The H 3K27 methylation mark occurs mostly outside the context of DNA methylation In contrast to the terminal quot 39 39 J thNA methvlation di rn edlater A genes that are silenced by PRCs in pluripotent tissues require repressive marks to be rapidly and exibly removed when differentiation begins Strikingly in cancer cells the genes targeted by PRCs often become DNA methylated which might result in a more permanent locking7in of a pluripotent state in cancer stem cells The second type of epigenetic regulation to be considered is whether A 4 39A y 391 quot1 l A V 39 inactivated in differeni tiated cell types Several genes that are required for early development or for germ7cell development only r xample those that encode plui A 7sustaining transcription factors such as OCT4 and NANOG 7 are known to be expressed byES cells but silenced on the This could indicate that after the repressive marks have been removed when expression of the components of PRCs are downregulated dur7 39 J 39 39 39 L 39 vised for transcrip7 tional activation through the H3K4 methylation mark It is important to note that ep igenetic silencing by PRCs might be mitotically heritable through an unknown mechanism 7 but these marks could presumably be rapidly removedby enzymatic demethylation of H3K27 by an unii f these cells with a defined kinetics of acquiring represi sive histone modifications and DNA methylation Fig 2b Silencing by both histone modifications and DNA methylation in somatic tissues seems to be typical of this group of genes and of those that encode cani v t quot quot L39 L A ddim n permato ene i 151tis probable that this permanent type of epigenetic silencing safeguards againt 39 1 A 39 394 ft 39 139 39 J quot emu e that might lead 1 quot and pprhap to AA quot 39 39 to r 3 1 1 1 1 g l l l l l i E H3KZ7 Ai154vlm l 3 u g H3K4methylation a A 7 a E u Oocyte Zygote Morula Blastocyst Embryo gt 39gt Adult Germcells nner 1 cellmass Sperm ES cells DNA methylation e 1 E l 3 u i rlgure 39 39 39 39 J 39 methylation U 39 39 dcvcluluuculOfPGCSDNA L 39 icy A Auge39he gLoILI Ao A 14 n 1 1 modi cations andgene7expression patterns Veryearly39 quot 39 A 39 m 39A 39 39 U A 39 U 39 39 that DNAmethylationiserased In additionAl 39A 39 39 4U quot U quot l U L 39 incuhure A 4 l l A A k A A A U A uupm A U A A U A A systemandH3K27methylationDuringthedifferentiationofpluripolent afterwards 39 39 quot 39 39 39 L39 IIIMIltIII x x 4 11 L B A U A A U a uuuumemA A y are uhnFDNA meihvlaiinn A 39 39 conLrasljDNA l39 39 l39 quot 39 39 39 39 potentially A developmental genes begin to be expressed and there is an increase in H3K4 426 L I 0 transposons and some pluripolency7associated genes 02007 Nituu Puhllnhlng Group INSIGHT canceng L L L L L L L L L tnn L L L cularlyofDNAmethylationquot quot 39 h T 39 l l quot39 39 39 39 1quot39 hfLLLLh L39 139 39 air L L L L 18 n L LL L II LL A mm LL L L L L L LLL L L c I A 41 I LI I I L L L I LI 4 L n 4 L L L l39 39 l 39 39 The RHMDID39 KL 39 39 39 1 hirha orir r l developmentardemethylation 139 rc amp M14 1 r 39 aLI wLHC repmssHaxrfamily genes and ocher somatic genes in PGCs19 Fig 2c 39 lL r L L L L L L L L L Tm quotof LLLL LL 7m L LL LLLLL L H L r L L I L L L L L H m m 117 ma LL Am LL 39 39 K nLechylauonm l l 39 r 39 39 I 39 A 4 Inprr In L n I L x u I 39 39 39 39 m w 14 quot Thereforemfter a Tempurary Lepressian uf develnpmenml genes by he PG pmein system P unpotant DLHeremLaLen CE eeH HLsLone dametm as HLstone mathy transfarasa P unpotant DLHeremLaLen CE eeH a HLsLone dametm as MQL U HLsLone mathy transfarasa DNMT c Maimenanee Msilencing ufsuma LegenesLLL earlygerm Lens P unpotant PRC Eany PGC CE 7 Q g HLsLone dametm asa QLQ Lani HLsLone mathy transfarasa h r 39 39 39 39 39 1quot I39m 1 LI ILL mrmcell quot1 39 39 39 39 39 4 n I L 39 L L L L L L L 5 LL L L L L L L L L L L L A Whethem L r r L m L LLLLILLL WLL e quot 1 39 l M II I u L L L L L L sileII 39 PRMTS 0mm NaIuIFuhIImng Gmup INSIGHT Fv li ati n 39 IUI I I development to take place discussed later 39 39 39 tw39r quot ch m tlrnlati n A T ofDNM1 1for one division cycle during very early prerimplantation development and then by DNMl ls the somatic form ofDNMTl in embryonic and adult tissues L Stability fortransposon silencing and imprinting 1 o H a I I u u i u r i n r Mm rue mutations Therefore man L L L L 39 39 cu m m u atedthemselvesand markedbyrepressiveL39 139 L 39 1 39 nonrcodingmnsplicednudearRNAE intn it Ina 39 r A39 WA inheritance across generations discussed later Imprinted genes are a class ofmammalian genes with possible promotersbecome methylated and in that silencing relies on long m and its cofactorDNM39ULlike DNM1 3LM25 Fig 33 How particular i i quot Tn n m rd I ram i Alternatively the RNA might coat the region to be inactivated simir x chromosomeam This might establish a physical structure from which DNAnlmn POIWH IndI quot 39 39 39 silencing 5 Fig 3b1n one case ofsilencing mediated by an imprinted A W it i i ii i r i Ram Mature gamete b Silenclngu hexhmmusumeandimprintedgenes Embmmhmgg Piurlpotant DNMT 52H Hlstone methyltmusterase7 Mmthqu PRC inartivatitm a quot 1 r Iquot 139 T3 lt Wm en t i i i i H PRMT i h Fmr i u in green H3K27 methylatiou inyellow histoue argiuiue methylatiouiu c t AnNA 423 mm NaturlFIlh lhlnl Gmup nonicoding RNAs Kenqlotl and Xz39st begin to be expressed from the paternal allele in the twoicell embryo and gene silencing in els and the acquisition of histone modi cations follow during the next few cleavage divisions and are largely complete by the blastocyst stage Fig 3b The second model of how imprinted genes are silenced involves an epigenetically regulated chromatin insulator In this model tissue7 speci c enhancers are located on one side of the DMR overlapping with the insulator whereas the silenced genes are on the other side Silencing occurs when the DMR is unmethylated and binds chromatiniorganizing proteins such as CTCF CCCTC 7binding factor resulting in a higher7 order chromatin structure that prevents interactions between remote enhancers and promoters Xichromosome inactivation is another example of a relatively stable epigenetic silencing event in this case large regions of a whole chroi osome are involved In mice imprinted Xichromosome inactivation is probably largely initiated by expression osz39st from the paternal chromosome at the twoicell stage The nature of the imprinting leading to paternal expression is still unknown but it is unlikely to be DNA methylation Imprinted Xichromosome inactivation is then stable even inthe absence of DNA methylation in the extraiembryi onic tissues Although the PcG protein system which confers H3K27 methylation marks has some39 1 quot 39 the e modii fications do not seem to confer heritable silencing Random Xichroi mosome inactivation is initiated in the epiblast after reprogrammin imprinted inactivation W This reprogramming might be initiated by the silencing oinst expression and if this is the case it is possible that the mitotic memory for inactivation simply resides in the expression oinst The subsequent upregulation oinst expression during the dif7 ferentiation of epiblast cells is again followed by coating gene silencing and acquisition of histone marks ver in contrast to imprinted Xichromosome inactivation CpG islands in inactivated genes on the X L L 1 although itha notbeentested genetically this might constitute longiterm memory for inactivation during embryonic and adult life 3 Fig 3b It is important to note that this methylation of CpG islands seems to be a dead end in that it does not need to be reprogrammed during the normal life cycle In the germ line the inactivated X chromosome does not become methylated o m a v r DNAimethylation patterns that have been acquired during develop7 ment are stable in somatic cells and during adult life DNAimethylation patterns are somatically heritable essentially through the action of DNMT1 the maintenance methyltransferase At most CpG sites the error rate of maintaining methylation 1 per division is low in relation to the number of cell divisions that are needed to pro duce a mammalian organism 44 for humans Indeed methylation of CpG islands is never erased during normal development By con7 trast methylation of CpG islands in imprintedigene DMRs needs to be erased in the germ line so that genderispecific methylation can be imposed subsequently during germicell development This erasure takes place in a defined period 7 from E105 to E125 in PGCs 7 in all imprinted genes that have been tested and it could occur by active demethylation of DNA by an unknown mechanism possibly involving DNA repair discussed later This mechanism for erasure might also underlie the demethylation and activation of noniimprinted genes such as Mvh Dazl and Sycp3 which takes place at about the same stage20 Fig 4a Epigenetic reprogramming in PGCs entails widespread loss of DNA methylation as well as H3K9 methylation In addition to the erasure of genomic imprints this epigenetic reprogramming might also help to return PGCs to a pluripotent state because at these stages ofPGC development pluripotent embryonic germ cells can be established in culture through the reactivation of genes such as Nanog Not all genomic methylation is lost however at L t such as IAPs remain fairly highly methylated Later in spermatogenesis de novo methylation occurs not only sexispecifically in imprinted genes but also in transposons and in singleicopy gene 4 INSIGHT sequences For example the Nanogpromoter becomes highly methyli ated in mature sperm Distinct genomeiwide reprogramming events also occur immedii may 39 M 391 a 39 1 39 J I Fig 4b Many sequences in the paternal enome become suddenly demethylated shortly after fertilization503953 This demethylation occurs after the removal of p rotamines basic proteins that are associated with DNA in sperm and the acquisition of histones by the paternal genome during the long G1 phase before DNA replication Methylation can be observed by staining cells with an immuno uorescently labelled antibody specific for Simethylcytosine Judged by the substantial loss of immuno uorescence signal together with the considerable loss of L1v rune I J t v JILWL39 139 j v 43 the paternal genome loses a signi cant amount of methylation although more precise measurements and more information about which sequences are affected and unaffected would be valuable Sequences that are known not to be affected include IAPs and paternally methi ylated DMRs in imprinted genes Fig 4c A recent study provides intriguing insight into a protein that might protect the genome from demethylation The protein stella also known as DPPA3 binds to DNA and was originally identified because expression of the encoding ene 39 I 1 during early PGC development Stella is present in large amounts in oocytes and after fertilization translocates to both pronuclei Deletion of the gene from the oocyte an therefore removal of the protein from the zygote results in early preiimplantation lethality of embryos as well as loss of methylation of the following sequences the maternally methylated genes Peg also known as Mest PegS also known as Nnat and Peg10 the paternally methylated genes H19 and Rasgrfl Ras proteinispecific guanineinucleotideireleasing factor 1 and IAPsS So stella might either directly or indirectly protect specific sequences from demethylation in the zygote but it is unknown how other sequences are protected Fig 4c The mechanism of active demethylation in the zygote is still unknown However the DNA deaminases AID and APOBECl have been shown in vitro to deaminate Simethylcytosine in DNA to thymi iness this results in ToG mismatches which can be repaired by the baseiexcision repairpathway Interestingly Aid and A170 bee are located in a cluster of genes with Stella growth differentiation factor 3 Gdf3 and Nanog Stella Gdf3 and Nanog are all expressed in pluripotent tis7 sues and Gdf3 and Nanoghave important roles in conferring stemicell identity on ES cells Indeed Aid and Apobeel are also expressed by oocytes stem cells and germ cells and recent work shows that in viva targeting ofAID to the methylated H19 DMR in the zygote results in efficient and substantial demethylation of this region C F Chan H Morgan F Santos D Lucifero S PeterseniMahrt W Dean and WR unpublished observations Although it is unclear whether AID andor APOBECl are responsible for the demethylation of the paternal genome in the zygote the evidence suggests that base7 excision or mismatch repair might have a role in this process I think that this suggestion is supported by the recent identification of a DNA glycosylase7lyase 7 DEMETER 7 that preferentially excises Simethyli cytosine from DNA inAmbldopsis thalimmssy DEMETER is required for the demethylation and activation of the imprinted gene MEDEA see page 418 Another DNAidamageiresp onsive gene the mouse gene Gadd45 growth arrest and DNAidamageiinducible 45 might also have a role in demethylationsg Although there have been suggestions that the methyl group could be directly removed from DNA by hydrolytic attack or by oxidation these mechanisms have not been substantiatedz The relative exibility of histone methylation might be brought about by th INSIGHT m 1 1 1 1 1 1 L 1 a Mn 1 n 1 1 1 a 1 1 1 r 1 1 nr 11mm r ri t t tn an i a I 9111 nt 39 39 r 39 39 39 L Z7 39 39 l quot 39 39 39 iti int r t t A n i 39 39 l m hiquot 39 39 U m 1 11 r 111 11 1 1 1 huh WMquot 1 L1 nmmh nn II 1 1 1 52 IL A 39 die pancn a Geis brokerf byerasureofmethylation ofthe paternal genome after of paternal imprints59 or to be a consequence ofDNAarepair processes fertilization 1i i r u r in Caenma General demethylation during this period could also havea role in h MW l n th L p 39 L 39 39 39 1 r amnl arl 139 139 39 4 4 ML r r r 39 39 uicc iabii he 39 39 quot 1 11 1 1 53 1 1 a 1 1 1 1 1 aquot 15 A A L I l 39 39 39 39 39 39 in needtobe 39 39 1 39 ladin t arl demethylatedfor proper expression to occur Fig 4b 39 39 l 39 39 action oern Epigenetic spillover across generations One other area that is unique to mammalian biology deserves 39 39 39 l maizequot a F r generation Atpresent we have no understanding ofhow molecular 39 39 u rquot r 39 quot 139 39 quot 554 rar ntt11d tion can spill overto thenextgeneration The ability ofsornaticcells 39 39 39 39 l 39 39 t39 439 394 39 inL r39 39Lquot 39 39 39 hhtmr 39 i ii 11a 1 parental germ cells isanieclnanisticexaniple ofthis Fig 4c Another 39 39 39 55 4 I L M M a L conferred on some genes by adjacent insertion ofLAPs This can alter L c L 1 1 1 1 1 1 1 1 1 1 r r 39 39 39 39 39 I uninen iaied embryo L a rasurenirnethylatinninPGCs LaterPGC EarinGC B Q Histonedemethyias DNAd tM 7 bibtbimz W W DNAreplieatiw b Erasure n1 methylatinn atand aiter fertilizatiun Plunpotent cell Mature gamete DNA demEtNias e l l m V DNArepliaation Histona methyitransferase Prnteatinn against DNA dernetliylatinn at fertilizatiun Plum potent cell a Mature gamete DNA dernetliylase7 J E A L i A Ati hitn nmknma 39 l quot quot 39 39 39 39 1 1 m 1 n m L 39 k 39 39 Thpnmtl in l m m 1 t l l quot 39 39l fertilizationH3K9methylationisshowningreenH3K4niethylationin L blue andDNA methylation in ed 02007 ununpuhlnhlng amp INSIGHT Conclusions and outlook 9 1 1 1 quot 1 1 1 1 1 1 11182012006 Development might be aoneeway street because ofthe somatic inherit 1O AZWPV 1 N HR R a 538 39 ance of epigenetic marks Whether there is a linear relationship between 2006 H I 1 1 A1 1 1 11 7 11Rn11nRF1139 39 1 e V 1 V A l l m39e 39039 Oubt embryomcsiemce11sCe11125315 32 2006 ful some key restrictions in developmental potential that are brought 12 K16 R 1 K llln F M R n Kimic doma39m about by epigenetic regulation might occur very early in development 13 geVVmechyElatJOIVigutfwe Rey 506271715VV7275V2006 d V 1 1 1 m e u s emce 1 ec roma 111 pa em maypre 1spose umorsuppressorgenes Judging from somatijcffell nucleajretransfer experiments 1tV1s far from m N V V P 242 2007 meal wuet er cells aVe r marks or 14 Feldman NYeiul39a med1a1ec1 1rrevers1b1e ep1gene11c inactivation o10c134dur1ng have marks that are more difficult for the oocyte to A V N l H R l 4 2 09 V V 1 1 1 b d dt tht 15 S1mpso 1 1111 n 01 ion Mum ClienT r1141 1 V Natum eplgenetlc reprogrammmg mlght e 1199 e 0 ensure a gametogenesls and cancer NulureRev Cunce15615 6252005 development can start afresh in every new generation Although various 16 Hochedllnger K Yamada Y Beard C ampJaenlsch R Ecto 1c expression of Oct 4 b1oc1lts 1 1 the 11 histone 1 v 1 lewudy Eg egtor ce11c1111eren11a11on and causes dysplasla 1n eplthellaltlssues Ce11121 465 477 been identified the mechanism ofDNA J till needsto 17 on P M F k a W H P A 1 Hquot K 1 1 be determined Recent work on the erasure of DNA methy1ation from mouse clones consequences 1or plurlpotency Genesoems 1209 1219 2002 1 1 1 1 SuramM MK Hum n 39 139 39 139 imprinted plant genes shows that baseeexcision repair has an important ng 747 762 2007 role and it is possible that this is also the case in mammals Because of 19 n 11 K 1 1 39 39 39 the generally accurate heritability of DNA methylation and because of 20 1601176 351 if Af ne C9 Bl l 31V 623 530V 2006 its chemical stability erasure of DNA methylation might only be pose 39 n5 11 i 11 1 sible eitherby replicating DNA inthe absence ofDNMTl orby breaking Deveopmen1133 3411 3418 2006 21 Suran1Aamp e1 Wln Ep1gene11csedsA111sCD1enuwe1n1ampRe1nbergD315 327 ColdS rln Harbortaboralor PressW00dbur 2007 It is fascinatingto seethatboth Udll LJJFUUkadLLUl J 21 RN 11 g m TH Y y epigenetic programming and reprogramming seem to be needed to germ ce11s1ack1ng Dnm13t Nature 431 96 99 2004 39 39 39 39 v 23 R ln 39 maintain plunpotench in early embryos and lVES Vcells Indeed experie 260V 309 310 1993 menial 1 nuclei Wiuiuut 24 Rm 111 1 11 1 lln Rnllm n R R 1n T H Dn 1 1 J 39 L 39 cell nuclear transfer or cell fusion has been achieved recently using 0 malemalgemmlc 1mpr1ntSSc139ence 29412536 2539 2001 amix of luri otenc factors1 It couldbe ex ected that forcin the 2539 K n A M f l V p F V V 39 V V p g materna11mpr1n11ngNo1o1e429900 9032004 expression of pluripotency transcrlptlonsfactor networks would also 26 Jellnlc P StehleJ c amp Shaw P Theleslls speci clactorCTCFLcooperates w391th the activate epigenetic reprogramming factors but whether this occurs is V39 1V1 1 39 V 39 39 V39 39 39 053 on11ne 4 e355 2006 do1101371iourna1pb1o0040355 unclear Perhaps combinations of transcription factors and epigenetic 27 Howe C reprogramming factors are needed for more complete reprogramming Dnm gene Ce11104 829 838 2001 ofsomatic cells toapluripotent state andthis would be ofgreat fundae 28 65 1993 l quot lquot P M l M N l 2 mental SCie ti C and medical interest 29 Sleutels F Zwart RampBarlow D P Thenon cod1ng411 RNAls required 1ors11enc1ng Inthe animal kinurlnm I39 39 iirh a imprintin 39 39 N 415810 8132002 have evolved only in mammals Many ofthe basic molecularbuilding 3039 gVlaflc m D quot l D 39 l l l l M 7 F T m 7 M ing blocks for epigenetics such as the enzymes for DNA methylation and De 20V 1263 123212006 histone modifications are highly conserved invertebrates but the regue 31 Lew1s 1 1 lation ofep1genet1c modifiers might evolve more rapidly together With 2004 N l l 2quot 1 q 1295 r 390 J A trategie Therefore 39 cygcnctl 32 11m1 1110 1 1 39 39 J 39 39 moms and epigenomics will have an important role in discovering links 6 39 39 V 39gt 39 N 1 v v v ene13s12961300 2004 between developmental adaptVationsandepigenetic regulators 33V KandUVVVCVVTP PVV N P M P P There is probably a con ict between the requirement for erase 39 1 39 1 1 F AR 1 71 19 2106 ing epigenetic marks between generations and the requirement for 2006 V 111 v 34Lew1s1 A v 11 maintaining others such as those in 1mpr1nted enes and some Deve pmem33V4203 4210 2006 transposons This con ict most probably underlies the observation that 35 ChaumellJ Le Baccon P W012 A amp Heard E A novel ro1e foerstRNA 1n the 1orma11on 39 39 notem e t39 39 39 39 39 Genes Dev 20 2223 2227 2006 1 1 a L r J 1 1 mgto V V V V nllul llldllf 19 V i s 6 mm R 1 M mm M R R r1n1nm 1 M 39 39 39 39 39 page 396 Epigenetic inheritance across generatlons 1s relatively come rego1a11on1n c1us1ersAnnu Rev CelDev B1o119 237 259 2003 moninplantsbut it isstillunclear OWWirle prea 39 l 3739 K W f T L A V 1 h th th 1 h 1 t 51 1nher1ledh1gher 0rderchr0mat1n conformation 1ores1r1c1 enhanceraccess to 1ng Pmc 1111113111ij VSOTW 9 V9 35 311V e 111 5 Vang 9V0 1 1011 V NutAcud so USA10310684 106892006 An exc1t1ng question for future work is whether segregation of 38 1 39 39 39 epigenetic marks in early developmenthas any primary role in deter 39 gl 219 jacl val m T m Fe Name 4381 339 3739005 a o mining cell and lineage commitment For example the mechanismby 1 r11 11 n 1 R39 1771 94 303 which the first two cell lineages are allocated in mammalian preeimplane 2000 tatvlon embryos although a matter thot debate is not really understood 40 Kohlmaler A e1o1 A chromosomal memorymggered by X151 regulates h1stone 39 1R1111 1H3911391 p1 1239111117 39 An epigenetic hypothesis might allow us to take a fresh look at a long pb100020171 J standing fundamental problem in developmental biology l 41 Mak 1 l 39 39 39 39 L 303666 669 200 1 Takahashl1ltamp rank 4 39 39 11 4 0 mnlnl 111 P 111 r 1th r H rlF 39 39 39 andadult 11brob1as1 cultures by de ned 1ac1ors Ce11125 663 676 2006 W W 39 39 39 39 J 39 m 44 649 2 n 11 mm F n K r n wampRe1k 39 39 2004 mamma1s Hum Mo Gene114 R47 R582005 43 11 A F 01 1 R M n 39 39 v 3 111 r 1 N1 in T R lnh r 39 mum oftheXchromosome GenesDev 201848 1867 2006 PressW00dburyZOO7 all M R 10 T 11 39 39 R1 1239 h 74 4 BlrdADNAmetliylatlon patternsand eplgenetlcmemoryGenesDev166 21 2002 481 5142005 5 HP i1 45H11mp 1 1 Ahn1117 NulureRev Gener 3 662 6732002 15 23 2002 6 Turner B M De11n1ngan ep1gene11c code Nature Ce1181o1 92 62007 46 1 1 1 1 7 R1quot 0 1 P n R 39 39 39 1 dayll5 pr1morc11a1 germ ce11s Deveopmen11291807 1817 2002 Trlthoraxgroup pro1e1nsAnnu Rev Gener 38 413 4432004 47 Sek1ve1o1 Extensive and orderly reprogramm1ngo1genome w1c1e chroma11n 8 Boyert L Chemistry amp Biology Vol 11 1037 1042 August 2004 2004 Elsevier Ltd All rights resented DOI101016jchembiol200406007 Protein Motions Promote Catalysis Audrey Tousignant and Joelle N Pelletier D partement de Chimie Universit de Montreal Montreal Quebec Canada HSC 3J7 A relationship between molecular dynamics motions of noncatalytic residues and enzyme activity has re cently been proposed We present examples where mutations either near or distal from the active site residues modify internal enzyme motion with resulting modification of catalysis A better understanding of internal protein motions correlated to catalysis will lead to a greater insight into enzyme function How does enzyme structure contribute to catalytic power1397 quot I 39 39 39 area of internal protein motions stemming from studies suggesting an intimate relation between molecular mo tion and enzyme activity The majority of studies regard ing l 1 independent from the event of ligand binding have been undertaken by direct modification of catalytic residues within the active site to study shortrange effects The distal environment which we will consider to be out side of the immediate area defined by the catalytic resi dues is generally neglected although a growing body of evidence indicates that it can be required in defining the body of molecular dynamics that governs catalysis This new outlook on the impact of the distal environment toward enzymatic reactivity has been made possible as a result of more efficient technologies particularly molecular dynamics simulations zyme active site generally corresponds to a fairly restricted location in the protein Its catalytic resi puxiiy m L quot r a 39 generally results in a dramatic loss of activity as classi cally illustrated by mutation of any residue constituting a catalytic triad 2 Mutation of catalytic residues is more frequently practiced than mutation of noncatalytic re idues because it is statistically easier to identify a residuem39 39 39 L quot her present at the active site than in the large number L n u t dues may directly contact the reactive substrate atoms or may participate in reactivity without direct contact For example the protonrelay mechanism of catalytic triads relies on residues Asp His that are a few ang stroms distant from the reactive substrate atoms How ever is there a catalytic role for residues that are not directly implicated in the chemical transformation be longing to what we will refer to as the distal environ ment Can residues that are located too far from the site of chemical transformation to participate directly in catalysis modulate catalysis otherwise Some recent studies on unrelated enzymes have re Minireview vealed mutations of distal residues ie residues that are not directly implicated in the catalytic mechanism that have effects on enzymatic catalysis without inter acting directly with the substrate lnstead their effect is a consequence of modified internal dynamics of the enzymes in question The internal motions were modi fied by specific mutations producing different subsets of conformations that can be attained by the mutants relative to the native enzymes These conformations are sampled through time until those resembling the transi tion state are achieved allowing catalysis to proceed Thus varying internal protein motions by mutation can affect the probability of attaining the transition state Although the mutations were of noncatalytic residues they affect catalysis because they result in conforma tional quot 39 g 39 the active site quot as different types of motions occur on time scales that may or may not be relevant to the reac tion of interest We will demonstrate providing specific examples from four systems that motions of certain 39 residues can help define the preorganiza tion of lization 8 These initial studies suggest that we must explore a large ensemble of internal fluctuations that participate in global enzyme molecular dynamics to comprehend the influence of dynamics on catalysis Molecular dynamics is a combination of all three dimensional motions in a molecule throughout time Protein motions occur on a time scale ranging between 10quot5 to 104 seconds and can cover amplitudes ranging between 001 and 100 A with an energy variation of 01 to 100 kcalmol 4 5 Three classes of internal motions have been identified in proteins The smallest local mo tions 10quot5 to 10quots include atomic fluctuations side chain motions loop motions and terminal arm motions 4 Rigidbody motions 10 9 to 13 constitute the sec ond type of internal motions where a small part of the protein moves in relation to another 4 These motions are responsible for altering the height of the activation free energy barrier at thetransition state 6 They include helix motions domain motions and subunit motions Finally largescale motions are similar to rigidbody mo tions but occur on a greater time scale 10 7 to 10 s 4 5 Helixcoil transitions dissociationassociation 39 quot 39 39 39 39 Iluuu angesy 1 ations and foldingunfolding transitions are examples quot 39 quot Allof L 39 39 39 ence protein organization The enzymatic reaction rate is directly influenced by the height of the activation free energy barrier and by the transmission coefficient Figure 1 Internal enzyme motions can modify the catalytic rate in two distinct ways First by influencing the height of the activation free energy barrier Figure 1 A which implies a modifica tion of the equilibrium between the transition state and the reactants and products Second by influencing the capacity of recrossing the barrier Figure 1B which Chemistry amp Biology 1038 Figure 1 The Free Energy Profile of an Enzymatic Reaction A represents the height of the activation free energy barrier and B represents barrier recrossing events which are characterized by the transmission coefficient K Adapted from 6 is characterized by the transmission coefficient K 6 These concepts have been thoroughly discussed in a recent review 6 the details of which are beyond the scope of this review We briefly present them below so as to introduce the concepts required for the following discussion Promoting motions which represent conformational changes occurring on the time scale of the overall reac tion along the reaction coordinate modify the height of the activation free energy barrier Dynamical motions on the other hand occur on the femtosecond time scale and specifically influence the transmission coefficient K These motions composed of local rigidbody and largescale motions contribute in different ways to the catalytic rate The reaction rate is defined as kdyn KkTsr where kGlyn is the overall rate constant kTST is the equilib rium transitionstate theory rate constant which ac counts only for crossing of the free energy barrier and K is the transmission coefficient which accounts for barrier recrossing kTST is defined by kBTgtAGikBT T kTST where h is Planck s constant k3 is Boltzmann s con stant AGi is the activation free energy barrier obtained from energetic profiles and T is the temperature of the system 6 From these equations we observe that a variation of free energy at the transition state AGi affects the reac tion rate exponentially while the transmission coeffi cient K which is responsible for barrier recrossing has an effect only as a prefactor Therefore promoting mo tions occurring on the time scale of the reaction should have a greater impact than dynamical motions fs time scale on enzymatic activity 6 However all of these types of motions whether of small or large amplitude or frequency and whether involving few noncatalytic resi dues or entire segments of the enzyme may influence catalysis We present recent studies on the influence of motions of noncatalytic residues on catalysis that have been performed on four enzymes dihydrofolate reductase DHFR triosephosphate isomerase TIM liver alcohol dehydrogenase LADH and BIactamase T EM1 which are all structurally and functionally unrelated While a wealth of research focuses on these wellcharacterized enzymes we have identified a restricted number of re search papers that specifically relate motions of noncat alytic residues to catalysis in each of these enzymes We now examine them as being the first examples in a new approach to a more detailed understanding of catalysis which are likely to serve as precursors to fur ther similar observations in many other enzymes in the future Dihydrofolate Reductase Dihydrofolate reductase is one of the bestcharacterized enzymes from a structural and a functional point of view Crystallographic analyses have demonstrated that E coli DHFR adopts different conformations during ca talysis which result from a subtle internal protein motion implicating an important fraction of the enzyme 7 The meticulous work of Benkovic and HammesSchif fer has related this important internal protein motion to catalysis 3 7 Mutagenesis studies showed that two major loops Met2O loop and BFBG loop Figure 2 containing conserved but noncatalytic residues are the most affected by this internal motion Their studies re veal a network of coupled promoting motions each of small amplitude 05 A existing throughout the enzyme which altogether modify the height of the activation free energy barrier 7 Their effect on catalysis is the conse quence of a network of hydrogen bonds present be tween the loops that induce a motion throughout the enzyme The motion leads to the formation of many structural conformations including those most similar to the transition state conformation Consequently these distal motions of noncatalytic residues influence cataly sis in DHFR by longrange structural perturbations whose effect is transmitted to the active site 8 Let us review the evidence that allowed the link between distal motions and catalysis to be established DHFR catalyzes the reduction of 78 dihydrofolate DHF to 5678 tetrahydrofolate l39 HF using nicotin amide adenine dinucleotide phosphate NADPH as the reducing cofactor NMR analyses of E coli DHFR have demonstrated that the Met2O loop constituted by resi dues 9 to 24 Figure 2 can adopt three different confor mations open occluded or closed depending on the nature of the bound substrate 8 When DHF and NADPH are bound the Met2O loop adopts a closed conformation resulting in a strong interaction between the loop and NADPH allowing hydride transfer to occur 7 In this form a more rapid ns to ps time scale dy namic motion in the BF3G loop residues 117 1 31 Fig ure 2 is proposed to be important for hydride transfer ms time scale although it contains no catalytic resi dues 3 Its importance was demonstrated by mutagen esis of the conserved Gly121 located 12 A from the site of hydride transfer to Val The mutation decreased the rate of hydride transfer by a factor of 163 because of an increase in the activation free energy barrier 9 Further investigation by molecular dynamic M D sim ulations identified important hydrogen bonds A hy drogen bond between Gly15 Met2O loop and Asp122 BF3G loop has been observed only in the closed con formation Ieading to the suggestion that the closed conformation stabilized by the GIy15Asp122 hydrogen bond favorably positions DHF and NADPH proximal to Minireview 1039 Figure 2 Internal Protein Motions in Dihydrofolate Reductase A Threedimensional structure of E coli DHFR 9 B A portion of a network of coupled promoting motions in DHFR Only the active site region is shown although the proposed network extends throughout the enzyme Arrows and a dotted arc indicate one potential motion of DHFR that may be conducive to catalysis of hydride transfer 27 each other to initiate the reaction 7 This hypothesis changes of the Met20 loop control substrate position ing in a manner that is critical for hydride transfer It thus appears that thejuxtaposition of DHF and NADPH in a catalytically productive arrangement is modulated y a change in protein motion provoked by contacts distal from the active site To con rm the importance of hydrogen bonds be tween the Met20 and BFBG loops 39 I were determined Replacement of residues 16 19 in the Met20 loop by only one Gly decreased substrate bind ing only 1 Ofold but decreased the rate of hydride trans fer 400fold due to a global alteration of the motion of both loops as detected by NMR 8 Also mutagenesis of Asp122 revealed a strong correlation between the presence of hydrogen bonds and the rate of hydride transfer 10 These results demonstrate that the motion of a noncatalytic residue such as Asp122 which is lo cated far from the active site m8 A Figure 2 can a 391 1 1 W It is interesting to note that several of these distal resi g u Jus As a result of these studies it was suggested that a network of coupled promoting motions is responsible for the internal dynamics of E coli DHFR The network includes the Met20 and the BFBG loops but extends yet farther from the active site and is comprised of many small amplitude motions No5 A Kinetic analysis of double mutants of other distal noncatalytic residues has shown a nonadditive effect on activity further sup porting the hypothesis of coupled promoting motions in catalysis This is the case for GIy121Met42 double mutants that are 20 A apart and GIy67Gly121 double mutants that are approximately 30 A apart These muta tions are spatially separated and distal to the active site yet enhance each other s effect on activity relative to the effects of the individual mutations 7 9 More re cently MD simulations were undertaken by Rod Rod kiewicz and Brooks III to visualize the effect of the proposed coupled motions on catalysis Mutants M42F G121S G1 21 V and M42FG121S were constructed in silico Correlated motions conformational changes hy studied Their work confirms that mutation of noncata lytic residues can affect correlated motions and cou pling of many structural elements throughout DHFR by longrange interactions and further points to internal mntinns39 39 39 39 quot that can have an important effect in enzyme catalysis This body of work reveals the importance of understanding the dy namic makeup of an enzyme to better understand enzy matic catalysis Triosephosphafe Isomerase L J of dihydroxyacetone phosphate DHAP to Dglyceral dehyde 3phosphate GAP TIM from chicken yeast and trypanosome sources controls active site access via the largescale motion of a loop 11 This conserved loop of 11 residues residues 166 176 behaves like a lid to the active site Figure 3A TIM thus has two forms an open and a closed form which differ by an N12 kcalmol energy difference and by a local internal movement of 7 A of loop 1 66 1 76 1 2 1 3 The amplitude of movement can be deduced by comparing the crystal structures of the two forms the distance between Gly178 and Ser211 is 977 A in the open form and 27 A in the closed form while the distance between Ala176 and Tyr208 is 505 A in the open form and 29 A in the closed form The potential role of noncatalytic residues of TIM in catalysis was examined by NMR following the observa tion that the time scale for the conformational transition of loop 166 176 correlated well with the measured rate of transformation of DHAP to GAP 12 Transition be tween the open and the closed conformations occurred at a rate in the range of 103 to 10A squot consistent with the turnover rate of 09 X 10A squot 12 14 Further more Chemistry amp Biology 1040 Figure 3 Noncatalytic Residues Proposed to Promote Catalysis via Protein Motions in Three Further Enzymes A TIM active site accessibility is controlled by movement of loop 166 176 The open and closed forms of TIM are illustrated 12 B Threedimensional structure of C Threedimensional structure of E coli TEM1 BIactamase 29 specific MD analyses have demonstrated that noncata lytic residues present at the active site Ala176 and Tyr208 are essential for enzyme reactivity 11 The hy droxyl group of Tyr208 forms an essential hydrogen bond with the amide nitrogen of Ala176 during loop closure Closure appears to stabilize the charged inter mediate by preventing water molecules from accessing the active site and also prevents elimination of phos phate1115 The most important catalytic residue is Glu165 which promotes proton transfer to produce GAP MD studies have demonstrated that Glu165 follows the motion of loop 166 176 When the enzyme is in its free form Glu165 hydrogen bonds with Ser96 the bond is broken upon substrate binding 12 Hence the loopclosing mechanism may be induced by substrate binding breaking the hydrogen bond and allowing Glu165 to LADH showing the central activesite cleft and position 203 28 move 2 A to interact with the substrate In addition the hydrogen bond formed between Ala176 and Tyr208 further stabilizes this closed conformation Recent stud ies concerning loop motions in TIM also propose that the open conformation compensates for the loss of in tramolecular hydrogen bonds Ala176Tyr208 Glu129 Trp168 and Ser21 1 Gly1 73 by forming new intramolec ular Glu165Ser96 and intermolecular hydrogen bonds with solvent molecules The solventenzyme hydrogen bonds considerably lower the barrier to transition and thus the energetic difference to transit from the closed to the open form 11 A more comprehensive model of TIMmediated catalysis at an atomic level has recently been published by Guallar et al 16 The relation be tween motion and catalysis documented in TIM may represent a model for similar largescale hingetype motions in other enzymes that have been suggested Minireview 1041 generally on the basis of crystallographic information to be related to catalysis although the correlation of motion to catalysis has not been established 17 18 TIM is therefore a further example demonstrating that protein dynamics generated by noncatalytic residues enhance the probability of attaining the transition state 11 12 Liver Alcohol Dehydrogenase Horse liver alcohol dehydrogenase represents a further example of an enzyme where the motion of a noncata lytic residue Val203 has a significant influence on reac tivity LADH is a Zn dependent metalloenzyme that catalyzes the reversible oxidation of various alcohols to their corresponding aldehydes or ketones using NAD as the hydride acceptor HammesSchiffer has reported on the motion of Val203 which is located 5 A from the reactive center and the hydrideacceptor C4 of NAD 6 within the activesite cleft yet on the face opposite the catalytic residues Figure 3B By dynamics simula tions the distance between Val208 0y and C4 of NAD was shown to increase as the transition state was ap proached This is the result of a thermally averaged promoting motion proposed to significantly decrease the activation free energy barrier to the reaction The motion of Val208 appears to favor the approach of the hydridedonating carbon on the alcohol being oxidized and the acceptor C4 of NAD via steric interactions This proposed role correlates well with the decrease in reaction rate observed upon mutation of Val208 to the smaller alanine The local motion of Val203 is of small amplitude No6 A and thus its investigation may be limited essentially to molecular modeling approaches The case of LADH further illustrates how enzyme mo tions by noncatalytic residues may promote catalysis BLactamase 3lactamases protect bacteria from the lethal effects of 3lactam antibiotics by hydrolyzing the amide bond of their 3lactam ring Class A 3lactamases are active quot 39 U r 2 1quot L 39 Blacta mase turnover which is not fully understood comprises a first step of enzyme acylation via the nucleophilic at tack by the activesite serine hydroxyl group Ser70 numbering according to E coli TEM1 3lactamase In the deacylation step an oxygen atom of Glu166 depro tonates a water molecule to provide a free hydroxyl group that is positioned to attack the carbonyl of the cleaved Blactam resulting in product release To return to the fully active state L r W transferred to the oxygen of Ser70 19 Hence Glu166 which is located on the loop residues 168 178 ap pears to act as the general base catalyst in deacylation 21 Clavulanate sulbactam and tazobactam are three mechanismbased inhibitors of TEM1 Blactamase They react with the active site serine creating long Iiu A II 39 1 quotIK A u lyticmechanismThemutation Met69Leu inTEM1 gives rise to TEM33 Blactamase an inhibitorresistant form of TEM1 The dissociation constant for the acylation complex is slightly greater in TEM33 than in the native TEM1 22 Therefore the probability of forming the preacylation complex between an inhibitor and the en zyme is reduced resulting in resistance The contribu tion of the mutation M69L to the loss of affinity toward inhibitors is not intuitive because residue 69 does not directly interact with the bound inhibitors but points away from bound ligand Only small changes in van der Waals and electrostatic energies were observed as a result of this mutation 22 The protein backbone and the side chain conformations are positioned almost identically in TEM1 and TEM33 forms the rmsd for the main chain is 057 A and for all protein atoms is 072 A Nonetheless the activities of both enzymes diverge im portantly with respect to inhibitor specificity Because residue 69 is not directly implicated in the substrate binding it was suggested that it could influ ence ligand specificity by provoking active site fluctua tions correlated motions via other residues or by pro voking a general alteration of the protein motion 22 Consequently MD simulations of both TEM1 and TEM 33 were performed Mobashery and coworkers identi fied two regions where the molecular dynamics of TEM 33 differ from thewildtypeTEM1 the loop residues 168 178 and the L1 loop residues 96 108 In both enzymes large fluctuations are observed for the first nanosecond of simulation but after 500 ps a greater conformational change is observed in the loop of TEM33 than TEM1 A more pronounced deviation was also observed in loop 96 108 The different conforma tions result in a small energetic increase 1 9 02 kcal mol for the formation of the preacylation complex in TEM33 relative to TEM1 which appears to be respon sible for the difference in ligand discrimination 22 Since the side chain of residue 69 points away from the active site 3lactamase constitutes a further exam ple where a noncatalytic residue that is not directly in volved in ligand binding affects protein motion with en suing effects on enzyme function 22 In DHFR TIM LADH and Blactamase the internal motions demonstrated to be implicated in enzyme func tion are either of the local or of the largescale type Different amplitudes of motions are observed in the four models the greatest being observed in TM Hydrogen L quot L 39 I 39 qunu to be an important factor in induction of motion and result in accessing many different structural conforma tions which may stabilize the transition state or may result in a conformational sampling that facilitates reaching a conformation that is similar to the transition state Hence the enzymatic organization defined by J enzyme The correlation between function and motion in non catalytic residues in 3lactamase is more subtle than in of interest M69 is the immediate neighbor of the cata lytic Ser70 Figure 3C Furthermore the role of motion in the neighboring residue has been implicated in ligand discrimination binding function rather than in the cata contributor to catalysis Although the correlation between the motions and catalytic activity in 3lactamase and LADH is not yet as well established as in DHFR and TM it is becoming 39 39 a clear that protein motions other than by m 39o a r and dynamics which may then have important effects in catalysis In the above examples specific noncatalytic residues of the Met20 and the BFBG loops for DHFR MBO I EIJOFI39S review Light and oxygenic photosynthesis energy dissipation as a protection mechanism against photo oxidation IIdikti S22 1115 Elisabetta Bergantino amp Giorgio Mario Giacometti University ofPadova Padova Italy Eff t photosynthesis is of fundamenhl impomnce f0 surviwl and tness However complex appamtus respon r plant in oxygenic photosynthesis the e for the conversion of Ii t into n s r r e e r synthetic organisms have therefore evolved seveml protective mechanisms to deal with light energy Rapidly inducible none photochemiml quenching NPQ a shortslerm response by which39 J 39 39 39 39 39 39 dissipated as heat ahd does hot take part ih photochemistry The phehomehoh ihvolves quehchihg of chlorophyll a Chla uoress cehce which is ihduced uhder steadysstate illumihatioh ahd which ih terms of p state trahsis tioh qT ApHsdepehdeht quehchihg qE ahd photoihhibitioh ql The maJOrlty of NPQ is believed to occur through qE ih the P80 ahtehha pigmehts bouhd to the lightsharvestihg proteihs LHCH D g 19 2 r r o m heat This review focuses o recent adwnces in the elucidation of the molecular mechanisms underlying this protective quenching pathwayi er Keywords photosyhthesis momsphotochemlcal quehchihg Psbs zeaxahthih llghtsharvestl g complex EMBOmpmt5a005 86207634 dot 10 103851 Ember 7400460 Introduction Durihg photosyhthesls photohs are abs rbed me as o by ahtehha plgr hts such protel sbou d chlorophylls Chl and Carote olds cehtres RCs Nield etal 2000 H charge separatioh which drives the electroh ow b tW p system H PSll ahd photosystem l PSl through the cytochrome bot complex The het result of this process is the oxidatioh otwater molecules the productioh of molecular oxygeh the reductioh of NADP ahd the geheratioh ota protoh gradieht ApH The ehergy stored as ApH is exploited for ATP syhthesis The ihterplay betweeh ulatioh This short review rocuses oh the molecular mechahisms that alloW sophisticated regulatioh or the amouht or excitatioh ehergytrahsrerred to the RC or PSH The photosyhthetic apparatus is highly dyhamic ahd able to u u iri lo rii light ahd the ioxide A shortsterm respohse is ehsured b h t h aio ri y r duehchihg NPQ a process ih which absorbed light ehergy is D2panmmtofoologyUnwExsity ofPadova vialsc Colomb0335121PadovaJtaly meespondlrgauthox Tel 30 040 32765241 30 040 8276300 Erm ail 11d1cw bio unlpd it Submitted 29 March znnsacoepted19 May 2005 2005 EUROPEAN MOLECULAR BlOLOGV ORGANlZATlON 9 J Nunphotochemical quenching stale trans on Rapid reorgahizatioh of the llghtsharvestl g apparatus termed 39state trahsitioh occurs lh respohse to chahges lh the availability Of C lexlde a d the reductlom state Of chloroplasts phosphorylated LHCH proteihs ahd their association With PSl take place vvollmah 2001 Allen amp Eorsberg 2001 Because the tluorescehce yield of Psll dimihishes durihg state trahsitioh due to ahtehha size reductioh this process also called qT is Np in i i u i u ru regulatory Kl ase STN7 recehtly idehtitied ih Arabldnpsr39s has eeh shoWh to be required for state trahsitioh Bellatiore et al 2005 ahd cytochrome b6f has beeh recoghized as a key parther ih lltihase activatioh ih Chlamydnmnnas Wollmah amp Lemaire 1988 lh previous models statetrahsitioh was cohsidered hecess apparatus is sWitched from the oxygehic type With two systems worlltihg ih series to ah ATPsgemerator type With cyclic electroh tloW arouhd PSl tor comprehehsive reviews see Wollman 200 Are amp Ohad 2003 M i i ii r 1 L a iI The photosyhthetic apparatus must deal With marllted chahges ih light ihtehsit i ectiohal movemehts of whole leaves ahdor chloroplasts Which may allow the plaht to optimize light absorps tioh are relatively slow processes Rather thah regulatihg light absorptioh a fast respohse is obtaihed through the regulatioh ot dissipative desexcitatioh of absorbed photohs lh hormal l7le tiohs most otthe ehergy ih sihgletsexcited chlorophyll hlquot is EMBOIepolts VOL 6 l NO 7 l 2005 m review Stroma Lumen Fig l r m mnl m Pnotoprotectlon ln pnotosyntnetlc ofgzl39llsrns l Szabo eta U Chl chlorophyll POH h l Mnecluster Fer rm used attne RC to drlve electron transport ln tnls case cnl uoresr cence quencnlng ls correlated to cnarge separatlon photochemlr cal quencnlng However wnen tne rate of formatlon of lcnla exceeds tne overall rate of lts energy converslon at tne RC l terr system crosslng leads to an lncreaslng populatlon of 3Cnlaquot trlplet state ln tne antenna molety wnlcn can actlvate molecular oxygen to lts nlgnly reactlve slnglet state 102 molecules as well as tne otner forms of reactlve oxygen specles R08 are known to lnduce oxldatlve damage to plgments pfor telns and llplds ln tne tnylalltold membrane tnereby lmpalrln overall pnotosyntnetlc efflclency pnotolnnlbltlon Tne uoresr cence quencnlng assoclated Wltn tnls pnenomenon wnlcn ls p tlon of N PQ lndlcated as ql By scavenglng tne trlplet exclted state of nl and dlsslpatlng assoclated energy by fast tnermallzatlon carotenolds prevent actlvatlon of oxygen tnus protectlng cmofor pnylleproteln complexes from phOtOrOxldath Vlltnout tne pfor tne entlre pnotosystems would occur Ll en tne acceptor qulnones are reduced at steady state cnarge separatlon at tne PSH RC ls followed by recomblnatlon wrtn a nlgn o nearby rcafoten close enougn Eerrelra eta 2004 For tnls reason regulatlon oftne 530 EMBOIepoIB VOL6 l Noumea Tne portlon of NPQ named qE nas a central role ln tnls con text and ls a ApHrdependent rapldly lnduclble component qE ls also called feedbacllt derexcltatlo because tnermal dlsslpar tlon of antenna lcnla ls stlrnulated by ApH wnlcn bullds up across tne tnylalltold membrane durlng pnotosyntnetlc electron nd ls tnerefore brougnt about by tne same excltatlon tnat qE contrlbutes to dlsslpatlon qE nas been snown to be lmportant for plant fltness ln varlable llgnt condltlons ratner tnan for tne lnductlon of to erance to hlghrlme slty llgnt ltself Kulnelm eta 2002 and mayeaslly be measured as tne qulcxly reverslble portlon of maxlmal Psll fluorescence quencnlng wnlcn ls not assoclated Wltn cnarge separatlon DemmlgrAdams ampAdams 2000 Durlng tne past decade our understandlng of qE nas been greatly advanced partlcularly bytne selectlon of NPQndeflCle t Arabldnpsr39s mutants by vldeo lmaglng of cnl fluorescence quencnlngleogl eta1998the successful appllcatlon offesr onance Raman spectroscopy Robert eta 2004 wnlcn ylelded structural lnformatlon about speclflc plgment molecules ln thyr lalltold membrane complexes in vl39vn and in vl39tru and tne ablllty to detect nonr uofescent optlcally darllt exclted states of plgn ments by femtosecond translent absorptlon lltlnetlcs ln lntact membranes Ma et al 2003 Hon eta 2005 The fesomtlon of tne crystal structure oftne lsolated antenna LHcll complex Llu tandfuss etal 2005 as well as in vl39trn feconstltur tlon of antenna protelns Sandona etal 1998 nas also Slg lfln cantly contrlbuted to tne ldentlflcatlon of tne lltey components responslble for qE Tney are plgments oftne xantnopnyll cycle a 3 U o m 2005 EUROPEAN MOLECULAR BlOLOGV ORGANlZATlON Photoprotecuon In photosynthetic organisms I Szab et a particularly zeaxanthin Zea the PSII S subunit PsbS protein and components of the LHCII lightrharvesting apparatus Fig 1 Despite the attention devoted to the qE component of NPQ the actual biophysical and biochemical mechanisms for energy disr sipation have only recently been proposed The hot topics39 in this respect are How does zeaxanthin contribute to qE and where is its site of action How is PsbS involved Are there parallel and independent mechanisms for qE Zeaxanthin andApHrdependent quenching The importance of the xanthophyll cycle Yamamoto 1979 in high light conditions became clear more than a decade ago DemmigrAdams ampAdams 1996 The most abundant xanthophyll pigment in the thylakoid membrane is lutein but rcarotenerderived violaxanthin Vio is also present Vio is synthesized from Zea via antheraxanthin in low light conditions by the stromal activity of zeaxanthin epoxir dase Under intense light lumenal pH decreases and ata critical threshold Vio derepoxidase converts Vio back to Zea Fig 2 The latter is absent in the thylakoid membrane in normal light condir tions but it is required for qE to occur as shown by the fact that npqi mutants lacking Vio derepoxidase have greatly reduced NPQ Niyogi eta 1998 A longrstanding debate has arisen over what feature of Zea might render this particular pigment essential for qE All carotenoids are able to dissipate excitation energy by rapid inter nal conversion but it has been calculated that only those with ten or more conjugated carbonrcarbon double bonds have an excited singlet state S1 at an energy level low enough to accept energy from Chla Although the S1 state of carotenoids is dipolerforbidden for direct onerphoton excitation it can be detected after rapid internal conversion of the S2 state However direct determination ofthe in vitro energy levels of the S1 state of Zea 11 double bonds and Vio 9 double bonds led to the dis covery that both pigments have an S1 state that enables direct quenching of Chl through singletisinglet energy transfer Polivka et al 1999 The S1 state of Zea has a particularly short lifetime 10 ps which allows for rapid thermal dissipation of excitation energy Accordingly an 11 ps lifetime has been found for Zea S1 in reconstituted LHCII Polivka et al 2002 and also as measured by femtosecond transient absorption TA kinetics in intact thylakoids under maximal qE Ma et al 2003 In the lat ter case excitation of the S1 state of Zea was observed after selective excitation of the first excited singlet state of Chla Qy nd Another distinctive feature of Zea with respect to other xanr thophylls is its presumed low ionization potential Dreuw et al 2003 Holt et al 2004 The kinetics of the process have been investigated by femtosecond TA measurements in the range 90071080 nm characteristic of carotenoid cation radical absorption Subtraction of quenched from unquenched TA data reveals a rise in the signal with a time constant of 11 ps folr lowed by a decay with a time constant of 150 ps Holt et al 2005 The same experiments performed on thylakoids from varir ous Arabidopsis mutants with different pigment composition allowed the authors to conclude that Zea is necessary in causing this phenomenon In the model proposed by Holt et al 2005 the 11 ps component was assigned to energy transfer from excited bulk Chl to a ChlarZea heterodimer This step was followed by a nonrresolved 0171 ps component corresponding to an 2005 EUROPEAN MOLECULAR BlOLOGY ORGANlZATlON review Lumen 0H Stroma O O HO Violamnlhln ZE VDE OH pH 7o W PH 50 HO Amheraxan un ZE VDE OH W HO leaxamhin FigZ T n VDE L39 A 1 7 zeaxanthin epoxidase electron transfer with the formation of a chargerseparated Chl lZea pair Charge recombination accounts for the 150 ps component of the observed kinetics This mechanism is proposed to be responsible for excess energy dissipation during qE These findings seem to resolve the longstanding issue of whether the role of Zea is that ofa direct quencher or not and indicate direct quenching The effective site of these events and the contribution of other factors known to be critical for qE such as that of PsbS were notdiscussed in this framework One interesting observation concerning the optical properties of the thylakoid membrane during qE is the presence ofa 535 nm band in the qE difference spectrum AA535 This band has been ascribed to a red shift to 525 nm of the absorption maximum of the Zea spectrum Ruban et al 2002 which is 503 nm in viva Apparently two redrshifted Zea molecules about 15 of the Zea pool can account for the observed band It has been proposed that a severe change in the environment of Zeaimost probably due to its binding to highly hydrophobic antenna proteinsi specifically alters the configuration of this carotenoid resulting in a red shift of its absorption spectrum Ruban et al 2002 In this respect it is important to note that the in vitro association of Zea with PsbS protein another key player of qE results in a red shift of Zea whereas Vio does not bind to the protein Aspinaler39Dea et al 2002 The activation of Zea by red shift of its excited S2 state presumably re ects a shift towards a lower energy level of the S1 state as well Whether this adjustment is a consequence of the formation of the Cherea heterodimer is still unclear but direct excitation of Zea as well as heterodimer formation are PsbSr dependent Ma et al 2003 Holt et al 2005 In addition to its role in direct quenching Zea has been pro posed to have a role in the regulation of the organization of LHClerSII complexes In the Arabidopsis double mutant IutZnqu in which Zea is the only xanthophyll present it has been shown to function in vivo as a lightrharvesting pigment to decrease LHCII size and stability and to induce LHCII monomerr ization which suggests a role for Zea in longrterm photoadaptation Havaux et al 2004 EMBO report vow NO 7 2005 631 review PSII PSII H Fig3I 39 F DsbSe rmenr hino pptexthr A 4 39u p minor antenna proteins For the sake of clarity only one chlorophyll Chl and one zeaXanthin Zea red zigzag per protein are indicatedYellow circles quenching sites Chl antenna Chl interacting with Zea Lightrharvesting complex II and ApHrdependent quenching An alternative to direct quenching by Zea involves carotenoidr mediated changes in the organization of antenna complexes resulting in Cherhl quenching A role in qE has been proposed for both minor LHCII proteins Lhcb4 5 and 6 also named CP29 CP26 and CP24 respectively and the peripheral LHCII trimers Lhcb1 2 and 3 Horton amp Ruban 1992 2005 Wentworth et al 2004 CP29 and CP26 bind dicyclohexylcarbodiimide DCCD a qE inhibitor that interacts with protonractive residues in a hydrophobic environment and all the LHCII proteins bind Zea in a pHrdependent manner and to varying extents Moreover aggregationrinduced in vitro quenching kinetics in LHCII CP26 and CP29 resemble qE kinetics in intact chloroplasts The role of LHCII proteins in qE has been questioned as repression of indie vidual LHCII enes does not induce phenotypic qE changes Andersson et al 2001 2003 However in field conditions each LHC protein seems to be important for plant fitness Ganeteg et al 2004 and recent data indicate that in the Chlrbinding pro tein CP26 a Zearinduced conformational change may be responsible for at least part oqu Dall39Osto et al 2005 In a different structurerbased model Liu et al 2004 aggrer gation of LHCII trimers mediated by digalactosyl diacylglycerol together with a protonrinduced conformational change of LHCII position the linker chlorophylls Chla 614 and Chlb 605 accord ing to the chlorophyll nomenclature used by the authors at the trimerrtrimer interface in the best orientation for promoting energy transfer to the closely located xanthophyllrcycle carotenoids Thus the actual quenching site proposed by these authors is the Chla 613614 pair together with xanthophyllrcycle carotenoids On the basis of a threerdimensional structure of pea LHCII that was recently determined by Kuhlbrandt and collaborators at 25 A resolution Standfuss et al 2005 a mechanism for qE at the level of the main antenna has been proposed in partial agreementwith the hypothesis of Liu et al 2004 The quenching site is similarly located in each LHCII monomer where the two most redrshifted Chla are in close contactwith a bound Vio On acidification ofthe lumen and production oneaVio is substituted by Zea with stronger binding due to its higher hydrophobicity The excitation energy collected in the monomer is funnelled to the neighbouring redrshifted Chlas and is finally dissipated after 632 EMBO repom VOL 6 I NO 7 I 2005 Photoprotectlon In photosynthetic organisms I Szab et a transfer to Zea By contrast to the model proposed by Liu et al 2004 this mechanism does not imply any conformational rearrangement of the LHCII moiety and is supported by the fact that the two structures were obtained from crystals grown at difr ferent pH 7775 in the structure from spinach resolved by the Chinese group and 5755 in that from pea by the German group The former condition is one in which no qE quenching is expected whereas the latter probably yields a quenched state Despite this the two structures overlap perfect y PsbS and ApHrdependent quenching PsbS has been identified as an essential participant in qE by isolation of npq4 Arabidopsis mutants which are defective in qE and either do notexpress PsbS proteins or express mutated versions Li et al 2000 npq4 mutants also lack lightrinduced AA535 but show no alteration in other PSII and LHCII proteins or in the xanthophyll cycle Li et al 2000 Peterson amp Havir 2000 Several properties of PsbS seem to be important for qE First PsbS contains eight conserved acidic aminoracid residues on the lumenal side two of which E122 and E226 have been shown to be essential for qE AA535 Li et al 2002 2004 and for binding ofthe qE inhibitor DCCD Li et al 2004 The two acidic residues are thought to enable PsbS to sense lumenal acidification and trigger qE A similar role had also been hypothesized for other antenna proteins Horton amp Ruban 1992 Second the ability of PsbS to bind Zea has been shown in vitro Aspinaler39Dea et al 2002 The fact thatAA535 which arises from activation of Zea molecules and the direct excitation of Zea and formation of the Cherea heterodimer see above are missing in the absence of PsbS strongly suggest that Zea can bind to PsbS in vivo as well Moreover neither phenomenon can be observed if PsbS is mutated at the E122 and E226 residues Ma et al 2003 Li et al 2004 which reinforces the viewpoint that protonation of PsbS is the first step in the quenching process A remaining question is whether protonation of PsbS is a prereqr uisite for Zea binding to the protein or whether protonation of the PsbSrZea complex induces a conformational rearrangement and allows Zea to quench Chl excitation In a model proposed by Li et al 2004 low lumenal pH is required for exposure of the two critical glutamate residues that on protonation are directly involved in generating two xanthophyll binding sites This pror posal is in good agreement with the light and pHrdependent reversible dimerization andor monomerization of PsbS observed in viva Bergantino et al 2003 In light of the above data and considerations binding of Zea to PsbS in vivo is highly probable However for the PsbSrbound Zea to be one of the Chi quenching centres there must be a Chl molecule close enough to form the heterodimer observed by Holt et al 2005 see above This Chl in turn must be at a distance suitable for energy transfer from other chlorophylls connected to the bulk antenna This suggests the presence of at least one Chl on PsbS Given the contrasting results obtained by various groups Eunk et al 1995 Dominici et al 2002 Chl binding to PsbS cannot be ruled out but it does need further confirmation Last a PsbSrdependent Zearindependent qE has been shown to occur in the PSII RC as a transient process Einazzi et al 2004 Although the authors do not propose a mechanism for this quenching at the RC they describe an interesting theory based on previous publications in which the various forms of NPQ qT 2005 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION Photoprotection In photosynthetic organisms I Szab eta q antenna qE and RC qE take place depending on the balance between the dissipation ability of the carbon fixation apparatus and lightflux Concluding remarks With reference to the three hot topics39 mentioned above some conclusions may be drawn Zea is certainly deeply involved in determining qE and its ability to quench chlorophyll excitation directly has been clearly demonstrated There is good evidence that this occurs in at least two different sites in the PSllrLHCII supercomplex in the hydrophobic pocket of LHCII monomers and in connection with the involvement of PsbS in producing qE On the basis of the literature various mechanisms can be envisaged for qE A Zeardependent qE may occur together with a conformational change in antenna proteins either on aggregar tion of LHCII trimers with each other Liu et al 2004 or by aggrer gation of minor antennae CP29 and CP26 with LHCII Horton amp Ruban 2005 A variant of this mechanism introduces the formation of a quenching site at the level of each LHCII monomer as the result of binding of Zea which displaces Vio with no need for conforr mational rearrangement of the LHCII trimers Standfuss et al 2005 In this view conformational rearrangements may still be important in relation to the Zeardependent contribution to qE of the minor antennae CP29 and CP26 which lie in a strategic locar tion inside the PSIIPLHCII supercomplex at the interface between the peripheral LHCII and the internal antennae CP43 and Other qE mechanisms depend on PsbS Some experimental evidence also positions this protein at the interface between LHCII and the PSII core The quenching mechanism associated with PsbS is also dependent on Zea but the detailed mechanism is still hypothetical In one model PsbS has a critical role in bringing activated Zea into close proximity with a Chl thus pror moting the formation of a Cherea heterodimer that is responsir ble for the quenching process Fig 3 Chi may be bound to PsbS itself Funk et al 1995 or more probably may be located on a neighbouring minor or major antenna protein PsbS has been proposed to bind Zea on protonation and consequent conformar tional change Aspinall O39Dea et al 2002 Li et al 2004 which may consist of protonrinduced monomerization followed by its association with an LHCII component Bergantino et al 2003 These mechanisms are all well supported by experimental data and their coexistence is highly probable some qE persists in PsbSrless Arabidopsis Li et al 2000 and PsbSrmediated quenching seems to be essential only in rapidly fluctuating light conditions Kulheim et al 2002 Instead inhibition of single antenna protein expression does not significantly affect feedback derexcitation in field conditions but it does affect overall plant fitness Ganeteg et al 2004 In the emerging scenario the interplay of these proposed mechanisms ensures the best photo protective performance in each different and variable light condition ACKNOWLEDGEMENTS The authors thankR Bassi and D Carbonera for f quot grateful to the EMBO Young Investigator Programme for financial support Thanks are due to G Walton and C Friso for revision of the text and figures respectively 2005 EUROPEAN MOLECULAR BlOLOGY ORGANlZATlON review REFERENCES Allen JF Forsberg J 2001 Molecular recognition In thylakoid structure and function Trend PlantSci 6 3177326 Andersson JWalters RG Horton P Jansson S 2001Antisense inhibition of I h a u P I the mechanism of protective energy dissipation Plant Cell 1 3 119371204 Andersson JWentworth M Walters RG Howard CA RubanAV Horton P Jansson S 2003Absence of Lhcb1 and Lhcb2 proteins ofthe Jlgl ll39 harvesting complex of photosystem Ilieffects on photosynthesis grana stacking and fitness Plant35 3507361 Aro EM Ohad 2003 Redox regulation of thylakoid protein phosphonylation Antioxid Redox SignalS 55767 Aspinaler39Dea MWentworth M PascalA Robert B Ruban A Horton P N 39 fthe am ated With energy dissipation in plants Proc NatlAcad Sci USA 99 16331 71 6335 Bellafiore S Barneche F Peltier G Rochaix JD 2005 State transitions and light I I T Nature 433 8927895 Bergantino E Segalla A Brunetta A Teardo E Rigoni F Giacom etti GM Szabb 2003 Light and pHrdependent structural changes in the PsbS subunit of photo stem Proc NatlAcad Sci USA 100 15265715270 conformational change in the antenna protein CP26 Plant Cell 17121771232 Demm igrAdams B Adams WW 3rd 1992 Photoprotection and other responses of plants to high light stress Annu Rev Plant PhyxiolPlant Mol Biol 43 5997626 DemmigrAdams B Adams AAN 3rd 1 996 The role of xanthophyll cycle carotenoids in the protection of photosynthesis Trend Plant Sci 1 2 6 Demm igrAdams B Adams WW 3rd 2000 Harvesting sunlight safely Nature 403 37 7374 Dom inici P Caffarri S Armenante F Ceoldo S Crimi M Bassi R2002 Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant BiolChem 277 22750722758 DreuwA Fleming GR Heaerordon M 2003 Chargertransfer state as a possible signature of a zeaxanthinrchlorophyll dimer in the non photochemical quenching process in green plantsPhyx Chem B107 650076503 Ferreira KN Iverson TM Maghlaoui K BarberJ Iwata S 2004 Architecture of the photosynthetic oxygenrevolving center Science 303 1831 71 838 Finazzi G Johnson GN Dall Osto L Joliot P Wollmann FA Bassi R2004 A 39ll lde quot 39 localized in the photosystem II core complex Proc NatlAcad Sci USA 101 12375712380 Funk C Schroeder WP Napiwotczki A TJus SE Renger G Andersson B 1995 The PSIIVS protein of higher plants a new type of pigmentrbinding protein Biochemixtry 34 11133711141 Ganeteg U Kulheim C Andersson J Jansson S 2004 Is each lightrharvesting complex protein important for plant fitness Plant Phyxiol 1 34 5027509 Havaux M Dall39Osto L Cuine S Giuliano G Bassi R2004The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopxix thaliana Biol Chem 279 13878713888 Holt NE Fleming GR Niyogi KK 2004 Toward an understanding ofthe mechanism ofnonphotochemical quenching in green plants Biochemixtry 43 8281 78289 Holt NE Zigmantas D Valkunas L LI XP Niyogi KK Fleming GR2005 4 r a rvesting Science 307 4337436 Horton P Ruban AV 1992 Regulation of photosystem II Photoxynth Re 34 37 8 r3 5 Horton P Ruban A 2005 Molecular design ofthe photosystem II Jlgl ll39 L I I Exp Bot56 3657373 Kulheim C rmanl 39 I light plant fitness in the field Science 297 91 793 EMBO reports VOL 6i NO 7 i 2005 633


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