GBME,SKKUMolecular&CellBiology

H.F.K.

Chapter3-I– ProteinStructureandFunction

Chapter3– ProteinStructureandFunction

Chapter3- ProteinStructureandFunction

3.1Hierarchical StructureofProteins3.2ProteinFolding3.3ProteinBinding andEnzyme Catalysis3.4Regulating ProteinFunction3.5Purifying,Detecting,andCharacterizingProteins3.6Proteomics

Protein?

• Oneofthefinalproductsincell• Variousfunctions

1.Structure

ProteinStructureandFunction

3.1HierarchicalStructureofProteins• Proteinsequencespecifiesfoldingintosecondaryandtertiarystructures thateitherarefunctionalunitsorcaninteractwithotherpeptidestoformquaternarystructurefunctionalunits.

• Homologousproteinsevolvedfromacommonancestor,havesimilarsequences,structures,andfunctions,andcanbeclassifiedintofamiliesandsuperfamilies.

Fourlevelsofproteinhierarchy.• (a)Primary structure– linearsequenceofaminoacidslinkedtogetherbypeptidebonds

• (b)Secondary structure– Foldingofthepolypeptidechainintolocalαhelicesorβsheets

• (c)Tertiary structure• structureofapeptidecomposedofsecondarystructuralelementsandvariousloopsandturns

• mayformdistinct,independentlystabledomains• (d)Quaternary structure– somefunctionalproteinsarecomposedofmorethanonepolypeptide

Structureofapolypeptide.• Proteins– unbranchedpolymersconstructedoutof20aminoacidswithdifferentRgroupsidechains.

• Aminoacidsidechainsdeterminethedistinctpropertiesofindividualproteins.

Primarystructure

AlphaBeta

The! helix,acommonsecondarystructureinproteins.• Secondarystructures– stablespatialarrangementsofpolypeptidechainsegmentsheldtogetherbyhydrogenbondsbetweenbackboneamideandcarbonylgroups

• ! helix:• Polypeptidebackbone(ribbon)foldsintoaspiral/helixwith3.6aminoacidsperturn(0.54nm).

• Helixisstabilizedbyhydrogenbondsbetweenbackboneoxygenandhydrogenatoms(morebonds-morestable).

• Rgroupsprojectoutward fromthesurfaceofthehelix– determinechemicalnatureofhelixfaces.

• Prolines – can’tparticipateinhydrogenbondingandusuallyareexcludedfroman! helix.

Secondarystructure

Doyouremembertheprolinestructure?Pleasecheckit!

Alphahelix

https://www.youtube.com/watch?v=eUS6CEn4GSA

The" sheet,anothercommonsecondarystructureinproteins.• βsheet– laterallypackedβstrands,eachofwhichisanearlyfullyextendedpolypeptidesegment

• (a)Three-strandedβsheet– antiparallelβstrandswithconnectingloops(topview):

• Stabilizedbyhydrogenbondsbetweenbackboneoxygenandhydrogenatomsinaminoacidsondifferentstrands

• (b)Antiparallelβsheet(sideview):• ! carbonbondanglesproduceapleatedpolypeptidebackbonecontour.

• AlternateRgroupsprojectaboveandbelowtheplaneofthesheet.

• (c)Parallelβstrandsheet:sameN-to-Cstrandorientationswithconnectingloops.

Secondarystructure

Betasheet

https://www.youtube.com/watch?v=wM2LWCTWlrE

Structureofa" turn.• Composedoffourresidues.• Reversesdirectionofapolypeptidechain(180° U-turn).

• Cαcarbonsofthefirstandfourthresiduesareusuallylessthan0.7nmapartandlinkedbyahydrogenbond.

• βturnsfacilitatethefoldingoflongpolypeptidesintocompactstructures.

• Glycine(smallestRgroup)andproline (builtinbend)arecommonlyfoundinβturns.

Secondarystructure

Thinkaboutthereasonindiscussion!

Proteinfolding?

Threemodelsofproteinfolding

• Frameworkmodel• Nucleationmodel• Hydrophobic-collapsemodel

https://www.youtube.com/watch?v=B275XtegrJ4

Nosinglemechanism…

Theoildropmodelofproteinfolding.• Tertiarystructure– stabilizedprimarilybyhydrophobicinteractionsbetweennonpolarsidechainsandhydrogenbondsinvolvingpolarsidechainsandbackboneaminoandcarboxylgroups

• Hydrophobicresidues(blue):clustertogetherlikedropsofoilinthefoldedproteincore,drivenawayfromtheaqueoussurroundingsbythehydrophobiceffect.

• Chargedandunchargedpolarsidechains(yellow):formstabilizinginteractionswithsurroundingwaterandionsontheproteinsurface.

Easyexample…

Fourwaystovisualizeproteinstructure.• Ras,amonomeric(singlepolypeptidechain)proteinthatbindstoguanosinediphosphate(GDP,inblue).

• (a)Cαbackbonetrace– depictshowthepolypeptideistightlypackedintoasmallvolume• (b)Ball-and-stickrepresentation– revealslocationsofallatoms• (c)Ribbondiagram– emphasizeshowβstrands(lightblue)andαhelices(red)areorganizedintheprotein

• (d)Water-accessiblesurfacemodel– revealsproteinsurfacetopologywithpositivecharge(purple)andnegativechargeregions(red)

Howtofind?• http://www.uniprot.org/

FindstructureofCREB!

• Structuralmotifs:• Regularcombinations ofsecondarystructuresusuallywithaspecifictypeoffunction

• Canbeencodedbyahighlyconservedsequencemotif

Similarfunctionsbysimilarstructures!!!

Motifsofproteinsecondarystructure.

• Twoαheliceswoundaroundeachother

• Atypeofhelix-loop-helixmotifinmanyproteins,includingmanycalcium-bindingandDNA-bindingregulatoryproteins

• presentinmanyDNA-bindingproteinsthathelpregulatetranscription

The Alpha-Helical SecondaryStructureofMyoglobin(Mb)

• Thesehelicalsegmentsarelabeled A-H andspanthefollowingresiduesofthis153aminoacidresiduepolypeptidechainofMb.Segments(residues): A (3-18), B (20-35), C (36-42), D (51-57), E (58-76), F (83-95), G (100-118),& H (124-149).

https://biosci.mcdb.ucsb.edu/biochemistry/tw-prt/myoglobin/alphahelixf.htm

Zincfingerdomain

https://www.youtube.com/watch?v=WyU2v7HT6bw

Leucin zipperdomain

https://www.youtube.com/watch?v=2-qFLfVymnw

Tertiaryandquaternarylevelsofstructure.

Tertiaryandquaternarylevelsofstructure.

Protein quaternary structure is the number and arrangement of multiple folded protein subunitsThe tertiary structure will have a single

polypeptide chain "backbone" with one or more protein secondary structures, the protein domains.

Modularnatureofproteindomains.

• Epidermalgrowthfactor(EGF)precursor:generatedbyproteolyticcleavagegeneratesmultipleEGFs(green)

• Neu:EGFdomainplusotherdomains• Tissueplasminogenactivator(TPA):EGFdomainplusotherdomains

SeveralproteinsworktogetherinCell!

Amolecularmachine:thetranscriptioninitiationcomplex.

Supramolecularcomplexes

Molecularevolution?

Evolutionoftheglobinproteinfamily.

Myoglobin (symbol Mb or MB)isan iron-and oxygen-binding protein foundinthe muscletissue ofvertebratesingeneralandinalmostallmammals.

Leghemoglobin (also leghaemoglobin orlegoglobin)isanitrogenoroxygencarrierandhemoproteinfoundinthenitrogen-fixingrootnodulesofleguminousplants.

ProteinStructureandFunction

• 3.2ProteinFolding• Proteinaminoacidsequencedeterminesits3Dstructureandfunction.

• ATP-dependentmolecularchaperones andchaperonins assist proteinfolding invivo.

• Misfolded/denaturedproteinscanformwell-organizedamyloidfibrilaggregatesthatcancausediseases,includingAlzheimer’sdiseaseandParkinson’sdisease.

Thinktheproteinfolding!

What’stheimportantphysicalmovementforthefolding?

Rotation!

Rotationbetweenplanarpeptidegroupsinproteins.

PolypeptidebackbonestericrestraintsandaminoacidsidechainpropertiesseverelyrestrictCα–aminonitrogenbond(theΦ angle)andCα–carbonylcarbonbond(theΨ angle)rotations.

Whatarethetransandcis?

• Planarpeptidebonds limittheshapesintowhichproteinscanfold.WhichRgroupofaminoacidhasthetras- andcis-forms?

Prolinecis/trans isomerizationsinfluenceprotein foldingandstructure.

• Cis/transisomerizationofasingleproline:

• AltersstructureofaproteinSH2domain.

• Caninfluenceaprotein’sactivity.• Prolineisomerasesmayactasswitchesthatregulateproteinactivity.

Hypotheticalprotein-foldingpathway

primary(a)→secondary(b–d)→tertiary(e)structure

But,thefoldingisnotalways‘easy-going’process

Molecularchaperone–mediatedproteinfolding.

Chaperonin-mediatedproteinfolding.It’saproteinchamber…

ChaperoneandChaperonin

https://www.youtube.com/watch?v=d1QIEQEyYRo

Misfoldedproteinscanformorderedamyloidaggregatesbasedonacross-"sheetstructure.

ProteinStructureandFunction3.3ProteinBindingandEnzymeCatalysis• Proteinfunctiondependsonbindingothermolecules(ligands).

• Enzymesaccelerateratesofcellularreactionsbyloweringactivationenergyandstabilizingtransition-stateintermediates.

• Enzymesoftenuseacid-basecatalysismediatedbyoneormoreaminoacidsidechains.

• Metabolicpathwayenzymesmaybeassociatedasdomainsofamonomericprotein,subunitsofamultimericprotein,orcomponentsofaproteincomplexassembledonacommonscaffold.

Protein-ligandbindingofantibodies.• Ligand:moleculetowhichaproteinbinds• (a)IgGantibody(ribbonmodel):twoidenticalheavy chains(lightanddarkred)andtwoidenticallight chains(blue)covalentlylinkedbydisulfidebonds

• CDR(complementarity-determiningregions):sixhighlyvariableloopsformtheantigen-bindingsites

• (b)Hand-in-glovefit:molecularcomplementaritybetweentheantibodyCDRandthesitetowhichitbinds(epitope)onitstargetantigen

Remindtheroleofenzyme

Activesiteoftheenzymetrypsin.• Enzymes(proteinsorRNAs)catalyzemakingorbreakingsubstratecovalentbonds.• (a)Trypsin (serineprotease)activesite:

• Substrate-bindingpocket – bindsspecificsubstrate• Catalyticsite– containssidechainsofthecatalytictriadSer-195,Asp-102,andHis-57thatbreakspeptidebonds.

• Insomeenzymes,thecatalyticandsubstrate-bindingsitesoverlap;inothers,thetworegionsarestructurallydistinct.

Schematicmodelofanenzyme’sreactionmechanism.

Nextclass…

• Realenzymeaction!• Structuralchangebyligandbinding&tech.• Practicalproteinworkslearnedbytext(?!?!)

the long-term sensitization of S-SW compared with theduration of siphon withdrawal before stimulation (pre-test) (not significant, p = 0.26, n = 9 and p = 0.85, n = 6,respectively; paired Student’s t test) (Figure 7C). How-ever, 30 min-interval pairing of SNS and a single tail

shock significantly increased the duration of S-SW com-pared with the duration of siphon withdrawal in pretest(p < 0.05, n = 13; paired Student’s t test) (Figure 7C). Incontrast, the tail shock before noxious stimulus, the re-verse order pairing, did not generate the long-term sen-sitization (not significant, p = 0.36, n = 8) (Figure 7C).These results indicate that SNS facilitates the long-term memory formation of Aplysia in a stimulus order-specific manner.

Discussion

Our experiments showed that neural activity induces theexpression of a novel nucleolar protein ApLLP, and thisexpression increases the ApC/EBP mRNA level requiredfor long-term synaptic plasticity. We also showed thata single 5-HT pulse can induce a long-term increase in

Figure 5. Blocking the Activity of ApLLP Blocks the Depolarization-Elicited ApC/EBP Induction

(A) Specificity test of the a-ApLLP antibody. In Western blotting,E. coli-expressed His6-ApLLP and Aplysia-ganglion-expressedApLLP-EGFP were detected by the a-ApLLP antibody but not bypreimmune serum.(B) In immunocytochemistry with the Cy3 antibody, only the nucleuswas observed in the preimmune serum-injected neuron. In contrast,the nucleolar structure was observed in the a-ApLLP antibody-in-jected sensory neurons. Upper white outlined box, magnified imageof the nucleolar structures (white arrow) stained by a-ApLLP anti-body. White dashed line indicates the plasma membrane of sensoryneurons. Scale bar, 30 mm.(C) The requirement of ApLLP activity on depolarization-elicitedApC/EBP induction. In comparison with the preimmune serum-in-jected neuron, ApC/EBP mRNA was not induced in the a-ApLLP-an-tibody-injected neurons. Scale bar, 30 mm.(D) The mean pixel intensity represents the blocking effect of the a-ApLLP antibody on ApC/EBP induction elicited by depolarization.Blocking the ApLLP activity by using a-ApLLP antibodies blockedthe depolarization-elicited ApC/EBP mRNA induction (n = 12) (aster-isk, p < 0.05, unpaired Student’s t test; compared with preimmuneinjected and high potassium treatment group [n = 14]; triple asterisk,p < 0.0001, unpaired t test; compared with noninjected and high po-tassium treatment group [n = 12]). Plus sign, treatment; minus sign,no injection or no treatment. Each bar represents the mean 6 SEM.

Figure 6. Treatment with a Combination of Depolarization and a Sin-gle 5-HT Pulse Increased the Long-Term Synaptic Strength

(A) The protocol combining treatment with 100 mM potassium solu-tion and a 5-HT pulse.(B) Relative mean percentage changes in the EPSP amplitude of co-cultured neurons treated with a combination of 100 mM potassiumsolution and 5-HT pulse (n = 16) compared with those in groupsthat were treated with either high potassium (n = 12) or 5-HT treat-ment alone (n = 9). EPSP changes for each group were normalizedwith the average amplitude of neurons treated with a 100 mM potas-sium solution. The EPSP amplitude of the group receiving the com-bination treatment of KCl and 5-HT was increased 24 hr after treat-ment (triple asterisk, p < 0.0001, ANOVA and Tukey’s multiplecomparison test). However, reverse order treatment of 100 mMpotassium solution and 5-HT pulse (n = 6) did not increase EPSP am-plitude after 24 hr. This long-term increase in EPSP was completelyblocked by either anti-ApLLP antibody (n = 7) or anti-ApC/EBP anti-body injection (n = 5) to the sensory neuron as compared with that inthe preimmune serum-injected sensory neurons cocultured with mo-tor neurons (n = 6) (asterisk, p < 0.05 and double asterisk, p < 0.01,unpaired Student’s t test). Each bar represents the mean 6 SEM.

The Role of ApLLP in Synaptic Plasticity713

Kimetal.,Neuron2006

What’sthis?

Discussionwithfriends

• Thinkaboutwhatcausemisfolding ofproteins.• Thendiscusshowthechaperoneandchaperoninhelptheproperfoldingofproteins(mechanisms).

• Practicallyproteinmisfolding canbeabigprobleminproteinproductioninbasicresearchfieldandindustry.Howcanyousolvethismisfolding problem?Pleasefindplausiblewaysexceptthechaperoneandchaperonin.

Furtherquestions

• Howdothechaperonandchaperoninrecognizethemisfolding proteins?

• Canthehumanchaperonbeusedinotheranimalssuchasratandmouse?

Chaperonin

• GroEL belongstothe chaperonin familyof molecularchaperones,andisfoundinalargenumberofbacteria.Tofunctionproperly,GroEL requiresthelid-likecochaperonin proteincomplex GroES.In eukaryotes theproteins Hsp60 and Hsp10 arestructurallyandfunctionallynearlyidenticaltoGroELandGroES,respectively.

• Unfoldedsubstrateproteinsbindtoahydrophobicbindingpatch ontheinteriorrimoftheopencavityofGroEL,formingabinarycomplexwiththechaperonin.

GroEL bindingThe EMBO Journal vol.13 no.13 pp.3192-3202, 1994

Conformational specificity of the chaperoninGroEL for the compact folding intermediates ofa-lactalbumin

Manajit K.Hayer-Hartl,Jonathan J.Ewbankl'2, Thomas E.Creighton1and FUirich HartiHoward Hughes Medical Institute, Cellular Biochemistry andBiophysics Program, Memorial Sloan-Kettering Cancer Center, 1275York Avenue, New York, NY 10021, USA and 'European MolecularBiology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, 69012Heidelberg, Germany2Present address: Department of Biology, McGill University, 1205 DrPenfield Avenue, Montreal, PQ, Canada H3A lBICommunicated by W.Neupert

The chaperonin GroEL binds unfolded polypeptides,preventing aggregation, and then mediates their foldingin an ATP-dependent process. To understand the struc-tural features in non-native polypeptides recognizedby GroEL, we have used a-lactalbumin (aLA) as amodel substrate. aLA (14.2 kDa) is stabilized by fourdisulfide bonds and a bound Ca2+ ion, offering thepossibility of trapping partially folded disulfide inter-mediates between the native and the fully unfoldedstate. The conformers of aLA with high affinity forGroEL are compact, containing up to three disulfidebonds, and have significant secondary structure, butlack stable tertiary structure and expose hydrophobicsurfaces. Complex formation requires almost the com-plete aLA sequence and is strongly dependent on saltsthat stabilize hydrophobic interactions. Unfolding ofaLA to an extended state as well as the burial ofhydrophobic surface upon formation of orderedtertiary structure prevent the binding to GroEL. Inter-estingly, GroEL interacts only with a specific subset ofthe many partially folded disulfide intermediates ofaLA and thus may influence in vitro the kinetics ofthe folding pathways that lead to disulfide bonds withnative combinations. We conclude that the chaperonininteracts with the hydrophobic surfaces exposed byproteins in a flexible compact intermediate or moltenglobule state.Key words: chaperonin/GroEL/a-lactalbumin/moltenglobule/protein folding

IntroductionMolecular chaperones of the Hsp6O family play an essen-tial role in mediating the folding and assembly of newlysynthesized polypeptides (Gething and Sambrook, 1992;Hartl et al., 1994). These 'chaperonins' (Hemmingsenet al., 1988) are large oligomeric ring-complexes con-taining 14 subunits of -60 kDa in stacked heptamericrings (Hendrix, 1979; Hohn et al., 1979; Langer et al.,1992a; Saibil et al., 1993). GroEL, the Escherichia colichaperonin, was discovered as a host protein required

for bacteriophage capsid assembly (Coppo et al., 1973;Georgopoulos et al., 1973). It interacts with newly synthe-sized polypeptides in the bacterial cytosol and is requiredfor their folding (Bochkareva et al., 1988; Goloubinoffet al., 1989a; Horwich et al., 1993). The GroEL homo-logues in chloroplasts and mitochondria are involved inprotein folding and assembly reactions within the respect-ive organelles (Barraclough and Ellis, 1980; Cheng et al.,1989; Ostermann et al., 1989).The basic function of the Hsp6Os is to assist the folding

of monomeric polypeptide chains (Ostermann et al., 1989;Laminet et al., 1990; Martin et al., 1991; Viitanen et al.,1991; Zheng et al., 1993). Hsp6O binds a partially foldedpolypeptide, preventing aggregation (Buchner et al., 1991),and releases it in an ATP-dependent reaction that canresult in folding (Goloubinoff et al., 1989b; Martin et al.,1991, 1993a). This process depends on the co-chaperoninHsplO, GroES in E.coli, a single heptameric ring of-10 kDa subunits that forms a complex with GroEL(Chandrasekhar et al., 1986; Viitanen et al., 1990; Saibilet al., 1991; Langer et al., 1992a; Landry et al., 1993).GroES regulates the ATPase activity of the GroEL sub-units, coordinating their action to allow the release ofbound polypeptide in a manner productive for folding(Gray and Fersht, 1991; Bochkareva et al., 1992; Jacksonet al., 1993; Martin et al., 1993a,b; Todd et al., 1993).During folding, the polypeptide is maintained in a shieldedenvironment (Martin et al., 1991), apparently provided bythe central cavity of the Hsp6O cylinder (Langer et al.1992a; Braig et al., 1993; Saibil et al., 1993).While there has been considerable progress in under-

standing the mechanistic principles of Hsp6O-mediatedfolding, the structural features recognized by the chap-eronins in unfolded polypeptides are still undefined.Although GroEL binds an amphiphilic N-terminal peptideof rhodanese with low affinity, stabilizing it in an a-helical conformation (Landry et al., 1992), the chaperoninalso interacts with the denatured state of an all-5 protein(Schmidt and Buchner, 1992). It is not known whetherGroEL recognizes primarily contiguous sequence elementsor hydrophobic surfaces typically exposed by partiallyfolded polypeptides. It has been suggested that GroELbinds the compact intermediates of proteins producedearly in folding by the partial collapse of hydrophobicresidues to the interior of the molecule (Laminet et al.,1990; H6ll-Neugebauer, 1991; Martin et al., 1991; vander Vies et al., 1992). These compact intermediates or'molten globules' (Kuwajima,- 1989) appear to be ofgeneral significance in the folding of globular proteins(Ptitsyn et al., 1990; Ptitsyn, 1992). They are characterizedby a hydrodynamic radius close to that of the native state,the presence of a significant amount of secondary structureand the partial or complete lack of stable tertiary structureinteractions (Kuwajima, 1989; Christensen and Pain,

(39)Oxford University Press3192

The EMBO Journal vol.13 no.13 pp.3192-3202, 1994

Conformational specificity of the chaperoninGroEL for the compact folding intermediates ofa-lactalbumin

Manajit K.Hayer-Hartl,Jonathan J.Ewbankl'2, Thomas E.Creighton1and FUirich HartiHoward Hughes Medical Institute, Cellular Biochemistry andBiophysics Program, Memorial Sloan-Kettering Cancer Center, 1275York Avenue, New York, NY 10021, USA and 'European MolecularBiology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, 69012Heidelberg, Germany2Present address: Department of Biology, McGill University, 1205 DrPenfield Avenue, Montreal, PQ, Canada H3A lBICommunicated by W.Neupert

The chaperonin GroEL binds unfolded polypeptides,preventing aggregation, and then mediates their foldingin an ATP-dependent process. To understand the struc-tural features in non-native polypeptides recognizedby GroEL, we have used a-lactalbumin (aLA) as amodel substrate. aLA (14.2 kDa) is stabilized by fourdisulfide bonds and a bound Ca2+ ion, offering thepossibility of trapping partially folded disulfide inter-mediates between the native and the fully unfoldedstate. The conformers of aLA with high affinity forGroEL are compact, containing up to three disulfidebonds, and have significant secondary structure, butlack stable tertiary structure and expose hydrophobicsurfaces. Complex formation requires almost the com-plete aLA sequence and is strongly dependent on saltsthat stabilize hydrophobic interactions. Unfolding ofaLA to an extended state as well as the burial ofhydrophobic surface upon formation of orderedtertiary structure prevent the binding to GroEL. Inter-estingly, GroEL interacts only with a specific subset ofthe many partially folded disulfide intermediates ofaLA and thus may influence in vitro the kinetics ofthe folding pathways that lead to disulfide bonds withnative combinations. We conclude that the chaperonininteracts with the hydrophobic surfaces exposed byproteins in a flexible compact intermediate or moltenglobule state.Key words: chaperonin/GroEL/a-lactalbumin/moltenglobule/protein folding

IntroductionMolecular chaperones of the Hsp6O family play an essen-tial role in mediating the folding and assembly of newlysynthesized polypeptides (Gething and Sambrook, 1992;Hartl et al., 1994). These 'chaperonins' (Hemmingsenet al., 1988) are large oligomeric ring-complexes con-taining 14 subunits of -60 kDa in stacked heptamericrings (Hendrix, 1979; Hohn et al., 1979; Langer et al.,1992a; Saibil et al., 1993). GroEL, the Escherichia colichaperonin, was discovered as a host protein required

for bacteriophage capsid assembly (Coppo et al., 1973;Georgopoulos et al., 1973). It interacts with newly synthe-sized polypeptides in the bacterial cytosol and is requiredfor their folding (Bochkareva et al., 1988; Goloubinoffet al., 1989a; Horwich et al., 1993). The GroEL homo-logues in chloroplasts and mitochondria are involved inprotein folding and assembly reactions within the respect-ive organelles (Barraclough and Ellis, 1980; Cheng et al.,1989; Ostermann et al., 1989).The basic function of the Hsp6Os is to assist the folding

of monomeric polypeptide chains (Ostermann et al., 1989;Laminet et al., 1990; Martin et al., 1991; Viitanen et al.,1991; Zheng et al., 1993). Hsp6O binds a partially foldedpolypeptide, preventing aggregation (Buchner et al., 1991),and releases it in an ATP-dependent reaction that canresult in folding (Goloubinoff et al., 1989b; Martin et al.,1991, 1993a). This process depends on the co-chaperoninHsplO, GroES in E.coli, a single heptameric ring of-10 kDa subunits that forms a complex with GroEL(Chandrasekhar et al., 1986; Viitanen et al., 1990; Saibilet al., 1991; Langer et al., 1992a; Landry et al., 1993).GroES regulates the ATPase activity of the GroEL sub-units, coordinating their action to allow the release ofbound polypeptide in a manner productive for folding(Gray and Fersht, 1991; Bochkareva et al., 1992; Jacksonet al., 1993; Martin et al., 1993a,b; Todd et al., 1993).During folding, the polypeptide is maintained in a shieldedenvironment (Martin et al., 1991), apparently provided bythe central cavity of the Hsp6O cylinder (Langer et al.1992a; Braig et al., 1993; Saibil et al., 1993).While there has been considerable progress in under-

standing the mechanistic principles of Hsp6O-mediatedfolding, the structural features recognized by the chap-eronins in unfolded polypeptides are still undefined.Although GroEL binds an amphiphilic N-terminal peptideof rhodanese with low affinity, stabilizing it in an a-helical conformation (Landry et al., 1992), the chaperoninalso interacts with the denatured state of an all-5 protein(Schmidt and Buchner, 1992). It is not known whetherGroEL recognizes primarily contiguous sequence elementsor hydrophobic surfaces typically exposed by partiallyfolded polypeptides. It has been suggested that GroELbinds the compact intermediates of proteins producedearly in folding by the partial collapse of hydrophobicresidues to the interior of the molecule (Laminet et al.,1990; H6ll-Neugebauer, 1991; Martin et al., 1991; vander Vies et al., 1992). These compact intermediates or'molten globules' (Kuwajima,- 1989) appear to be ofgeneral significance in the folding of globular proteins(Ptitsyn et al., 1990; Ptitsyn, 1992). They are characterizedby a hydrodynamic radius close to that of the native state,the presence of a significant amount of secondary structureand the partial or complete lack of stable tertiary structureinteractions (Kuwajima, 1989; Christensen and Pain,

(39)Oxford University Press3192

GroEL bindingJ. Biochem. 124, 319-325 (1998)

The Region of ƒ¿-Lactalbumin Recognized by GroEL

Akio Shimizu,' Takahiro Tanba, Isamu Ogata, Masamichi Ikeguchi, and Shintaro Sugai

Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577

Received for publication, February 17, 1998

The binding constants between disulfide-intact or various disulfide-reduced bovine ƒ¿-lactalbumins and an Escherichia coli chaperonin, GroEL, were determined by using the

equilibrium dialysis method. The disulfide-intact and one-disulfide (Cys6-Cys120)-re

duced ƒ¿-lactalbumins were shown not to bind with GroEL both in the presence and absence of Call. The two-disulfide (Cys6-Cys120 and Cys28-Cyslll)-reduced ƒ¿-lactalbumin, which

has the native-like tertiary structure in its ƒÀ-domain region and an unfolded a-domain in the presence of Call, showed considerable binding with GroEL. The binding free energy of

the two-disulfide-reduced ƒ¿-lactalbumin in the presence of Call is close to that of the

molten globule state of disulfide-intact ƒ¿-lactalbumin. This result suggests that GroEL binds to the unfolded a-domain of ƒ¿-lactalbumin regardless of the conformation of the

,ƒÀ-domain. The fully disulfide-reduced and two-disulfide-reduced ƒ¿-lactalbumins were found to bind more strongly with GroEL in the absence of Ca" than the two-disulfide-

reduced ƒ¿-lactalbumin in the presence of Ca", thus indicating that the unfolding of the ƒÀ-domain of ƒ¿-lactalbumin leads to stronger interaction with GroEL.

Key words: equilibrium dialysis, GroEL, ƒ¿-lactalbumin, binding constant, binding site.

The Escherichia coli chaperonin GroEL is one of the most

extensively studied molecular chaperones that is able to

assist the refolding of proteins (1-6). GroEL is a large homotetradecamer composed of 60 kDa subunits (7-10)

and seems to prevent off-pathway reactions of the protein folding that lead to protein aggregation (1). The initial step

in the process of GroEL action is capture of the target

protein. It has previously been reported that GroEL recognizes the a-helical conformation (11, 12), although its interaction with an all ƒÀ-protein (13) has also been report-

ed. Recently, the apical domain of GroEL has been suggest-

ed to be responsible for its interaction with substrate

proteins (14), and this domain contains many hydrophobic residues (7). Many substrate structures recognized by GroEL have been shown to have considerable hydrophobic

regions exposed to the solvent (like that in the molten

globule or in more disordered states), from which it has been suggested that hydrophobicity is important for the

GroEL recognition (15, 16). Also, because the binding strength between GroEL and substrate protein is influ

enced by salt concentration (17, 18), electrostatic interaction has also been suggested to affect the binding strength

between GroEL and substrate proteins. Although consider-

able progress has been made in understanding the interaction between substrate proteins and GroEL, important

questions remain concerning the conformation of the target protein recognized by GroEL. What kinds of the structural elements and features of substrate protein are recognized by GroEL? One way to elucidate this problem is to study

the exact binding affinity of the protein substrate, which

can assume various conformations, to GroEL.

1 To whom correspondence should be addressed.

(c) 1998 by The Japanese Biochemical Society.

The equilibrium dialysis method is most appropriate for studying the protein-ligand interaction. Compared to gel

filtration chromatography, by which many binding affinities

of GroEL to substrate proteins have previously been studied, the equilibrium dialysis method allows exact determination of the binding constant and detection of

weak interactions. Also, bovine ƒ¿-lactalbumin (BLA) may be a suitable substrate for the study of the recognition

mechanism of the protein by GroEL (4, 6, 17-22). This

protein has four disulfide bonds (Cys6-Cys120, Cys28-Cys111, Cys61-Cys77, and Cys73-Cys9l) and binds a single Ca2+ (23) in the native state (Fig. 1). Its structure

changes easily between the native and unfolded states by treatments other than changes in pH, temperature, and

concentrations of denaturants and salts: (a) selective reduction of disulfide bonds, (b) the chemical modification

of the reduced disulfide bonds (24-26), and (c) control of Call binding (25, 27). The separate contributions of

hydrophobic and electrostatic interactions to the binding of

GroEL with the target protein can be studied by skillful use of these processes of conformational change.

In this paper, to clarify the structural elements and

features of BLA recognized by GroEL, the binding

strengths of intact, and one-, two-, and four-disulfide-reduced (the reduced disulfide bonds were S-carboxyamido-

methylated by iodoacetamide or S-carboxymethylated by

iodoacetate) BLAs with GroEL were studied by the equilibrium dialysis method in the presence and absence of Call at

a low temperature, 4•Ž. By comparing the conformations of the derivatives of BLA determined mainly from the CL

data, the region of BLA that is important for recognition of

GroEL was investigated.

Vol. 124, No. 2, 1998 319

HSPbindingRoles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones inPost-translational Translocation of Presecretory Proteins into theEndoplasmic Reticulum*

Received for publication, October 15, 2002Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210544200

Jantra Ngosuwan, Nancy M. Wang, Katie L. Fung, and William J. Chirico‡From the Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center,Brooklyn, New York 11203

Hsp70 molecular chaperones and their co-chaperoneswork together in various cellular compartments toguide the folding of proteins and to aid the translocationof proteins across membranes. Hsp70s stimulate proteinfolding by binding exposed hydrophobic sequencesthereby preventing irreversible aggregation. Hsp40sstimulate the ATPase activity of Hsp70s and target un-folded proteins to Hsp70s. Genetic and biochemical evi-dence supports a role for cytosolic Hsp70s and Hsp40s inthe post-translational translocation of precursor pro-teins into endoplasmic reticulum and mitochondria. Togain mechanistic insight, we measured the effects ofSaccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p(Hsp40) on the translocation of histidine-tagged prepro-!-factor (pp!F6H) into microsomes. Radiolabeledpp!F6H was affinity purified from wheat germ transla-tion reactions (or Escherichia coli) to remove endoge-nous chaperones. We demonstrated that either Ssa1p orYdj1p stimulates post-translational translocation bypreventing pp!F6H aggregation. The binding and/or hy-drolysis of ATP by Ssa1p were required to maintain thetranslocation competence of pp!F6H. To clarify the con-tributions of membrane-bound and cytosolic Ydj1p, wecompared the efficiency of chaperone-dependent trans-location into wild-type and Ydj1p-deficient microsomes.Neither soluble nor membrane-bound Ydj1p was essen-tial for post-translational protein translocation. Theability of Ssa1p, Ydj1p, or both chaperones to restore thetranslocation competence of aggregated pp!F6H wasnegligible.

Hsp701 molecular chaperones and their co-chaperones worktogether in a variety of cellular compartments to guide thefolding of proteins and to aid the translocation of proteinsacross membranes (reviewed in Ref. 1). The binding and hy-drolysis of ATP regulate the action of Hsp70s (2). In the ATP-

bound form, peptide substrates undergo cycles of rapid bindingand release from Hsp70 (3). In the ADP-bound form, the inter-actions are slower resulting in higher affinity. Hsp70s stimu-late protein folding by binding to exposed hydrophobic se-quences (4–6) and preventing their irreversible aggregation(7). The activity of Hsp70s is regulated by Hsp40s and otherco-chaperones. Hsp40s stimulate the ATPase activity ofHsp70s (8) and can target unfolded proteins to them (7).Hsp40s preferentially bind hydrophobic polypeptides (9) andcan prevent the aggregation of some unfolded proteins (7, 10).

Genetic and biochemical evidence supports a role for cytoso-lic Hsp70s and Hsp40s in the post-translational translocationof precursor proteins into the endoplasmic reticulum and mi-tochondria (reviewed in Ref. 11). Deshaies et al. (12) reportedthat presecretory proteins and mitochondrial precursors accu-mulated in the yeast cytosol when the concentration of cytosolicHsp70s was lowered. A temperature-sensitive mutant of theyeast cytosolic Hsp70 Ssa1p rapidly accumulated presecretoryproteins at the nonpermissive temperature (13). Chirico et al.(14) showed that yeast cytosolic Hsp70s stimulated the in vitrotranslocation of prepro-!-factor (pp!F) into yeast microsomes.Complexes containing Hsp70s and presecretory and mitochon-drial precursor proteins have been identified in wheat germextracts and reticulocyte lysates (15–17). The ability to stimu-late post-translational translocation is not shared by allHsp70s or by other stress protein families. Neither Hsp60 (18),Kar2p (19), nor Hsp90 (18) stimulates translocation. However,DnaK can partially substitute for yeast cytosolic Hsp70s invitro (19, 20).

Several laboratories have explored the role of DnaJ homologsin protein translocation. Caplan et al. (21) reported that atemperature-sensitive mutant of YDJ1 was defective for trans-location at the nonpermissive temperature. However, Atencioand Yaffe (22) and Becker et al. (13) showed that pp!F istranslocated normally in deletion mutants of YDJ1. Transloca-tion of pp!F was defective in a strain expressing a mutant formof Ydj1p that cannot be farnesylated indicating that the lipid-modified version of Ydj1p plays a role in protein translocation(21). Hendrick et al. (23) showed that Escherichia coli DnaJcompletely inhibited post-translational translocation of pp!Fin vitro. DnaK and GrpE together relieved the DnaJ-dependentinhibition, but neither alone had any effect. Export of certainprecursor proteins was defective in some dnaK and dnaJ mu-tant strains of E. coli (24).

Functional interactions between yeast Hsp70s and DnaJhomologs have been studied in vitro and in vivo (13, 25–27).Ydj1p stimulates the ATPase activity of Ssa1p (25) and to-gether they constitute a protein folding machinery capable ofrefolding denatured luciferase (26). Becker et al. (13) exploredthe in vivo interactions of SSA1 and YDJ1 in protein translo-

* This work was supported by National Science Foundation GrantMCB-9905988 and a grant from the American Heart Association (toW. J. C.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 718-270-1308;Fax: 718-270-3732; E-mail: william.chirico@downstate.edu.

1 The abbreviations used are: Hsp70, 70-kDa heat shock protein;AP-pp!F6H, affinity purified, histidine-tagged prepro-!-factor; H, hex-okinase; Hsc70, 70-kDa heat shock cognate protein; NEM, N-ethylma-leimide; NEM-Ssa1p, N-ethylmaleimide-modified Ssa1p; Nt, nondena-tured pp!F6H; PRS, postribosomal supernatant; p!F, pro-!-factor;p!F6H, histidine-tagged pro-!-factor; pp!F, prepro-!-factor; pp!F6H,histidine-tagged prepro-!-factor; S, Ssa1p; Y, Ydj1p; Ni2!-NTA, nickel-nitrilotriacetic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 9, Issue of February 28, pp. 7034–7042, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Roles of Cytosolic Hsp70 and Hsp40 Molecular Chaperones inPost-translational Translocation of Presecretory Proteins into theEndoplasmic Reticulum*

Received for publication, October 15, 2002Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M210544200

Jantra Ngosuwan, Nancy M. Wang, Katie L. Fung, and William J. Chirico‡From the Department of Anatomy and Cell Biology, State University of New York Downstate Medical Center,Brooklyn, New York 11203

Hsp70 molecular chaperones and their co-chaperoneswork together in various cellular compartments toguide the folding of proteins and to aid the translocationof proteins across membranes. Hsp70s stimulate proteinfolding by binding exposed hydrophobic sequencesthereby preventing irreversible aggregation. Hsp40sstimulate the ATPase activity of Hsp70s and target un-folded proteins to Hsp70s. Genetic and biochemical evi-dence supports a role for cytosolic Hsp70s and Hsp40s inthe post-translational translocation of precursor pro-teins into endoplasmic reticulum and mitochondria. Togain mechanistic insight, we measured the effects ofSaccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p(Hsp40) on the translocation of histidine-tagged prepro-!-factor (pp!F6H) into microsomes. Radiolabeledpp!F6H was affinity purified from wheat germ transla-tion reactions (or Escherichia coli) to remove endoge-nous chaperones. We demonstrated that either Ssa1p orYdj1p stimulates post-translational translocation bypreventing pp!F6H aggregation. The binding and/or hy-drolysis of ATP by Ssa1p were required to maintain thetranslocation competence of pp!F6H. To clarify the con-tributions of membrane-bound and cytosolic Ydj1p, wecompared the efficiency of chaperone-dependent trans-location into wild-type and Ydj1p-deficient microsomes.Neither soluble nor membrane-bound Ydj1p was essen-tial for post-translational protein translocation. Theability of Ssa1p, Ydj1p, or both chaperones to restore thetranslocation competence of aggregated pp!F6H wasnegligible.

Hsp701 molecular chaperones and their co-chaperones worktogether in a variety of cellular compartments to guide thefolding of proteins and to aid the translocation of proteinsacross membranes (reviewed in Ref. 1). The binding and hy-drolysis of ATP regulate the action of Hsp70s (2). In the ATP-

bound form, peptide substrates undergo cycles of rapid bindingand release from Hsp70 (3). In the ADP-bound form, the inter-actions are slower resulting in higher affinity. Hsp70s stimu-late protein folding by binding to exposed hydrophobic se-quences (4–6) and preventing their irreversible aggregation(7). The activity of Hsp70s is regulated by Hsp40s and otherco-chaperones. Hsp40s stimulate the ATPase activity ofHsp70s (8) and can target unfolded proteins to them (7).Hsp40s preferentially bind hydrophobic polypeptides (9) andcan prevent the aggregation of some unfolded proteins (7, 10).

Genetic and biochemical evidence supports a role for cytoso-lic Hsp70s and Hsp40s in the post-translational translocationof precursor proteins into the endoplasmic reticulum and mi-tochondria (reviewed in Ref. 11). Deshaies et al. (12) reportedthat presecretory proteins and mitochondrial precursors accu-mulated in the yeast cytosol when the concentration of cytosolicHsp70s was lowered. A temperature-sensitive mutant of theyeast cytosolic Hsp70 Ssa1p rapidly accumulated presecretoryproteins at the nonpermissive temperature (13). Chirico et al.(14) showed that yeast cytosolic Hsp70s stimulated the in vitrotranslocation of prepro-!-factor (pp!F) into yeast microsomes.Complexes containing Hsp70s and presecretory and mitochon-drial precursor proteins have been identified in wheat germextracts and reticulocyte lysates (15–17). The ability to stimu-late post-translational translocation is not shared by allHsp70s or by other stress protein families. Neither Hsp60 (18),Kar2p (19), nor Hsp90 (18) stimulates translocation. However,DnaK can partially substitute for yeast cytosolic Hsp70s invitro (19, 20).

Several laboratories have explored the role of DnaJ homologsin protein translocation. Caplan et al. (21) reported that atemperature-sensitive mutant of YDJ1 was defective for trans-location at the nonpermissive temperature. However, Atencioand Yaffe (22) and Becker et al. (13) showed that pp!F istranslocated normally in deletion mutants of YDJ1. Transloca-tion of pp!F was defective in a strain expressing a mutant formof Ydj1p that cannot be farnesylated indicating that the lipid-modified version of Ydj1p plays a role in protein translocation(21). Hendrick et al. (23) showed that Escherichia coli DnaJcompletely inhibited post-translational translocation of pp!Fin vitro. DnaK and GrpE together relieved the DnaJ-dependentinhibition, but neither alone had any effect. Export of certainprecursor proteins was defective in some dnaK and dnaJ mu-tant strains of E. coli (24).

Functional interactions between yeast Hsp70s and DnaJhomologs have been studied in vitro and in vivo (13, 25–27).Ydj1p stimulates the ATPase activity of Ssa1p (25) and to-gether they constitute a protein folding machinery capable ofrefolding denatured luciferase (26). Becker et al. (13) exploredthe in vivo interactions of SSA1 and YDJ1 in protein translo-

* This work was supported by National Science Foundation GrantMCB-9905988 and a grant from the American Heart Association (toW. J. C.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence should be addressed. Tel.: 718-270-1308;Fax: 718-270-3732; E-mail: william.chirico@downstate.edu.

1 The abbreviations used are: Hsp70, 70-kDa heat shock protein;AP-pp!F6H, affinity purified, histidine-tagged prepro-!-factor; H, hex-okinase; Hsc70, 70-kDa heat shock cognate protein; NEM, N-ethylma-leimide; NEM-Ssa1p, N-ethylmaleimide-modified Ssa1p; Nt, nondena-tured pp!F6H; PRS, postribosomal supernatant; p!F, pro-!-factor;p!F6H, histidine-tagged pro-!-factor; pp!F, prepro-!-factor; pp!F6H,histidine-tagged prepro-!-factor; S, Ssa1p; Y, Ydj1p; Ni2!-NTA, nickel-nitrilotriacetic acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 9, Issue of February 28, pp. 7034–7042, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org7034

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HSP70overexpression

Heat Shock Protein 70 Chaperone OverexpressionAmeliorates Phenotypes of the Spinal and Bulbar MuscularAtrophy Transgenic Mouse Model by Reducing Nuclear-Localized Mutant Androgen Receptor Protein

Hiroaki Adachi,1 Masahisa Katsuno,1 Makoto Minamiyama,1 Chen Sang,1 Gerassimos Pagoulatos,2

Charalampos Angelidis,2 Moriaki Kusakabe,3 Atsushi Yoshiki,4 Yasushi Kobayashi,1 Manabu Doyu,1 and Gen Sobue1

1Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya 466-8550, Japan, 2Department of GeneralBiology, University of Ioannina, School of Medicine, Ioannina GR-45110, Greece, 3ANB Tsukuba Institute, ALOKA Company, Ltd., 1103 Fukaya,Kasumigaura, Niihari, Ibaraki 300-0134, Japan, and 4Experimental Animal Division, Department of Biological Systems, BioResource Center, The Instituteof Physical and Chemical Research (RIKEN) Tsukuba Institute 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ)tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine withmany components of ubiquitin–proteasome and molecular chaperone pathways, raising the possibility that misfolding and altereddegradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP)chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whetherincreasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with miceoverexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motorfunction of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the largecomplex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting theenhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice byreducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for thedevelopment of an HSP70-related therapy for SBMA and other polyQ diseases.

Key words: HSP70; chaperone; polyglutamine; SBMA; transgenic mice; protein degradation

IntroductionPolyglutamine (polyQ) diseases are inherited neurodegenerativedisorders caused by the expansion of a trinucleotide CAG repeatin the causative genes (Zoghbi and Orr, 2000). To date, ninepolyQ diseases have been identified (Ross, 2002). Spinal and bul-bar muscular atrophy (SBMA) is a polyQ disease, characterizedby proximal muscle atrophy, weakness, contraction fascicula-tions, and bulbar involvement (Kennedy et al., 1968; Sobue et al.,1989; Takahashi, 2001). In SBMA, a polymorphic CAG repeatwith 14 –32 CAGs expands to 40 – 62 CAGs in the first exon of theandrogen receptor (AR) gene (Tanaka et al., 1996) and has so-matic mosaicism (Tanaka et al., 1999). There is an inverse corre-lation between the CAG repeat size and the age at onset or thedisease severity in SBMA (Doyu et al., 1992; Igarashi et al., 1992;La Spada et al., 1992). In SBMA, nuclear inclusions (NIs) con-taining mutant AR have been observed in the brainstem motor

nuclei, spinal motor neurons, and some visceral organs (Li et al.,1998a,b). Such neuronal inclusions are common pathologicalfeatures in polyQ diseases and are also colocalized with manycomponents of ubiquitin–proteasome and molecular chaperones(Chai et al., 1999; Huynh et al., 2000; Adachi et al., 2001; Zanderet al., 2001; Schmidt et al., 2002), raising the possibility that mis-folding and altered degradation of the mutant protein may beinvolved in the pathogenesis of SBMA as well as other polyQdiseases (Stenoien et al., 1999; Waelter et al., 2001). Furthermore,these chaperones and proteasomes would facilitate refolding orproteolysis of the mutant protein and may play a role in protect-ing neuronal cells against the toxic properties of the expandedpolyQ (Cummings et al., 1998; Kobayashi et al., 2000). We haveshown recently that overexpression of heat shock proteins(HSPs) decreases the aggregate formation of truncated AR withthe expanded polyQ and markedly prevents cell death in the neu-ronal cell model of SBMA (Kobayashi et al., 2000; Kobayashi andSobue, 2001). HSP70 overexpression has been reported to en-hance the solubility and degradation of mutant AR (Bailey et al.,2002). HSPs have also been shown to suppress aggregate forma-tion and cellular toxicity in a wide range of polyQ disease models(Cummings et al., 1998; Warrick et al., 1999; Carmichael et al.,2000). Recently, overexpression of the inducible form of rat

Received Nov. 5, 2002; revised Dec. 31, 2002; accepted Dec. 31, 2002.This work was supported by a Center of Excellence grant from the Ministry of Education, Culture, Sports, Science,

and Technology of Japan and by grants from the Ministry of Health, Labor, and Welfare of Japan. We thank SugikoYokoi for her technical assistance.

Correspondence should be addressed to Dr. Gen Sobue, Department of Neurology, Nagoya University GraduateSchool of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya, 466-8550, Japan. E-mail: sobueg@med.nagoya-u.ac.jp.Copyright © 2003 Society for Neuroscience 0270-6474/03/232203-09$15.00/0

The Journal of Neuroscience, March 15, 2003 • 23(6):2203–2211 • 2203

Heat Shock Protein 70 Chaperone OverexpressionAmeliorates Phenotypes of the Spinal and Bulbar MuscularAtrophy Transgenic Mouse Model by Reducing Nuclear-Localized Mutant Androgen Receptor Protein

Hiroaki Adachi,1 Masahisa Katsuno,1 Makoto Minamiyama,1 Chen Sang,1 Gerassimos Pagoulatos,2

Charalampos Angelidis,2 Moriaki Kusakabe,3 Atsushi Yoshiki,4 Yasushi Kobayashi,1 Manabu Doyu,1 and Gen Sobue1

1Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya 466-8550, Japan, 2Department of GeneralBiology, University of Ioannina, School of Medicine, Ioannina GR-45110, Greece, 3ANB Tsukuba Institute, ALOKA Company, Ltd., 1103 Fukaya,Kasumigaura, Niihari, Ibaraki 300-0134, Japan, and 4Experimental Animal Division, Department of Biological Systems, BioResource Center, The Instituteof Physical and Chemical Research (RIKEN) Tsukuba Institute 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of the polyglutamine (polyQ)tract within the androgen receptor (AR). The nuclear inclusions consisting of the mutant AR protein are characteristic and combine withmany components of ubiquitin–proteasome and molecular chaperone pathways, raising the possibility that misfolding and altereddegradation of mutant AR may be involved in the pathogenesis. We have reported that the overexpression of heat shock protein (HSP)chaperones reduces mutant AR aggregation and cell death in a neuronal cell model (Kobayashi et al., 2000). To determine whetherincreasing the expression level of chaperone improves the phenotype in a mouse model, we cross-bred SBMA transgenic mice with miceoverexpressing the inducible form of human HSP70. We demonstrated that high expression of HSP70 markedly ameliorated the motorfunction of the SBMA model mice. In double-transgenic mice, the nuclear-localized mutant AR protein, particularly that of the largecomplex form, was significantly reduced. Monomeric mutant AR was also reduced in amount by HSP70 overexpression, suggesting theenhanced degradation of mutant AR. These findings suggest that HSP70 overexpression ameliorates SBMA phenotypes in mice byreducing nuclear-localized mutant AR, probably caused by enhanced mutant AR degradation. Our study may provide the basis for thedevelopment of an HSP70-related therapy for SBMA and other polyQ diseases.

Key words: HSP70; chaperone; polyglutamine; SBMA; transgenic mice; protein degradation

IntroductionPolyglutamine (polyQ) diseases are inherited neurodegenerativedisorders caused by the expansion of a trinucleotide CAG repeatin the causative genes (Zoghbi and Orr, 2000). To date, ninepolyQ diseases have been identified (Ross, 2002). Spinal and bul-bar muscular atrophy (SBMA) is a polyQ disease, characterizedby proximal muscle atrophy, weakness, contraction fascicula-tions, and bulbar involvement (Kennedy et al., 1968; Sobue et al.,1989; Takahashi, 2001). In SBMA, a polymorphic CAG repeatwith 14 –32 CAGs expands to 40 – 62 CAGs in the first exon of theandrogen receptor (AR) gene (Tanaka et al., 1996) and has so-matic mosaicism (Tanaka et al., 1999). There is an inverse corre-lation between the CAG repeat size and the age at onset or thedisease severity in SBMA (Doyu et al., 1992; Igarashi et al., 1992;La Spada et al., 1992). In SBMA, nuclear inclusions (NIs) con-taining mutant AR have been observed in the brainstem motor

nuclei, spinal motor neurons, and some visceral organs (Li et al.,1998a,b). Such neuronal inclusions are common pathologicalfeatures in polyQ diseases and are also colocalized with manycomponents of ubiquitin–proteasome and molecular chaperones(Chai et al., 1999; Huynh et al., 2000; Adachi et al., 2001; Zanderet al., 2001; Schmidt et al., 2002), raising the possibility that mis-folding and altered degradation of the mutant protein may beinvolved in the pathogenesis of SBMA as well as other polyQdiseases (Stenoien et al., 1999; Waelter et al., 2001). Furthermore,these chaperones and proteasomes would facilitate refolding orproteolysis of the mutant protein and may play a role in protect-ing neuronal cells against the toxic properties of the expandedpolyQ (Cummings et al., 1998; Kobayashi et al., 2000). We haveshown recently that overexpression of heat shock proteins(HSPs) decreases the aggregate formation of truncated AR withthe expanded polyQ and markedly prevents cell death in the neu-ronal cell model of SBMA (Kobayashi et al., 2000; Kobayashi andSobue, 2001). HSP70 overexpression has been reported to en-hance the solubility and degradation of mutant AR (Bailey et al.,2002). HSPs have also been shown to suppress aggregate forma-tion and cellular toxicity in a wide range of polyQ disease models(Cummings et al., 1998; Warrick et al., 1999; Carmichael et al.,2000). Recently, overexpression of the inducible form of rat

Received Nov. 5, 2002; revised Dec. 31, 2002; accepted Dec. 31, 2002.This work was supported by a Center of Excellence grant from the Ministry of Education, Culture, Sports, Science,

and Technology of Japan and by grants from the Ministry of Health, Labor, and Welfare of Japan. We thank SugikoYokoi for her technical assistance.

Correspondence should be addressed to Dr. Gen Sobue, Department of Neurology, Nagoya University GraduateSchool of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya, 466-8550, Japan. E-mail: sobueg@med.nagoya-u.ac.jp.Copyright © 2003 Society for Neuroscience 0270-6474/03/232203-09$15.00/0

The Journal of Neuroscience, March 15, 2003 • 23(6):2203–2211 • 2203

HumanHSP70expressioninmouse

genic mice, whereas testosterone administration enhanced thenuclear localization of mutant AR and caused significant motordysfunction in the female transgenic mice (Katsuno et al., 2002).In particular, the large aggregated complexes of the mutant ARprotein detected in the stacking gel or slowly migrating species inthe Western blot analysis in the nuclear fraction were well corre-lated with the phenotypic expression in this mouse model (Kat-suno et al., 2002). This suggested that oligomeric or polymericmutant AR large complex molecules positively associated withother molecules would exert the toxicity rather than monomericmutant AR (Katsuno et al., 2002).

In the present study, we demonstrated that the amount ofnuclear-localized mutant AR protein, particularly that of thelarge complex form present in the stacking gel or trapped by thecellulose acetate membrane, was significantly reduced in the AR-97/HSP70 double-transgenic mice. Thus, the overexpression ofHSP70 is suggested to exert its amelioration of the phenotypicexpression by diminishing the amount of nuclear-localized mu-tant AR protein. However, in the previously reported SCA1transgenic mouse model, NIs of the mutant protein were notapparently decreased in the double-transgenic mice with ratHSP70 overexpression, although the neurological deficit andneuronal degeneration were ameliorated (Cummings et al.,2001). Because the gain of amelioration for phenotypic expres-sion in the model mice of Cummings et al. (2001) was mild evenin the double-transgenics with HSP70 homozygotes, the changein the frequency of the NIs would not have been significantenough to detect. In our mouse model, NIs were present only inthe small subpopulation of neurons and muscles, particularly inthe early phase of phenotypic expression, whereas the 1C2-positive nuclei were abundant (Katsuno et al., 2002). In addition,1C2-positive neurons are more extensive than those of NI-bearing neurons in the tissues of the autopsied samples frompatients with polyQ diseases, and the distribution of 1C2-positiveneurons is well correlated with the neurological symptoms(Yamada et al., 2001). These observations suggest that 1C2 stain-ing is a more sensitive histological marker for the detection of the

nuclear localization of the mutant protein with an expandedpolyQ stretch compared with NIs detected by antibodies for theresponsible protein.

The interesting observation in our study was the diminutionof monomeric mutant AR in the double-transgenic mice withoverexpression of HSP70. Recently, HSP70 overexpression in thecell culture model has revealed enhanced solubility of mutant ARwith an expanded polyQ and degradation through the ubiquitin–proteasome system (Bailey et al., 2002). Overexpression of chap-erones generally enhances the function of the ubiquitin–protea-some pathway and subsequently accelerates protein degradation(Bukau and Horwich, 1998). The ubiquitin–proteasome path-way, particularly its activity, is known to be related to chaperoneexpression levels (Bukau and Horwich, 1998). The molecularmechanism for this relationship remains unsolved, but recentlyCHIP (C terminal of HSC70-interacting protein), U-box-type E3ubiquitin ligase, has been shown to interact with HSP90 orHSP70 (Connel et al., 2001) and ubiquitylate unfolded proteinstrapped by molecular chaperones and degrades them, thus actingas a “quality control E3” (Murata et al., 2001). Furthermore, thereis a cofactor of HSC70/HSP70, Bcl-2 associated athanogene 1,which possesses a ubiquitin-like domain and promotes bindingof HSC70/HSP70 to the proteolytic complex (Luders et al., 2000).Although such coupling factors between the HSP70 chaperonesystem and the protein degradation machinery for mutant AR areunknown at present, if the similar E3 for mutant AR is present, itcould ubiquitylate and degrade mutant AR as a result of interact-ing with HSP70. In this scenario, the overexpression of HSP70may accelerate E3-dependent capture of mutant AR and its deg-radation through the proteasome pathway. A remarkable reduc-tion of the monomeric mutant AR in the double-transgenics withHSP70 overexpression can be the reflection of the accelerateddegradation of mutant AR through the HSP70-mediated E3–proteasome system. Interaction between mutant AR and HSP70detected by coimmunoprecipitation and Western blot analysis inthe double-transgenic mice would support this view. The over-expression of HSP70 could enhance the degradation of the mo-

Figure 4. HSP70 decreases nuclear-localized mutant AR in double-transgenic mice. Immunohistochemical study of the spinal anterior horn ( A–C) and muscle ( D–F) of AR-97Q/HSP70 (!/!) andAR-97Q/HSP70 double-transgenic mice stained with a monoclonal antibody (1C2) against abnormally expanded polyQ (16 weeks old). AR-97Q/HSP70 (!/!) mice have intense and frequent stainingfor 1C2 in the nucleus (A, D). AR-97Q/HSP70 (tg/!) (B, E), and AR-97Q/HSP70 (tg/tg) (C, F ) mice exhibit low levels of 1C2 staining in the nucleus. G, H, Quantitative assessment of diffuse nuclear stainingfor 1C2 in the spinal ventral horn ( G) and muscle ( H ). Positively stained nuclei were estimated by counting in the thoracic spinal ventral horn and muscle using six transgenic mice (16 weeks of age).There are significantly more 1C2-positive cells in AR-97Q/HSP70 (!/!) mice than in AR-97Q/HSP70 (tg/!) mice or AR-97Q/HSP70 (tg/tg) mice in both tissues. Results are expressed as means " SD forsix mice. The differences in 1C2-positive cell populations are not statistically significant between AR-97Q/HSP70 (tg/!) and AR-97Q/HSP70 (tg/tg) mice. *p # 0.05; **p # 0.01; ***p # 0.001.

2208 • J. Neurosci., March 15, 2003 • 23(6):2203–2211 Adachi et al. • HSP70 Overexpression Ameliorates SBMA Phenotypes