Journal of Cell Science ta. 06.W aepeiul sltda nmlmdlo TPI of model animal Gnerer an 2006; isolated al., previously have et We (Celotto 2006). al., neurological patients et the human recapitulate in to seen shown phenotypes been have mutants which ucincuilt eairadlongevity Roland P. and Bartholomew behavior to crucial non-catalytic function isomerase triosephosphate a of Evidence Article Research rae l,19) P eiinyhsavr orgenotype- poor systems. are very animal pathogenesis in a elucidate especially has to limited, studies extremely deficiency and TPI correlation Holla to 1997). phenotype 1986; reported al., al., catalysis et et been in the Arya (Daar have changes stability to affect all enzyme owing and/or activity and can TPI sequence reduced mutations dramatically coding deficiency or TPI promoter as al., Pathogenic et such Orosz 2000; 2006). (Schneider, symptoms death premature the by and and degeneration, dysfunction characterized neurological in cardiomyopathy, anemia, clinically hemolytic mutations is missense deficiency from resulting and aerobic both extra of metabolism. bioenergetics an of the anaerobic in of enhancement gain important This production is glucose. net glycolysis of the of molecule the in per as pyruvate steps for of well remaining molecule responsible as the ATP is by glycolysis-derived TPI utilized be Therefore, converting can glycolysis. aldolase; GAP the enzyme glycolysis, only from the produced by however, 1,6-bisphosphate are of 3- fructose GAP of and glyceraldehyde step catabolism DHAP into Both (GAP). non-linear (DHAP) phosphate phosphate a that dihydroxyacetone enzyme in homodimeric a functions is (TPI) isomerase Triosephosphate Introduction this in dysfunction neurological the to words: significantly Key contributes which of loss the TPI, model. for function animal non-metabolic a demonstrate results was activity catalytic enzyme that the whether demonstrate question for to of us system led pathogenesis This engineering 3- proteasome. the the glyceraldehyde by these The to degraded Of symptoms. into prematurely crucial neurological enzymopathies. (DHAP) protein of glycolytic a severity as phosphate encodes the known and dihydroxyacetone in collectively deficiency unique diseases converts is metabolic that deficiency to TPI leads enzyme enzymopathies, dysfunction glycolytic enzyme Glycolytic a (GAP). is phosphate (TPI) isomerase Triosephosphate Summary 10.1242/jcs.124586 doi: 3151–3158 126, ß Science Cell of Journal 2013 April 4 Accepted ( correspondence for *Author 4 3 2 1 nrwP VanDemark P. Andrew oeua hraooyGaut riigPorm nvriyo itbrhSho fMdcn,Ptsug,P 56,USA USA 15261, 15260, PA PA Pittsburgh, USA Pittsburgh, Medicine, 15261, Pittsburgh, of PA of USA School Pittsburgh, University 15261, Pittsburgh Medicine, Sciences, PA of of Biological Pittsburgh, University School of Medicine, Program, Pittsburgh Department of Training of School Graduate University Pittsburgh Pharmacology Diseases, of Molecular Neurodegenerative University for Biology, Institute Chemical Pittsburgh and Pharmacology of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. Drosophila disease human loss-of-function recessive a is deficiency TPI P,Tispopaeisomerase, Triosephosphate TPI, r h nymdlsse dniidt aein date to identified system model only the are P sugarkill TPI [email protected] 4 lcaM Celotto M. Alicia , TPI 1,2,3 a egntclycmlmne by complemented genetically be can P sugarkill TPI ) ibryA Stuchul A. Kimberly , ou htealsteefcetgnrto fnovel of generation efficient the enables that locus rspiamelanogaster Drosophila erlgclpeoye.T td P deficiency TPI study To phenotypes. neurological TPI ´ ta. 1993; al., et n 1,2 ee TPI gene. n ihe .Palladino J. Michael and 1,2 aataB Larsen B. Samantha , lclss ogvt,Lcmtrfunction Locomotor Longevity, Glycolysis, , eiinykonas known deficiency usiuini TPI in substitution eems motn othe the to important of most influence were the of result cooperation. the dimeric a that on predict is we mutation sites, efficiency catalytic catalytic TPI the reduced of one to proximity the M0) h fetdmtinn speetna h dimer monomer-dimer the in shift near a in present result to is populations seem not methionine does yet affected interface a conditional substitution The threonine from to (M80T). resulting and methionine a 2006) causing al., mutation neurodegeneration, et missense (Celotto lifespan, abnormalities behavioral shortened by eatnae yoeepesn uatTPI protein mutant TPI overexpressing Interestingly, can by 2010). total mutation attenuated loss-of-function Palladino, this proteasomal be reduced that and shown Hrizo abnormal previously in have 2008; we induce resulting al., et to TPI (Seigle shown of been degradation has mutation nyeo t aayi ciiywsms motn othe to important most was activity the of catalytic presence of the its pathogenesis whether question or to us enzyme led result This 2006). aayial ncievrato h rti.Ls1o P sa is TPI of Lys11 protein. with the phenotypes catalytic of disease variant of the inactive rescue independent catalytically to able a pathogenesis be would to we activity, crucial was efficient highly enzyme using locus gene the attB modify to used the be an can of establishes process (GE) This locus. engineering genomic developed TPI oadeswehrtepeec fteezm riscatalysis its or enzyme the of presence the whether address To noigactltclyiatv nye utemr,our Furthermore, enzyme. inactive catalytically a encoding rngnss ehpteie hti h rsneo the of presence the if that hypothesized We transgenesis. nvivo in 1,2, sugarkill rspiasugarkill Drosophila sgk * TPI 1,2 Sil ta. 08.Hwvr the However, 2008). al., et (Seigle eue t smrs ciiyad ae on based and, activity isomerase its reduces hitpe .Amrich G. Christopher , TPI eei ains sn hssse we system this Using variants. genetic eew eosrt htteM80T the that demonstrate we Here . uakl (sgk) sugarkill TPI nvivo in sgk ies phenotypes disease attP uatcoeymdl TPI models closely mutant edvlpdagenomic a developed we . D TPI TPI sgk one iewhich line founder sgk scharacterized is rspiaTPI Drosophila Cltoe al., et (Celotto 4 , nvivo in 3151 TPI attP/ ,we sgk Journal of Cell Science TPI eiusaecuilfrezmtcfnto nTI(abi tal., et These (Lambeir TPI 1B,C). in (Fig. function enzyme enzymatic the for crucial of are H94 site residues and active using K11 the both within hydrogen- network positions properly and coordinated ultimately hydrophobic that a both interactions is bonding mediate interactions to residues these respectively. conserved R97, of and These H94 sum (also with are Q76 interact 1B). The which and and (Fig. loop), T74 third loop, residues subunit the between on third bonding neighboring the hydrogen by the position aided help of dimerization and interactions N14 M81 hydrophobic the between and contacts important within M13 including TPI loop interactions This of 1A). interface located Fig. numerous 86; is to 63 (M81) (residues coordinates TPI methionine of loop homologous third the the within and (PGH), acid phosphoglycolohydroxamic analog transition-state the of presence with conservation TPI Drosophila ogvt n eairo the of behavior and longevity k ahgnsso P eiiny oivsiaeti hypothesis the this recombinant to investigate generated crucial To we was deficiency. further catalysis TPI TPI behavioral of reduced led data and pathogenesis that These hypothesize 2008). survival to al., et mutant us (Seigle overexpressed rescue if to phenotypes function sufficient ta. 08.Gntcdt ugse htTPI that suggested data Genetic (Seigle levels 2008). TPI al., reduced by et characterized mutation function al .Kntcprmtr of parameters and Kinetic 1. Table from – TPI 1994) of structure crystal the analyzed S1). Fig. Michaelis– material typical (supplementary displayed a kinetics enzymes Menten in Both resulted efficiency. enzyme ultimately and This affinity substrate enzyme. in decrease activity. 33% isomerase a , has in protein reduction dM80T substantial The a exhibits (dM80T) that established studies Previous activity enzyme TPI Recombinant have We 2010). al., et for Wierenga required 1994; abolishes be the al., completely generated Met et to to (Lodi known substitution catalysis and residue binding catalytic substrate conserved fully k 3152 K rspiasugarkill Drosophila eiiny utemr,ctltclyiatv P complements TPI TPI inactive catalytically Furthermore, deficiency. o ahezm Tbe1.Teedt eosrt htTPI that demonstrate data These 1). (Table enzyme each for edmntaethat demonstrate we ugs ucino P needn fisctltcactivity catalytic Results longevity. its and of behavior independent to crucial TPI is of which data function these a Collectively, stress. suggest metabolic associated the reducing cat cat m 1fl euto nctltcatvt oprdt WT to compared activity catalytic in reduction 11-fold dWT, ausfor Values oass h oeo h 8 oiinwti P ucin we function, TPI within position M80 the of role the assess To / GP m)2.8 (mM) (GAP) K GP (s (GAP) sgk sgk m sugarkill GP (s (GAP) d8T n xmndtekntc fioeaeactivity isomerase of kinetics the examined and (dM80T) hntpswtotehnigissaiiy aayi or catalysis stability, its enhancing without phenotypes Drosophila Drosophila ora fCl cec 2 (14) 126 Science Cell of Journal 2 k 1 a and cat 1454 ) attP 2 d8T roehsht smrs enzymes isomerase triosephosphate (dM80T) 1 K11M M idtp roehsht smrs;dM80T, isomerase; triosephosphate wild-type K roehsht isomerase. triosephosphate 2 .gallus G. D m 1 5.2 ) noigactltclyiatv P.Here TPI. inactive catalytically a encoding , P hw 7 dniyad8%functional 80% and identity 67% shows TPI TPI r means are TPI one ieadhv sdG ocreate to GE used have and line founder hssrcuewsdtrie nthe in determined was structure This . K11M 6 s.e.m. TPI eeial opeet the complements genetically Drosophila TPI Drosophila W dM80T dWT 6 6 6 sgk sgk 10 . 3.7 0.4 6. 129.6 163.6 5 , nmlmdlo TPI of model animal .gallus G. sarcsieloss-of- recessive a is 5fl euto in reduction 15-fold idtp (dWT) wild-type T(W)and (dWT) WT Zage al., et (Zhang sgk 3.5 retained 6 6 6 10 0.5 1.5 4 sgk dniidTInl allele, were null lines TPI identified HR methods. positive molecular and three genetic using positives, verified and putative identified the Among 97 oldee l,20)adetbihacneto between connection a establish and in 2001) M80 al., et Norledge 1987; hoooebetween chromosome niaigancsiyfrTIctlssdrn development. during catalysis TPI for necessity a indicating TPI ihncntuteeet.Tecnimdfudrln was line and founder arms confirmed remove homology The to the elements. reduced outside homology PCR construct right directed and within primers included left of using targeting validation arms proper confirm Molecular to analyses lethality. homozygous N n lndit the into cloned and DNA Reet eeietfe fe screening after identified outlined were previously events as positive putative HR hundred induced four Approximately 2009). was al., et (Huang HR chromosome. second aayi xlnto forpeoyi teuto i TPI via attenuation phenotypic our of explanation catalytic a TPI TPI tlmrc.Hmlg rsdrcigtedlto fteentire the of deletion the TPI directing arms Homology (telomeric). functional S2A). a Fig. material with (supplementary endogenous through recombination generated the was homologous line of founder GE The replacement 2008; 2009). al., al., et et (Huang efficient Huang more have considerably technology process GE the in advances made recent but 2003) Golic, and Gong Drosophila the for of allele approach inactive catalytically a create To by line recombination knockout homologous founder the of Generation activity. isomerase changing this without of assessment rigorous hypothesis a However, overexpression. transgenic ots hte P smrs ciiywscuilt disease to crucial was activity isomerase TPI pathogenesis, whether test To longevity and behavior Animal for homozygous Animals stress material (supplementary S3). substantially longevity are Fig. reduced substitution exhibit and M80T sensitive the bearing animals ( P rdcdb n leeof allele one by produced TPI eitdrdcint remove to reduction CRE- by followed mediated integration site-directed phiC31 standard using TPI lcmn fteG lmnsadteitgiyo h homology the of integrity the and elements arms. GE the of placement entire designated td,contrasting study, i.SE n h ou a eune.Ipraty GE Importantly, sequenced. was locus PCR the using and verified S2E) Fig. molecularly the were of animals analyses These S2). Fig. lmn rngnsswsue oobtain to used was transgenesis element attR eei aiainicue alr ocmlmn previously a complement to failure included validation Genetic Ewspromdt create to performed was GE salsigta TPI that Establishing sgk JS10 sgk M80T ou eePRapiidfo the from amplified PCR were locus nml r ooyosval n eaenral while normally behave and viable homozygous are animals ) /TPI /TPI attP Drosophila sanl leeoigt . bdlto,therefore deletion, kb 1.6 a to owing allele null a is ( attR JS10 K11M nvivo in a rvosybe eeoe Rn n oi,2000; Golic, and (Rong developed been previously had attP D TPI ), TPI nml ersn prxmtl h mutof amount the approximately represent animals TPI attR D nml eecletdadae t25 at aged and collected were animals TPI einwssqecdt ofr h correct the confirm to sequenced was region TPI sgk TPI ol eur upeetto fTIlevels TPI of supplementation require would K11M P n eue aayi o TPI for catalysis reduced and TPI /TPI osrc lmn splmnaymaterial (supplementary element construct w ou.Hmlgu eobnto H)in (HR) recombination Homologous locus. sgk sgk mc+ splmnaymtra i.SB.The S2B). Fig. material (supplementary ( pGX-attP /TPI tl eandioeaeatvt supported activity isomerase retained still attR CG31029 + , TPI sn R eobns n is and recombinase CRE a using TPI ), JS10 n TPI and JS10 TPI sgk TPI w with agtn etr rdtoa P- Traditional vector. targeting mc+ /TPI sgk TPI Cltoe l,20) and 2006), al., et (Celotto + TPI cnrmrc and (centromeric) TPI o h upsso this of purposes the For . ( pGX-attP sgk splmnaymaterial (supplementary K11M TPI attR w TPI K11M slctdo h third the on located is 1118 ou sn ends-out using locus , sgk ), -CFP TPI edvlpdaGE a developed we /TPI idtp genomic type wild , TPI ( attR sgk 000animals. 60,000 neto nthe on insertion K11M + ( /TPI attP attR -CFP r lethal, are ) sgk JS10 animals ) . provides element ( and , attR AdoR TPI ˚ sgk C. ), + Journal of Cell Science n yca imns h oiino the of position The diamonds. cyan by melanogaster estvt tbt a n a 0 ( 20. day and 3 day both at sensitivity niae ssik.VndrWasitrcin ewe 8 n eiusM3adN4aeidctda lc otdlns yrgnbnsbtenresid ( between the methionine. bonds on initiator based Hydrogen numbered the lines. are lacking Residues dotted lines. standard black dotted numbering green as as TPI indicated melanogaster shown the are are D. core using N14 catalytic are the and to we connecting M13 clarity and residues loop for and third the M81 of between end interactions distal the Waals on der Van sticks. as indicated eoey(i.2) ncontrast, In 2A). (Fig. recovery stress, and inactive catalytically The 2. Fig. While assay. each recovery in temperature- measured to were Time and paralysis 20. to and time 3 mechanical- and days on for defects locomotor examined dependent and population individuals animal each as active catalytically comparison, one contains informative most the gr (PGH, acid phosphoglycolohydroxamic analog state transition the of position ( The pocket. orange. catalytic in the highlighted loop within third pocket. the catalytic with the blue, to colored interface dimerization cartoon the connecting network interaction an forms from loop dimer third the of Positioning 1. Fig. TPI osgso tessniiiy(i.2) rvosy ehave we Previously, 2A). (Fig. sensitivity stress of signs no . aeadfml nml eehue nvaso 10–20 of vials in housed were animals female and Male 5prgntp o l eairlasy,and assays, behavioral all for genotype per 15 TPI + hw osg fprlsso ezrsuo mechanical upon seizures or paralysis of sign no shows sgk TPI /TPI alsgallus Gallus sgk eiusta eit P ieiainaeidctdb rycrls eiuswti h yrgnbnigntokcuilfrctlssaeindi are catalysis for crucial network hydrogen-bonding the within Residues circles. grey by indicated are dimerization TPI mediate that Residues . K11M /TPI rti lgmn a efre sn h lsaWmto,with method, ClustalW the using performed was alignment Protein . nml eentprlzd ( paralyzed. not were animals sgk and PoenDtbs D TH.MnmrAo h ie srnee sasraeadclrdwie hra ooe ssona a as shown is B monomer whereas white, colored and surface a as rendered is dimer the of A Monomer 1TPH). ID: Database (Protein B oa niomn ftetidlo.Clrda eoe ihrsde aigipratcnrbtost h ieiaininterface dimerization the to contributions important making residues with before, as Colored loop. third the of environment Local ) TPI sgk TPI B /TPI ) sugarkill TPI TPI K11M TPI JS10 sgk K11M allele. ece eairladlneiypeoye in phenotypes longevity and behavioral rescues /TPI uat(8,rd n t oiin ihntetidlo r niae ya rnebox. orange an by indicated are loop third the within positions its and red) (M81, mutant xii la ea in delay clear a exhibit xrsincomplements expression C n K11M . ) TPI 5 e eoyefrallfsas ro asindicate bars Error lifespans. all for genotype per 150 K11M nml display animals opeet h ogvt eetof defect longevity the complements TPI sgk TPI / sgk hra tessniiiy gi tbt a n a 20. day and 3 day both at again sensitivity, stress thermal w iepit,dy3addy2,t eelwhether reveal to 20, day and at behaviors 3 these day measured We points, 2008). time al., two et Seigle 2006; al., et ettepsiiiyo ahgncdvrec Fg B.Here, 2B). (Fig. divergence pathogenic of possibility the test 2A). (Fig. phenotype disease the of progression the the complements fully hw that shown ne.Tectltclyiatv nyewssonto time both shown at was sensitivity enzyme the stress that indicating inactive mechanical points the catalytically complement The onset. .sapiens H. C hra tessniiiyo h nml a loeautdto evaluated also was animals the of sensitivity stress Thermal naineto P rti eune from sequences protein TPI of alignment An ) and TPI TPI sgk .gallus G. TPI sgk . ( sgk A hntpsaeporsiei aue(Celotto nature in progressive are phenotypes .** ) TPI 6 o-aayi P ucin3153 function TPI Non-catalytic hrn 35 n 73 dniywith identity 67.3% and 63.5% sharing P TPI s.e.m. , K11M .1ad*** and 0.01 TPI sgk complements K11M hntpso ipydly their delays simply or phenotypes leede o ipydelay simply not does allele P , ( A .0 eaieto relative 0.001 TPI h rsa tutr fTPI of structure crystal The ) alsgallus Gallus .sapiens H. sgk { ehnclstress mechanical indicates e)i indicated is een) , .gallus G. euneand sequence TPI TPI D. TPI + sgk /TPI ues /TPI and , K11M cated + ; + Journal of Cell Science importantly, tes while stress, htptoeei of pathogenesis that 3A). independent (Fig. is activity complementation isomerase genetic of the that demonstrating xrsigtectltclyiatv allele inactive catalytically the at expressing initiated phenotypes , paralytic distinct ieegtcsau Cltoe l,21) Pag/ag aisin ratios [P-arg]/[arg] 2011). al., of et TPI assay sensitive (Celotto extremely status an to is bioenergetic this as (Viant [P-arg] 2001), described al., previously arginine et as ratios phosphorylated concentration [arg] measured arginine we longevity relationship and behavioral the phenotypes deficiency evaluate TPI significant further and a To metabolism detect 2006). between to al., failed et have (Gnerer mutant change same measuring this studies in Previous levels stress. ATP metabolic animal to unrelated ciiysmlrto similar activity eei opeetto with complementation Genetic TPI deficiency. TPI of model described previously 2003). as al., lifespans et performed (Palladino we catalytically stress longevity the 2B). thermal (Fig. the of time points complement expression time two both to and at at shown 20, sensitivity behaviors was day induction enzyme these and stress inactive 3 measured thermal day we after points, Again, paralysis of 2B). (Fig. signs no displayed TPI 3154 fezm smrs ciiy P nyeatvt a outin robust was activity enzyme both TPI activity. isomerase enzyme of TPI aaso htepeso factltclyiatv P is TPI inactive the in catalytically of adult longevity and a the behavior These absence of animal whole. of rescue expression a to the as behavior sufficient that genotype show the the suggesting genotypes of data that representative all rates is and population Finally, Mendelian deficits S1). developmental at Table eclosed material supplementary euto nmda ogvt htwsas complemented inactive also was catalytically that the longevity median with in reduction ubro osbemcaim.Tu,w xmndwhether a examined by activity we isomerase Thus, of mechanisms. restoration TPI possible a enzyme of from the result number could of it function or non-isomerase unidentified previously 0scns epciey(i.2) ncnrs,animals contrast, In 2B). (Fig. respectively seconds, 90 h rmtcrdcini yaeioeaeatvt suggested activity isomerase lysate in reduction dramatic The ots hte eei opeetto xeddto extended complementation genetic whether test To sgk sgk sgk K11M sgk TPI /TPI /TPI uataiaswr erse oprdt idtype, wild to compared depressed were animals mutant nmlbonreisadlst nyeactivity enzyme lysate and bioenergetics animal + rvddgntccmlmnaintruharestoration a through complementation genetic provided ora fCl cec 2 (14) 126 Science Cell of Journal /TPI sgk + hw osgso aayi rsiue pnthermal upon seizures or paralysis of signs no shows TPI + TPI and and sgk sgk /TPI TPI /TPI TPI TPI TPI K11M sgk sgk sgk sgk sgk /TPI /TPI /TPI TPI nml xii eeso isomerase of levels exhibit animals and erlgclpeoye ol be could phenotypes neurological + JS10 sgk sgk yae e akdyrdcdin reduced markedly yet lysates TPI TPI and nml xii marked a exhibit animals TPI nml Fg A.Most 3A). (Fig. animals sgk K11M /TPI K11M TPI sgk JS10 , ol niaea indicate could 4 eod and seconds 240 /TPI lee(i.2C; (Fig. allele TPI nml show animals JS10 sgk Drosophila /TPI animals TPI K11M sgk atclritrs htTPI that interest particular ooyoe.OsrigTPI Observing homozygotes. Pag/ag aiscmae owl yeaias(i.3B). (Fig. animals collectively that and type results catalysis indicate lysate wild the corroborate to data These compared their in ratios depression similar [P-arg]/[arg] a showed also activity catalytic of levels ytectltclyiatv sfr,adntmrl increasing TPI active merely performed catalytically not still being a and of likely isoform, presence the inactive is catalytically rescue the phenotypic by any that suggests TPI ees iia ohwmmasuiiepoporaie(Celotto Importantly, ATP phosphocreatine 2011). their utilize buffer al., mammals to et rate how higher the to a that similar at suggests levels, pool and [P- this available, utilizing of [arg] are pool total animals available the the to in glycolysis normalized reduction in arg] a represents mutation This loss-of-function 3B). strong (Fig. a with consistent erddfo eeoie opee;o 3 htTPI that (3) or complexes; heterodimer from degraded bec fa soito ewe nyectlssand catalysis enzyme between association patients. human the in an to complications in neurological as explanation TPI of an of provide absence role may well-established and bioenergetics, the cellular of independent mechanism eeso TPI of levels aphenotypic the in seen that to levels TPI fioeaeatvt n nmlmtbls togyspot the supports that strongly conclusion metabolism animal and activity independent isomerase phenotypes of longevity and behavioral of all allele complement inactive catalytically a of capacity The TPI uatTPI mutant complementing Interestingly, 4A,C). (Fig. nepeain:()TPI (1) interpretations: rti ees o euto nTIctltccpct.To capacity. a without or catalytic with TPI of TPI presence TPI the total for in immunoblotted rescues we TPI levels, reduction inactive catalytically the a whether determine not levels, protein emnlCPde o nlec h aaiyof capacity the influence C- this not of does addition CFP The to isoforms. terminal us allowed protein tag the between of use discriminate the tag; (CFP) protein fluorescent cyan eeoieiain 2 TPI (2) heterodimerization; aayial ncieallele inactive catalytically ta. 08 rz n aldn,21) n P eeswere levels TPI and 2010), Palladino, and when Hrizo reduced further 2008; al., et oTPI to a ese htTPI that seen be can complement h nblt fTPI of inability The sgk K11M-CFP sgk TPI activity diinly complementing Additionally, ohsoe eue aiso -r/riieidctn bioenergetic indicating P-arg/arginine *** of stress. ratios reduced showed both nyeactivity. enzyme i.3. Fig. yae nt htteuisaetesm u h cl a been has scale the but same the are ( units changed). the that (note lysates sgk nml ipa eue P rti levels protein TPI reduced display animals n TPI and /TPI sgk /TPI sgk TPI eaiet TPI to relative TPI nvivo in JS10 TPI K11M-CFP K11M P TPI K11M B sgk rti osntidc h erdto of degradation the induce not does protein K11M , ) rTPI or K11M .0 eaiet T ro asindicate bars Error WT. to relative 0.001 TPI sgk ne:acmaio between comparison a Inset: . hntps(upeetr aeilFg S4). Fig. material (supplementary phenotypes yaekntc eel o ciiylvl nboth in levels activity low reveals kinetics lysate K11M-CFP al orsu nmlbonreisadTPI and bioenergetics animal rescue to fails TPI rti levels protein ( sgk hntpsaedet hnei oa TPI total in change a to due are phenotypes A hs eut ugs he possible three suggest results These . sgk ) /TPI complements sgk sgk TPI K11M-CFP TPI sgk sdgae ro obtsal upon stable but to prior degraded is + /TPI JS10 a ardwt the with paired was sgk /TPI K11M-CFP TPI al ocag TPI change to fails osntsgiiatyifunethe influence significantly not does sgk sgk xiisltl ciiyi nmllysates. animal in activity little exhibits nml and animals TPI sgk /TPI K11M-CFP K11M sgk Fg AC n,conversely, and, 4A,C) (Fig. ooesaeselectively are monomers omdlt TPI modulate to K11M with n TPI and ece oa P protein TPI total rescues TPI TPI Fg 4B,C). (Fig. nml xiiiglow exhibiting animals TPI TPI sgk sgk K11M sgk TPI sgk + hntpsb a by phenotypes TPI eody ti of is it Secondly, . TPI TPI /TPI /TPI sgk neednl,it independently, sgk osntrsu its rescue not does nvivo in /TPI sgk JS10 K11M eesrelative levels ogenetically to + TPI 6 JS10 sgk idtype wild ulallele null ihthe with animals s.e.m. K11M sgk (Seigle and levels and to Journal of Cell Science nierd(E the (GE) engineered with *** aube hyd o rvd h lgn noeosgenetic endogenous elegant the cellular provide not mammalian are do paradigms and they experimental valuable, these yeast Although systems. erythrocytes, expression patient utilized expression. primarily have miss human deficiency and TPI function analyzing overexpression its studies in gene’s transgenic Previous born to is a GE due of study artifacts power The to 2011). 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Fig. eut ofr that confirm results A uniiaino h eeso nagdTI TPI TPI. untagged of levels the of Quantification ) P K11M-CFP sgk K11M K11M TPI , sugarkill /TPI aiuain ne noeosrgltr control, regulatory endogenous under manipulations .0 oprdt T sidctsn infcn hnecompared change significant no indicates ns WT; to compared 0.001 sgk hw lvtdlvl fttlTIsmlrt T Additionally, WT. to similar TPI total of levels elevated shows JS10 /TPI ee xs sahtrdmr ae oehr these together, Taken heterodimer. a as exist never K11M uniiainadcmaio ftelvl fTPI of levels the of comparison and Quantification . a iia eeso P to TPI of levels similar has ´ JS10 ta. 93 aaaee l,19;Ay tal., et Arya 1996; al., et Watanabe 1993; al., et n oe fTIdfcec sdi hs experiments these in used deficiency TPI of model xrsinde o rvn h erdto fTPI of degradation the prevent not does expression . n 5 TPI .( 4. t ts a sdt opr h w rusadfound and groups two the compare to used was -test B K11M TPI TPI ) n 5 Drosophila .Errbr indicate bars Error 4. sgk opeet the complements ou omk targeted make to locus osntidc h erdto of degradation the induce not does TPI sgk nvivo in /TPI ehv genomically have we K11M-CFP sgk TPI 6 opeetdwith complemented hl reducing while ...( s.e.m. sgk .** phenotypes P K11M C , nvivo in )A .1and 0.01 -CFP TPI sgk . nyeo t aayi ciiywsms motn othe to important TPI in enzyme most substitution assessing M80T was the experiments that Our identified activity activity mutant. our catalytic of its pathogenesis or enzyme longevity. and behavior of as examination the such preclude and GE of control eeial opeetalbhvoa n ogvt phenotypes longevity to and of TPI behavioral of all catalytic isoform complement isomerase-inactive a genetically an experimentation for catalytic supported further capacity the but initially several revealed pathogenesis, results to disease of These proximity mechanism 1). its (Fig. taken through residues structure enzyme activity TPI the isomerase of analysis 2006; al., An from et 2010). 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Yang Dr from obtained were stocks ; Esmlrt rvosypbihdmtos(un tal., et (Huang methods published previously to similar GE w and 1118 y 9 1 n 3 and attR w nml sn rdtoa -lmn transgenesis. P-element traditional using animals Drosophila 67c23 + 9 TPI n eosiuetetre ou.Sc events Such locus. target the reconstitute and CP pGE-attBTPI -CFP, , fthe of P{y[+mDint2] . bhmlg rswr eeae by generated were arms homology kb 2.6 ou a lndit the into cloned was locus tc etr(loigo,I,USA). IN, (Bloomington, Center Stock TPI ou.Teehmlg rswere arms homology These locus. 5 w 1 + ry1;D/M,Sb D*/TM3, Crey}1b; and M80T hntp n verified and phenotype ,/,h-i;hs-FLP, hs-hid; y,w/Y, pGE-attBTPI , vasa-phiC31 pGE-attB TPI attP TPI sgk TPI and /TPI sgk vector 1 JS10 ZH-2A K11M was , attB sa is JS10 is , otiigNtria His N-terminal containing TPI of expression directs plasmid resulting techniques. The standard using pLC3 vector h oigsqec for was sequence coding Mutagenesis The protein. the Drosophila of 80 position USA). Met- a the affecting and CA, on 11 change performed position Clara, affecting Coralville, change codon codon Technologies, Santa Lys-to-Met to-Thr a DNA introduce Technologies, (Integrated to USA) Site- generated IA, (Agilent Lightning were QuikChange primers Kit the Mutagenesis using Mutagenesis performed was Directed mutagenesis directed Site Mutagenesis Gntk n u 92.Temlsrs estvt a sesdb acutely by to assessed similar a was recovery, in sensitivity to stress animals time Thermal the measuring 1982). and vortexing Wu, seconds by 20 and for determined (Ganetzky vial was media sensitivity standard stress analysis Mechanical lifespan and testing Behavioral a in nm 340 CA, at Sunnyvale, NADH Devices, 80 of (Molecular using performed absorbance reader was three assay the microplate performed The monitoring USA). 384 were measurements by Plus kinetic triplicate SpectraMax All MO, in Louis, mM. St. 0.94–5.64 (Sigma-Aldrich, times different GAP of a three range over with calculated USA) was measured enzyme previously was the as of activity velocity purified assay TPI both of NADH-linked 1999). concentrations al., an enzyme et using (Williams determined detailed was activity Isomerase assays Enzyme osprt h His the separate to A.AoewyAOAwspromdt sesvrac n aast were sets data and variance assess analysis. to post-hoc without performed Tukey’s performed was using reactions compared ANOVA to one-way normalized A and triplicate GAP. in and times dehydrogenase three glycerol-3-phosphate performed unit 1 NADH, mM 30 0.42 GAP, mM 0.47 – gm n2 MTi H88 5m al .%gyeo n mM 1 and glycerol was 2.0% purity NaCl, The mM Healthcare). 25 (GE 8.8, concentrator pH . Vivaspin Tris a mM using 20 mercaptoethanol in mg/ml 4–8 S- concentrated to (Sephacryl were fractions Peak filtration UK). England, Chalfont, Healthcare, Little gel GE 200, by followed (HiTrap-QP) chromatography cation-exchange Pec,Rcfr,I,UA.Tehmgntswr c ahsnctdfor bath-sonicated ice were homogenates 4 The at centrifuged USA). then minutes 1:100 IL, 10 PMSF (Sigma-Aldrich), Rockford, 1:1000 pepstatin (Pierce, Belgium), Geel, Organics, (Acros rtae P rti a xrse nB2(E)CdnPu (RILP) Codon-Plus BL21(DE3) in expressed was protein TPI protease. h ierpaeo ahrato,adtedt eeftt h Michaelis–Menten the to Software, (GraphPad fit 5.0b USA). were Prism CA, data Graphpad Jolla, the in La and regression reaction, nonlinear using each during without taken equation of performed velocities phase initial reactions assessing linear to (TEA), by the normalized determined were triethanolamine were kinetics curves Enzyme mM GAP. activity 100 glycerol-3- Enzyme in unit 7.6. 1 pH (Sigma-Aldrich) and (Sigma-Aldrich), dehydrogenase NADH phosphate mM 0.42 concentrations, enzyme akn y imnswr sdt euebcgon bobne h bodies The absorbance. 1 Tissues background of ml animals. reduce 0.75 frozen to in the thorax) used homogenized of and were were shaking (abdomen pigments appendages mechanical eye and vigorous lacking head after the isolated lacking were Bodies nitrogen. liquid ulndaoe h sa a efre sn 80 using that performed to similar was assay, assay linked-enzyme a The using above. assessed was outlined activity 1 enzyme to TPI diluted were Lysates debris. M80T 5m rsp .,50m al 0 lcrl Miiaoe mM 1 imidazole, mM 5 glycerol, 10% NaCl, mM 500 30,000 in centrifugation at homogenization 8.0, by via cleared pH mercaptoethanol and lysed centrifugation, Tris by temperature mM harvested room were 25 at Cells 2005) hours. (Studier, 24–30 media for auto-induction ZY in grown Technologies) fiiycrmtgah olwdb vrih E rtaetetett laethe cleave to treatment protease TEV overnight His by followed chromatography affinity NaH 9 svrfe ySSPG.Epeso n uiiaino the of purification and Expression SDS-PAGE. by verified as 99% o yaeasy,aiaswr olce n gdt a t25 at 3 day to aged and collected were animals assays, lysate For 6 m MPtgfo P.Ascn on fnce fiiyprfcto a performed was purification affinity nickel of round second A TPI. from tag -MBP 2 flst rti n10m E,p ..Ezm ciiycre were curves activity Enzyme 7.6. pH TEA, mM 100 in protein lysate of g PO sgk 4 ,10mMNa uatwspromda ecie above. described as performed was mutant P purification TPI pGE-attBTPI 6 MPadTVpoes.TIpoenwsfrhrprfe using purified further was protein TPI protease. TEV and -MBP 2 HPO nele erptoeei nTIdeficiency. function TPI this in of neuropathogenesis loss underlies that suggests normal and for function required neural function TPI non-isomerase a of presence the demonstrate to data (leads The degradation). sgk protein and activity) catalytic (abolishes studied K11M was (yellow) function TPI of catalytic Impairment functions. has (blue) non-catalytic TPI and that (orange) suggest study pathogenesis. this and of function results TPI of Model 5. Fig. 6 Drosophila n B as oho hc a ermvdwt TEV with removed be can which of both tags, MBP and - 4 H74 ihpoes niiosluetn1:1000 leupeptin inhibitors protease with 7.4) pH + ˚ o iuea 5,000 at minute 5 for C lsi n ofre ysequencing. by confirmed and plasmid m g/ 6 m P a lndit h atra expression bacterial the into cloned was TPI n10m E,p . niiosand inhibitors + 7.6 pH TEA, mM 100 in l B 27m C,17m al mM 2 NaCl, mM 137 KCl, mM (2.7 PBS Drosophila m itrscnann aidGPand GAP varied containing mixtures l TadM0 nye Initial enzyme. M80T and WT g P a uiidb nickel by purified was TPI . m g itrscontaining mixtures l ormv exoskeletal remove to nvivo in ˚ n rznin frozen and C Drosophila .coli E. using (Agilent The TPI b b - - Journal of Cell Science vrtm,adlfsaswr oprdwt o-ak(atlCx uvvltests. survival (Mantel–Cox) Log-rank with compared were behavior lifespans genotype and compare time, to over test post Bonferroni’s with performed were ANOVAs PCpoporiieassay phosphoarginine HPLC 38 to animals shifting ra . alz .R,Bliga,A .adLyo,D M. D. Layton, and J. A. Bellingham, R., M. Lalloz, R., Arya, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.124586/-/DC1 online available material Supplementary after release for PMC in R01AG027453, months. Deposited 12 Achievement B.P.R.]. numbers to A.P.V. [grant to from T32GM8424-17 R01GM097204 and Health M.J.P., fellowship to and of R01GM103369 B.P.R.]; a R01AG025046, Institutes [to Chapter National by Pittsburgh the Scientists: supported College for was Rewards work This paper. the Funding wrote M.J.P. and and C.G.A. M.J.P. B.P.R. and and research; A.P.V. data, C.G.A., the the B.P.R., analyzed B.P.R., performed reagents, research; new A.M.C. contributed the A.P.V. and designed M.J.P. S.B.L., and K.A.S., A.M.C., K.A.S., B.P.R., contributions Author genomic the with assistance for Olsen Brian the Alicia engineering. Baumgartner, perform and Matt strains; Patel to Hensel, fly Krupa clones Mallory Kosik, for Liu, and center Henry strains Mula, Stock fly Samula Bloomington for the Hong engineering; Yang genomic Dr thank We Acknowledgements 29 at aged and collected were Animals Immunoblots 95:5 a with separated and KH Japan) mM (Kyoto, 20 system (HPLC) chromatography liquid 250 25 at 3 day to aged and collected were genotype per animals 25 of sets Three t60scns etn aswr eetdbsdo rvoseprec ihTPI 25 with experience at previous performed on based selected in were progression capped were days deficiency responses Testing behavioral seconds. All 2003). 600 al., at et Palladino 2002; al., et (Palladino oass aineaddt eswr oprduigTkysps-o analysis. post-hoc were performed Tukey’s using was phospho-arginine compared ANOVA were and one-way sets data compared A arginine and respectively. and variance for minutes, arginine assess 5.4 to total times and described to software minutes Retention 7.3 normalized 3.7 previously EZStart were genotypes. al., using as levels et performed across was P-arg (Celotto generated analysis triplicate and Peak in (Shimadzu), were 1957). measured al., were et standards samples (Morrison all were Phospho-arginine standards and (P-arg) nm, 2011). 205 phospho-arginine at and Organics) detected (Acros Arginine minute. olce ntilct rmec eoyeadpoesda ulndpreviously outlined 80 in as pestle by processed ground were and heads fly genotype the Briefly, 2010). each Palladino, and from (Hrizo triplicate in collected 01.Teeetat eeijce noaPeoee ua5 Luna Phenomenex a onto injected were extracts These 2011). itrdtruhaPD 0.45 PVDF a through filtered n-a NV a efre oass aineo P eesaddt eswere sets data analysis. and post-hoc A levels Tukey’s TPI Health. of using of variance compared Institutes performed assess National to were performed the was films from ANOVA scanned one-way available the software developed ImageJ of PBST, using and analyses in digitally Densitometric antibody, washed (Pierce). secondary were ECL HRP-conjugated blots using appropriate The USA). the IA, in City, incubated Developmental Iowa alpha5; Bank, monoclonal Hybridoma mouse with Studies (1:10,000; incubated Cruz, anti-ATPalpha were Santa or Biotechnology, blots USA) PVDF 5000 Cruz CA, the Santa onto FL-249; at PBST, polyclonal transferred milk rabbit minutes (1:5000; and 1% anti-TPI 5 in SDS–PAGE treatment for by Following centrifuged resolved membrane. were and Proteins glycerol) exoskeleton. 20% 4% SDS, 6.8, pH (4% buffer sample SDS–PAGE rzni iudntoe.Teaiaswr ooeie n200 in homogenized were animals The 25 with neutralized nitrogen. and acid liquid perchloric in frozen N S)a 12,000 at USA) TN, eete etiue t12,000 at centrifuged then were one feto h l14s usiuinadietfcto fnwmttosin mutations new of identification and deficiency. substitution isomerase Glu104Asp triosephosphate the of effect founder 6 . mclm Trac,C,UA sn hmduhg performance high Shimadzu a using USA) CA, (Torrance, column mm 4.6 2 PO 4 ˚ H26 ctntielna oiepaea lwrt f06ml/ 0.6 of rate flow a at phase mobile linear acetonitrile 2.6: pH speiul ecie Plaioe l,20) Two-way 2003). al., et (Palladino described previously as C g ˚ n esrn iet aayi,a rvosydescribed previously as paralysis, to time measuring and C o iue t4 at minutes 5 for Drosophila m pnclm Ntoa cetfc Rockwood, Scientific, (National column spin m g o 0mntsa 4 at minutes 10 for Cltoe l,20) nmllfsaswere lifespans Animal 2006). al., et (Celotto u.Mutat. Hum. m f2Mptsimcroae h lysates The carbonate. potassium M 2 of l ˚ speiul ecie Cltoe al., et (Celotto described previously as C ˚ .Atr2 or,tnfyhaswere heads fly ten hours, 24 After C. b mratehnl 3 MTris–HCl mM 130 -mercaptoethanol, 10 290-294. , ˚ n h supernatant the and C 19) vdnefor Evidence (1997). g ople the pellet to m m . M 0.6 l mNH ˚ and C m l2 6 2 un,J,Zo,W,Wto,A . a,Y .adHn,Y. Hong, and N. Y. Jan, M., A. Watson, W., Zhou, J., Huang, J. M. Palladino, and L. S. Hrizo, ux .X,IlRg,G,Bao . aaa . eFbiis . oa M., Coma, G., Fabritiis, de Holla T., Nakaya, R., Bravo, G., Ill-Raga, X., F. Guix, og .J n oi,K G. K. Golic, and J. W. Gong, ikag .B,Dvnot .C,Ptk,G .adKols .R. J. Knowles, and A. G. Petsko, C., R. H. Davenport, A. Ennor, B., and E. E. Nickbarg, D. Griffiths, F., J. Morrison, o .K,Kuek,A,Ays .L n ihr,J P. J. Richard, and L. T. Amyes, A., Koudelka, K., M. Go, ink,Y,Pk,S,Hg,A,Wtnb,H n a,A. Raz, and H. Watanabe, A., Haga, S., Paku, Y., Niinaka, K., Gottlob, E., J. Skeen, E., P. Coy, P., Bhaskar, V., Nogueira, N., Majewski, L. S. Gluck, and S. L. Holliday, Y., Y. Sautin, M., Lu, nrr .P,Kee,R .adGntk,B. Ganetzky, and A. R. Kreber, P., J. Gnerer, olde .V,Lmer .M,Aayn .A,Rtmn,A,Fradz .M., A. Fernandez, A., Rottmann, A., R. Abagyan, M., A. Lambeir, V., B. Norledge, L. S. Gluck, and F. Albrecht, H., Ives, D., Ammar, M., Lu, A. E. Komives, and R. J. Knowles, C., L. Chang, J., P. Lodi, G. Kim, and S. Kim, H., N. Won, C., B. Yoo, Y., Lim, C., Kim, aezy .adW,C F. C. Wu, and B. Ganetzky, E. L. Maquat, and C. D. Phillips, J., P. Artymiuk, O., I. Daar, e,W . hi .S,Bu,M . o,K . hn . e,S .adSr,Y J. Y. Surh, and K. S. Lee, S., Shin, T., K. Koo, R., M. Byun, S., J. Choi, H., W. Lee, K. R. Wierenga, and R. F. Opperdoes, M., A. Lambeir, V. C. Dang, B. and W. M. J. Kim, Feany, and V. Khurana, Y. Hong, and M. A. Watson, W., Dong, W., Zhou, J., Huang, oa . ux .X,Uieag,I,Espun I., Uribesalgo, X., F. Guix, M., Coma, J. M. Palladino, and W. Voorhies, Van K., W. Chiu, M., A. Celotto, J. M. Palladino, and L. J. Seigle, C., A. Frank, M., A. Celotto, Lambeir, K., Augustyns, I., El-Sayed, K., Groebel, M., Alahuhta, G., M. Casteleijn, un,J,Hag . hn .J,Asi,E,Dvr .E,Reir,F n Hong, and F. Roegiers, E., C. Devor, E., Austin, J., Y. Chen, L., Huang, J., Huang, u eetreigi Drosophila. in targeting gene out Drosophila. in pathogenesis 676-683. sugarkill TPI underlies degradation icoe .P,Villa P., G. Miscione, eiiny w eeeyafce rtesoewt n n ihu neurological without one and with one brothers affected symptoms. severely two deficiency: Gyo glycation induces fibrillation. nitrotyrosination tau and isomerase triosephosphate Amyloid-dependent Drosophila. rpriso riiephosphokinase. arginine of properties aayi ytispopaeioeae w-atsbtaeapproach. substrate two-part a 49 isomerase: triosephosphate by catalysis roehsht smrs:rmvlo uaieyeetohlchsiieresidue mechanism. histidine catalytic in electrophilic change putatively subtle a a in of results removal isomerase: Triosephosphate the in apoptosis inhibit to Bak. required and is N. Bax Hay, of Akt and absence by B. or R. presence mediated Robey, B., interaction C. mitochondria Thompson, S., N. Chandel, uaini roehsht smrs,cue aayi,nuoeeeain n early and neurodegeneration, paralysis, death. causes isomerase, triosephosphate in mutation iioo,V . ee,M .adWeeg,R K. R. loop Wierenga, phosphate-binding and conserved fully G. the on M. studies Peter, structural and V., mutagenesis, V. Filimonov, autocrine as cells. factor tumor by isomerase/maturation factor motility neuroleukin/phosphohexose of secretion assembly the for pump. essential proton is the H+-ATPase of vacuolar activity and and aldolase between interaction H+-ATPase. vacuolar of activity Chem. and expression, assembly, mediates aldolase lysine-12. of role the site: active 33 charged positively a requires isomerase hsht smrs eiiny igeaioai usiuinrslsi a in melanogaster. Drosophila results in substitution excitability nerve acid altered with amino single a enzyme. deficiency: thermolabile isomerase phosphate uigeooieidcdaotssi eacls oeta oeo Cdk2. of role potential cells: HeLa 278 in phosphorylation apoptosis through etoposide-induced isomerase during triosephosphate of inactivation Functional (2010). cycle. cell HeLa during methylation Acta Biophys. arginine protein post-translational Sci. Biochem. u opoennitrotyrosination. protein to due J. F. enzymopathy. glycolytic deficiency isomerase triosephosphate Genetics inherited human of isomerase. triosephosphate of K. proline R. site Wierenga, and active P. Neubauer, M., A. roepopaeioeaefo rpnsm rcibue.Acmaio ihthe enzymes. with comparison yeast A and brucei. muscle brucei rabbit Trypanosoma from isomerase triose-phosphate Drosophila. in neurodegeneration polarization. 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Proc. 92 124 c,M. ´cs, 132 18) nietsprsinivligbhvoa mutants behavioral involving suppression Indirect (1982). o-aayi P ucin3157 function TPI Non-catalytic 486-490. , 20) utfctdrlso lcltcenzymes. glycolytic of roles Multifaceted (2005). 4001-4013. , 1335-1345. , acrRes. Cancer 20) nsot rrpaeet eetreigin targeting gene replacement, or Ends-out, (2003). 6 Brain e25823. , .Bo.Chem. Biol. J. 19) eeiaytispopaeioeae(TPI) isomerase triosephosphate Hereditary (1993). Genetics ici.Bohs Acta Biophys. Biochim. 21) s7-adHp0mdae proteasomal Hsp90-mediated and Hsp70- (2010). u.J Biochem. J. Eur. 103 20) oncigce Connecting (2007). o.Cell Mol. ice.J. Biochem. 14987-14993. , 128 100 106 a . Sole G., ˜a, 1613-1621. , 180 2556-2561. , 58 20) ucinlrl fteconserved the of role Functional (2006). 8284-8289. , 2667-2674. , 703-707. , 16 282 20) atdaa,aDrosophila a away, wasted (2006). 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