ARMENISE-HARVARDSYMPOSIUM1999
ARMENISETHIRDSYMPOSIUM
3rdAnnualSymposium
June28-July1,1999,CastelvecchioPascoli,Lucca,Italy
AbouttheSymposium
WhentheArmenise-HarvardFoundationconvenedits3rdAnnualSymposiumatIlCiocco,a
conferencecenterhighintheAlpiApuane,breathtakingviewsoftawnymountainsandlushvalleys
remindedparticipatingscientiststhattheirworkunlocks""theimpressivebeautyofthe
biosphere,""inthewordsofFoundationPresidentandCEODanielC.Tosteson,formerdeanofthe
HarvardMedicalSchool.
Inhisopeningremarkstomorethan100researcherswhogatheredfortheSymposium,HMSDean
JosephMartinnotedthatithasbeenonlythreeyearssincetheFoundationbeganstimulating
collaborationamongHarvard'ssixbasicsciencedepartmentsandbetweenscientificcentersinItaly
andHMS.Inhisview,theFoundationhassucceededonbothfronts.Today,itsgenerositysupports
notonlytheannualSymposiumbutalsoabroadrangeofjointventures,includingexchangesof
technologyandpersonnelandanincreasingnumberofsmallseminars.Armenise-sponsored
researchisconductedatfiveItalianinstitutions:theEuropeanInstituteofOncologyinMilano,the
UniversityofPadova,theInstituteforCancerResearchandTreatmentattheUniversityofTorino
SchoolofMedicine,theDipartmentodiRicercaBiologicaeTecnologica(DIBIT)atScientific
InstituteSanRaffaeleinMilano,andUniversita'DiRomaLaSapienza.AtHarvard,thefour
Armenisecentersarestructuralbiology,neurobiology,cellsignaltransduction,andhumancancer
viruses.RepresentativesfromalltheArmeniseprogramsparticipatedinthe3rdAnnual
Symposium.
ImpressedwiththeproductivityoftheItaliancenters,theFoundationrecentlyinvitedallofthemto
applyforatwo-yearextensionoftheiroriginalaward.DeanMartinannounced,duringhisopening
remarks,thatonthepreviousdaytheFoundation'sboardvotedtoextendfundingforallfive.""This
isverygratifyingfortheItalianpartoftheenterprise,""Dr.JacopoMeldolesiofDIBITsaidin
responsetoDeanMartin'snews.TheArmenisefundingbringsnotonlymoney,hesaid,butalso
scientificchallengeandtheopportunitytodevelopmoremature,ongoingrelationshipswith
collaborators.
Aconsiderableexchangeofhumantalentisalreadyunderway.Forexample,DeanMartincitedthe
recentmoveofArmenisefellowAndreaMusacchiofromHMStotheEuropeanInstituteof
Oncology,wherehewillleadanewstructuralbiologydepartment.InDr.DanielaPietrobon's
closingremarks,thePadovaresearcherreminiscedaboutthreeyearsshespentatHMSearlyinher
careerandcreditedtheFoundationwithmakingitpossiblefortwoyoungscientistsfromhergroup
tofollowinherfootsteps.TheArmenisegranthasbeeninvaluableforthesupportofyoung
investigatorsandthestrengtheningoflaboratoryinfrastructure,shesaid,andotherscientists
agreed.
Thestrengthofthe3rdAnnualSymposium'sscientificprogramisonemeasureoftheFoundation's
successinfulfillingitsmission.Theeventdrew112basicscientists,slightlymorethantwo-thirdsof
themfromItaly.Thisyeartherewere46posterpresentationsand20formallecturesgroupedinto
fivesessions:
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NeurobiologyI
BiomedicalResearch
PlantDefense/Pathogenesis
NeurobiologyII
Proteolysis/Apoptosis/CellCycle
Session1:NeurobiologyI</strong>
<strong>Overview</strong>
SomanyArmenise-sponsoredinvestigatorsareengagedinexcitingneurobiologyresearchthatthis
year'sSymposiumdevotedtwofullsessionstothisvitalandfast-movingfield.Theconference's
openingsessionintroducedfourissuesthatarefundamentalforunderstandingneuronalandbrain
function,accordingtoDr.JacopoMeldolesi,whomoderatedtheprogram.Asdistinctastheseareas
ofresearchare,allareinfast-movingareaswhereevenmoreinterestingnewscanbeexpectedin
thenearfuture.
Thefirstpresentationexploreshowthehumannervoussystemtransducessmellfromthebinding
ofodorantstospecialreceptorsinthenosetothetransmissionofinformationtothebrain.The
secondspeakerhomedinonalargefamilyofreceptors,theSEX/plexins,thatinmammalsappear
toplayessentialrolesinthedevelopmentofnervetissue.Thefocusshiftedtothecellmembrane
forthethirdtalk,whichintroducedanewprocessofmembraneregulationinneuronsthatmolds
theexcitabilityofthosecells.Thefinalpaperdealtwithafascinatingtranscriptionfactorthat
appearstoexertitselfduringembryogenesisbysuppressingneuronalfatedetermination.
<strong>Presentations</strong>
<em>Deconstructingsmell
</em>LindaBuck,AssociateProfessor
DepartmentofNeurobiology,HowardHughesMedicalInstitute,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">LindaBuck</a>
Smellshaveshapes.Butinsteadofhavingonereceptordedicatedtosmellingbananasandanother
topinetrees,Dr.Buck'sresearchdemonstratesthatindividualodorantreceptors(ORs)recognize
specificpartsofanodorant'sstructure,ratherthanthewholemolecule.Theolfactorysystem
combinesdatafromnumerousORstodeterminewhatit'ssmelling,whichexplainshowhumans
andothermammalscandiscriminateanenormousvarietyofodorsaswellaspheromones
(chemicalsthatelicitsexualorotherinnatebehaviors).Thiscombinatorialcodingschemerelieson
about1,000ORsexpressedbythemillionsofsensoryneuronsintheolfactoryepitheliumliningthe
nose.EachneuronexpressesoneORgeneandneuronsexpressingthesameORarescattered
throughoutoneoffournasalzones,anarrangementthatdecentralizesinformationprocessing.
Whenavolatilechemicalentersthenose,differentpartsofitsstructurearerecognizedbyscattered
neuronsequippedwithvariousORs.Axonsfromtheseneuronstraveltotheolfactorybulb,where
inputfromneuronswiththesameOR-nomatterwheretheymaybelocatedinthenasal
epithelium-ischanneledintospecificglomeruliwithfixedlocations,muchasscatteredlightis
focusedbyalens.
Thevomeronasalorgan,wherethesubconsciousdetectionofpheromonesbegins,isequippedwith
twodistinctfamiliesofcandidatepheromonereceptors,oneofwhichwasfirstidentifiedbyDr.
Buck'slaboratory.InputsfromthesereceptorsappeartobeprocessedseparatelyfromORs,
ensuringthatsignalsforprimalbehaviorssuchasmatingandaggressionwon'tbeconfusedwith
commonplacesmells.
Inrecentexperiments,Dr.Buckandhercolleagueshavecombinedcalciumimagingandsinglecell
RT-PCR(atechniqueforamplifyingpartsofthegenomethatencodeproteins)toidentifyORsfor
odorantsthatpeopleperceivequitedifferentlyeventhoughtheyarestructurallysimilar.The
combinationofreceptorsthatcodesforafloralaroma,theyfound,mayoverlapsubstantiallywith
thecombinationthatpicksuparancidsmell-butthetwowillbechanneledtodifferentglomeruliin
theolfactorybulb.Theseobservationsofsinglecellssupportthreeimportantconclusions:asingle
ORrecognizesmultipleodorants,asingleodorantisrecognizedbydifferentORs,andmost
odorantscanbedetectedbydifferentcombinationsofORs.
<em>ThehumanSex/plexingenefamilyencodessurfacereceptorsforsemaphorinsandcontrols
cellrepellingcues
</em>LucaTamagnone
InstituteforCancerResearch,UniversityofTorinoMedicalSchool
Email:<ahref=""mailto:[email protected]"">LucaTamagnone</a>
Severalyearsago,whenDr.Tamagnonediscoveredanunusualfamilyofreceptortyrosinekinase
genes,henamedtheprototypeSEXbecauseitwaslocatedontheXchromosome.Additional
researchhasexpandedtheuniverseofhumanSEX/plexinstoincludeninemembersgroupedin
fourfamilies.Thesegeneswerefirstidentifiedinthenervoussystem,andtheycontainhighly
conservedsequencesthatarethesameinnematodesasinhumans.TheSEX/plexinsencodecell
surfaceproteinshomologoustotheextracellulardomainofthereceptortyrosinekinasethatbinds
hepatocytegrowthfactor(HGF),akeygrowthfactorinembryonicdevelopment.Earlyon,
researchersdeterminedthatSEX/plexinsappeartoinfluencethemigrationofaxonprocesses
towardsynapseformation.Nowotherrolesarecomingtolight.
Dr.Tamagnone'steamhasobservedthattheextracellulardomainsofSEX/plexins(thepartsthat
protrudefromthecellsurface)bearastrongresemblancetosemaphorins,alargefamilyofsoluble
andmembrane-boundligands.InCell,theydescribedaninteractionbetweenplexin-Aand
semaphorin-1inDrosophila;sincethatreporttheyhaveidentifiedtwosemaphorinsthatbindto
humanSEX/plexinreceptors.ThisisthefirsttimethatligandsfortheSEX/plexinshavebeen
identified.
Morerecently,Dr.Tamagnone'slaboratoryhasexploredthepossibilitythattheplexinsare
importantincell-to-cellsignalinginepithelialandendothelialcells,inadditiontotheirestablished
roleinneuronaldevelopment.Whentheyusedsemaphorinproteinasaprobe,theresearchers
foundthatitboundtoplexinsthatinteractwithneuropilinsincellsignaling.Futureexperiments
willexplorewhethersemaphorin-bindingplexinsmayalsocollaboratewithneuropilinsinsignal
transduction,actingviasomenovelcytoplasmic(insidethecell)structure.
<em>Activechloridecurrentsmoldtheelectricalpropertiesofthe""resting""sympatheticneuron
</em>RiccardoFesce
DIBIT-InstituteSanRaffaele
Email:<ahref=""mailto:[email protected]"">RiccardoFesce</a>
There'smoretoarestingneuronthanmeetstheeye.Althoughtheconventionalviewisthat
nothingmuchgoesonwhenthemembranepotentialisbelow-60mV,Dr.FesceandOscarSacchi,a
biologistattheUniversityofFerrara,havedetectedaseriesofactiveconductancesinthe-60to80mVrange.Theynowsaythatinteractionsbetweenfluctuatingcurrentsofpotassiumand
chloridemoldtheexcitabilityofthecellandhelpdeterminehowmuchsynapticinputisrequiredto
triggeranactionpotential.
Workingwiththeisolated,intactsuperiorcervicalganglionofrats,Dr.Sacchiusedatwo-electrode,
voltage-clampsystemtocharacterizetheconductancesandelectricalpropertiesofthesecells.Dr.
Fesceusedtheseexperimentalmeasurementstobuildacomputationalmodelofacomplete
sympatheticneuron,whichrevealedsurprisinginteractionsbetweenapotassiumcurrentthatacts
justabovethe-60mVthresholdandasubthreshholdchloridecurrent.Apotassiumcurrentcalled
IAisactivatedatabout-60mVandbecomesfullyactivatedatabout-30/-40mV,causingtransient
depolarizationsthatrapidlyrelaxtotheinitialvalue.Duringtheupstrokeofexcitatorypotential,
Dr.Fescesaidthatthispotassiumcurrentapparentlygivesrisetoacountercurrentofchloride
ions,whichpushtowardrepolarization.Allthishappensinaneuronthatappearstobe""resting,""
withtheresultthatahigherlevelofsynapticinputwillberequiredtotriggeranactionpotential.
Whenaneuronisatrest,thereissupposedlynonetflowofionsacrosstheplasmamembrane.
Nevertheless,Dr.Fescefoundremarkablechloridecurrentsthatoperatebelow-60mV.These
currentsrespondtotransientchangesinmembranepotentialandreturntoasteadystateafter
hundredsofseconds.Chlorideconductanceincreaseswithdepolarization,andat-60/-80mVthe
chlorideequilibriumpotentialsustainsasmallinwardcurrent.Whateverhappenstomembrane
restingpotentialsparkschangesinchlorideredistributionandconductances,Dr.Fescesaid,which
challengesthenotionthatmembranepotentialreflectsasimplebalancebetweensodiumand
potassium.
<em>Transcriptionfactorsincellfatedeterminationanddifferentiation
</em>YangShi,AssociateProfessor
DepartmentofPathology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">YangShi</a>
Scientistshaveknownforyearsthatregulationoftissue-specificgenesduringembryonic
developmentiscontrolledprimarilyattheleveloftranscription.Dr.Shi'sexperimentsdemonstrate
thatin<em>Caenorhabditiselegans</em>,certainenzymesareinvolvedbothinnormal
transcriptionandinabnormalcellgrowth.NaturehasmanystrategiesforreleasingDNAfromthe
compactchromatinpackagingthatenablesittofitintothecellnucleus.Onesuchmethodisthe
attachmentofanacetylgrouptohistones,whichbindthechromatinpackage.Acetylationis
determinedbygive-and-takebetweenhistoneacetyltransferases(HATs),suchasmammalianCBP
andp300,andhistonedeacetylases(HDAs).Earlierworkrevealedthattheadenovirusoncoprotein
E1AcannotinduceimmaturecellstodivideendlesslysolongasCBP/p300arefunctioning,
suggestingthattheseproteinsareneededfornormalcellgrowthanddifferentiation.Morerecently,
Dr.Shiexpandedonthisideabymanipulatingcbp-1,thegeneencodingCBP-1,the<em>C.
elegans</em>proteinthatcorrespondstoCBP/p300.
<em>C.elegans</em>maybeasimpleorganismwithlessthan1,000cells,""butitdoesallthe
thingsthatwedo,prettymuch,""Dr.Shisaidofhisfavoritemodel.Manyofits20,000proteincodinggenesarehomogolouswithhumangenes.Asinhumans,cellfateisdeterminedinpartby
transcriptionfactors.WhentheresearchersusedanewtechniquecalledRNA-mediated
Interference(RNAI)toinhibitexpressionofCBP-1,theysawundifferentiatedembryoswithnosign
ofnormalmorphology.Atthestagewhengutandmuscletissueareapparent,nosuch
differentiationhadoccurred.Asexpected,<em>C.elegans</em>genesthatcorrespondto
mammalianhistonedeacetylaseappeartorepresssomaticdifferentiation.Inakindoftugofwar,
CPB-1appearstopromoteendodermdifferentiationbyantagonizingtherepressiveeffectsofHDA.
TheseexperimentsarethefirsttoshowhowahomologofthehumanproteinsCPBandp300
functionsinaliveanimal.Theseresultsalsoprovidecritical<em>invivo</em>evidencethatthe
histoneacetylaseactivityofCBP-1maybeimportantforitsbiologicalactivity.Inadditionto
confirmingDr.Shi'shypothesis,theseexperimentsyieldedanunexpectedresultaswell.When
monoclonalantibodieswereusedtostudytheseeminglyundifferentiatedcellsinwormswithout
CPB-1activity,thosecellsappearedtobeneuronsÐsuggestingthatneuronaldifferentiationmay
beakindofdefaultsettingfor<em>C.elegans</em>cellsintheabsenceofCBP-1.Similar
observationshavebeenmaderecentlyin<em>Xenopuslaevis</em>,suggestingthatHATsmaybe
ahighlyconserved,essentialplayerinthedifferentiationofnon-neuraltissue.
<strong>Session2:BiomedicalResearch</strong>
<strong>Overview</strong>
AschairmanoftheFoundation'sScientificAdvisoryCommittee,oneofDr.PeterHowley's
responsibilitiesistoshapetheprogramfortheannualsymposium.Asheorganizedindividual
abstractstocreatethisyear'ssessions,Dr.Howleydiscoveredthatsomeofthecutting-edge
investigationssponsoredbytheFoundationaren'teasytocategorize.Hegatheredfourofthem
togetherforthissession,whichhedubbedBiomedicalResearch.Theopeningpresentationdealt
withviralvectorsforgenetherapy,followedbyatouroftheenzymaticassemblylinesthat
microbesusetogrowantibiotics.Thethirdspeakerintroducedthefirstmammalianmutationgene
thatappearstoincreasestressresistanceandextendlifespan,andthefourthtookhislisteners
insidea""chamberofdoom""whereproteinsaredestroyed.Asdisparateasthesetopicsappear,
Dr.Howleysaidthat""theyhaveasimilartheme:allareinareasofresearchthatinterestme.""
<strong>Presentations</strong>
<em>Newgenerationsoflentiviralvectorsforexperimentalandhumangenetransfer
</em>LuigiNaldini
LaboratoryforGeneTransfer&amp;Therapy,InstituteforCancerResearch,UniversityofTorino
MedicalSchool
Email:<ahref=""mailto:[email protected]"">LuigiNaldini</a>
Penetratingcellsisado-or-diepropositionforviruses,whichcan'treplicateuntilthey'vegotten
insidehostcells.Researcherswhoaremindfulofthisspecialviralskillareseekingtousethemas
vectorsforgenetherapyorvaccines.Manyviruses,however,aren'twellsuitedtothistaskbecause
theycanonlyenterdividingcells,theydon'tpenetrateverymanycells,ortheydon'texpressthe
transfectedgeneathighenoughlevels.Togetaroundtheselimitations,Dr.Naldiniandhis
colleagueshavedesignedhybridlentiviralvectorscapableoftransferringandexpressinggenesin
severalrodenttissuesinvivo,andinprimitivehumanhematopoieticstemcells<em>exvivo</em>.
TheyhaveaccomplishedthisbycombiningcoreelementsofHIV-1,thepathogenthatcausesAIDS,
withtheenvelopeofalessharmfullentiviruscalledvesicularstomatitisvirus(VSV).Thesafety
profileforthesevectorshasimprovedastheresearcherscutbackontheamountofHIVgenetic
materialtheyuse,they'veincreasedtransgeneexpressionthroughselectiveuseofHIVltr(long
terminalrepeat)andpackagingsignals,andVSVelementspermitentryintoavarietyofcelltypes.
Dr.Naldini'slatestandsafestvectorsinactivateupontransductionandincludeonlyaminimalsetof
HIVgenes.
Inearlierexperiments,hisgroupdemonstratedefficientdeliveryandsustainedexpressionof
markergenesbythesevectors,both<em>invitro</em>withhumandonorlymphocytesand
<em>invivo</em>wheninjectedintothebrainsofadultrats.Morerecently,Dr.Naldinihasbeen
workingwithamousemodelformetachromaticleukodystrophy(MLD),aninheritedliposomal
storagedisorderthatininfantscausesrapid,dramaticdeathaslipidsaccumulateinthecentral
nervoussystemandothermajororgans.Histeamdesignedahybridlentivirusvectorcarryingthe
geneforASA,theenzymethatislackinginthiscondition,whichtheyaretestingintwoways.When
thevectorisinjecteddirectlyintothebrainsofMLDmice,thereispreliminaryevidencethatthe
transgeneappearstoexpresswellandinastablefashion.Asecondsetofexperimentsinvolves
<em>exvivo</em>reconstitutionofhematopoeticstemcellswiththetransgene,whichthe
researchersspeculatewillrepopulateandreplacethemissingenzymeinanimals.Earlyresults
appearpromising,andadditionalresearchisunderway.
<em>Assemblylineenzymology:thebiosynthesisofpolyketideandnonribosomalpeptide
antibiotics
</em>ChristopherT.Walsh,Professor
DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">LucaTamagnone</a>
Becausetheyliveinabug-eat-bugworld,fungusesandothermicrobeshaveevolvedthousandsof
antibioticsthattheyusetodisabletheirenemiesandcompetitors.Becausemanyofthese
substancesdisruptonlythefunctionsofprocaryoticcells,leavingeucaryoticcellsalone,doctorsuse
themasinvaluableweaponsagainstmicrobesthatcausehumandisease.Allaremadebyaprocess
thatDr.Walshcalls""assemblylineenzymology.""Hislaboratoryseekstounderstandthe
molecularlogicofantibioticassemblyand""theflipside,bywhichbacteriaproducenonribosomal
peptidevirulencefactors,whichallowthemtoinfectyouandme.""Ultimately,thegoalistouse
combinatorialelaborationtoassemblenewantibiotics,blockvirulencefactors,andfightgrowing
problemsofantibioticresistance.
Atagestaltlevel,polyketideantibioticssuchaserythromycinandtetracyclineandpeptideslike
penicillinsandvancomycinlooknothingalike.Yetallaretemplatednaturalproductswhere
assemblyinstructionscomefromthedomainorderofgiantmegasynthases.Thesemoleculeshave
""waystations,""modulesthatenzymes,suchaspolyketideornonribosomalpeptidesynthetases,
usetoinitiate,elongate,andterminatethenaturalproductchains.Atsomeofthesewaystations,
Dr.Walshandhiscolleagueshavebeenabletosubstituteonemoduleforanother,an
accomplishmentthatcouldleadthewaytocombinatorialbiosynthesisofnewantibiotics.If
investigatorscanbuildapartslistfortheantibioticassemblyline,Dr.Walshpredictsthatitwillbe
possibletoswapmodulesaroundandcreateantibioticsthathavenotyetbeenmadeinnature,but
whichmightbepowerfulweaponsagainstresistantbacteria.
<em>Thep66<sup>shc</sup>adaptorproteincontrolsoxidativestressresponseandlifespanin
mammals
</em>EnricaMigliaccio
EuropeanInstituteofOncology
Email:<ahref=""mailto:[email protected]"">EnricaMigliaccio</a>
Althoughgenemutationsthatextendlifespanandenhanceresistancetoenvironmentalstresses
suchasultraviolet(UV)lightorreactiveoxygenspecieshavebeenidentifiedin
<em>C.elegans</em>andotherinvertebrates,nosuchgenesareknowninmammals.Inthis
presentation,Dr.Migliaccioannouncedthatsheandhercolleagueshavefoundamutationinthe
mousep66<sup>shc</sup>genethatappearstohavesuchproperties.Thisgeneisasplice
variationofp52<sup>shc</sup>/p46<sup>shc</sup>,acytoplasmicsignaltransducerinvolvedin
thetransmissionofmitogenicsignalsfromtyrosinekinasestotheRasoncogene.Unlike
p52<sup>shc</sup>/p46<sup>shc</sup>,whichareknowntocausemalignantcellchanges,
p66<sup>shc</sup>fortunatelydoesnottransformfibroblasts.
Theresearcherscreatedap66<sup>shc</sup>knockoutmousethatretained
p52<sup>shc</sup>/p46<sup>shc</sup>,then<em>invitro</em>subjectedcellsfromthat
mousetoUVstress.Afterfourdays,wild-typecellswerealldead,whereasthecellswiththe
deletionwerealive.Additionalinvivoexperimentsshowedtheknockoutmicetobemoreresistant
toparaquat-inducedoxidativestressthanwild-typemice.Theresearcherssuspectthat
p66<sup>shc</sup>ispartofasignaltransductionpathwaythatregulatesoxidativestress
response,andthathypothesizethatdisruptingthispathwaywillprotectagainstthiswell-known
causeofaging.Furthersupportcomesfromanobservationalstudy,inwhichagroupofmicewitha
doublep66<sup>shc</sup>deletionoutlivedthosewhoweremissingonecopyandthosewithtwo
normalcopiesofthegene.
<em>MechanismsofProteinDegradationwithinEukaryoticProteasomes
</em>AlfredL.Goldberg,Professor
DepartmentofCellBiology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">AlfredL.Goldberg</a>
Forcells,animportantpartofviabilityisthepromptandappropriatedegradationofintracellular
proteins.Inmammaliancells,largestructurescalledproteasomesdegradeproteinsthathavebeen
markedfordestructionbyubiquitin.Someofthefragmentsthatemergefromthisprocessare
convertedtoaminoacids;othersareantigenicpeptidesthattriggercytolyticT-cellactivityafter
presentationonMHC-class1molecules.
Unliketypicalproteases,mammalian20Sand26Sproteasomesdegradeproteinsinahighly
processivefashionthatDr.Goldbergdescribesasa""bite-chew""model.Onceaproteinsubstrate
hasbeenlabeledbyubiquitin,anATPaseshepherdsitintotheproteasome'scentral""chamberof
doom,""whereitwillbeunfoldedandmethodicallychoppedintosmallpieces.Eachmoleculeis
completelychoppedupbeforetheproteasomemovesontothenext.Dr.Goldbergandhis
colleaguesweresurprisedtofindthatalltheproductsofthisprocessarethesamesize,whether
theystartedoutassmallpolypeptidesorbigproteins,whichtheyseeasanindicationthat
proteolysiscontinuesuntiltheproductsaresmallenoughtodiffuseoutoftheproteasome,then
stops.About99%ofthesefragmentsaresmallerthan25residues,withmostinthe3-20residue
range.Only10-15%oftheproductsare8-9residuesinlength,thesizerequiredforMHC-class-1presentation.
Eukaryotic20Sproteasomescontainactivesitesthatcleaveproteinsinthreedistinctways:twocut
likechymotrypsin,twoliketrypsin,andtwolikecaspase.Theresearchersweresurprisedtofind
thatinsteadofactingindependently,chymotripsinappearsto""bite""thesubstratefirst,which
initiates""chewing""bytheotherprocessingsites.Caspasesubstratessignalchymotripsinwhen
it'stimetotakethenextbite.Theresultingprocessisahighlyefficientmethodfordestroying
abnormalproteinsandforalertingtheimmunesystemtothepresenceofvirusesandother
undesirables.Inthefuture,proteasomeinhibitorsmayholdpromiseastreatmentsforcancerand
otherhumandiseases.
<strong>Session3:PlantDefense/Pathogenesis</strong>
<strong>Overview</strong>
Themorethatscientistslearnabouthowplantsprotectthemselvesagainstdisease,themore
parallelstheyseebetweenplantandmammaliandefensesystems.Muchofwhatisknownabout
howplantsrespondtopathogenattackhascomefromstudiesof<em>Arabadopsisthaliana</em>
orrice.Thistypeofresearchwillgetaboostin2000,whenthegenomesofthesemodelsystemsare
expectedtobefullysequenced,Dr.BrianStaskawiczpredictedinhisintroductiontothissession.
Plantshavemanyenemies,includingbacteria,fungi,viruses,andnematodes.Agriculturistshave
beendevelopingdisease-resistantplantssincetheturnofthecentury,relyingalmostentirelyon
classicalbreeding,hybridization,andrecurrentselectionfordesirabletraits.Longbeforegenes
couldbeisolated,itwasobviousthatasingleresistancegenecouldmakethedifferencebetweena
bountifulharvestandafailedcrop,Dr.Staskawicznoted.Aftergeneticmappingandmap-based
cloningbecameavailable,scientistsbegancloningspecificgenesfordiseaseresistance.Manyplant
resistancegeneshaveleucine-richrepeat(LRR)domains,whichareimportantforrecognizingnonselfproteinsintheenvironmentandsettinginmotionasignaltransductionpathwaythat
ultimatelyleadstodefensiveaction.Themachineryforrecognitionandresponsecloselyresembles
themammalianimmunesystem.
Inrecentyears,studiesofbacteriathatpreyonplantshaverevealedthattheysharemany
characteristicswithhumanpathogens,includingsimilaritiesineffectorproteinsknownas
virulencefactors.Nowthatbothhostplantsandtheirpathogenscanbegeneticallymanipulated,
scientistsaregoingtofindbetterwaystogiveplantsanedgeovertheirenemies.""Thefieldof
plantpathogenesisanddefenseisexplodingrightnow,""Dr.Staskawiczsaid.
<strong>Presentations</strong>
<em>Exploitingpolygalacturonase-inhibitingproteins(PGIPs)toengineernovelplantreceptors
</em>GiuliaDeLorenzo,Professor
LaboratoryforGeneTransfer&amp;Therapy,InstituteforCancerResearch,DepartmentofPlant
Biology,Universita'diRomaLaSapienza
Email:<ahref=""mailto:[email protected]"">GiuliaDeLorenzo</a>
Plantscanberesistanttodiseaseonlywhenthereisamatchbetweenaplantresistancegeneand
anavirulence(Avr)geneinthepathogen.Resistancegenesarethoughttocodeforreceptorsthat
recognizespecificAvrproducts.Withoneexception,alltheplantresistancegenesthathavebeen
identifiedhaveleucine-richrepeats(LRR),whichencodespecificreceptorsforawidevarietyofAvr
proteins.Dr.DeLorenzosuspectsthatrecognitionofspecificpathogenshingesonahypervariable
regioninresistancegenesthatcodesforvariationsinaspecificregionwithinLRRproteins.Thisis
theoreticalatpresent,however,becauseherteamhasnotyetobservedadirectinteraction
betweenLRRandAvrproteins.Polygalacturonase-inhibitingproteins(PGIPs),presentinthecell
wallofmanyplants,belongtothelargefamilyofLRRproteinsandarestructurallysimilartoother
knownproductsofresistancegenes.PGIPrecognizesendopolygalacturonases(PG),enzymesthat
disease-causingfungiusetobreachthecellwallsofplants.PGIPsandPGsofferaunique
opportunitytoanalyzehowLRRproteinsrecognizespecificattackers.Dr.DeLorenzo'slaboratory
hasbeenusingsite-directedmutagenesistoexplorehowtherecognitioncapacityofPGIPscanbe
manipulated.Inoneexperiment,alterationofasingleaminoacidresiduecausedaPGIPtolose
function,shereported.Nowherteamisworkingwithmutationsthatmayincreaserecognition,
seekingtocreatechimericproteinsthatwillenableplantstoidentifyandresistawiderrangeof
pathogens.
<em>Plantextracellularmatrixanddevelopment:targetingpectins
</em>FeliceCervone,Professor
DepartmentofPlantBiology,Universita'diRomaLaSapienza
Email:<ahref=""mailto:[email protected]"">FeliceCervone</a>
Dr.Cervone'slaboratoryfocusesoneventsintheplantcellwall,whichistheorganism'sfirstlineof
defenseagainstpathogenicinvaders.Forsomeyears,theyhavepursuedgeneticmutationsinthe
cellwallthatmaybeimportantinbothdefenseandnormaldevelopment.Whentheseproved
elusive,theresearchersdecidedtoseeifcell-wallmutationscouldbedetectediftheyusedwellcharacterizedbacterialenzymestomanipulateextracellularmatrixarchitecture.Dr.Cervone's
teamused<em>Agrobacterium</em>-mediatedtransformationtoproduceplantsthat
overexpresspolygalacturonase(PG)andpolygalacturonase-inhibitingproteins(PGIPs).Theywere
abletogeneratetwokindsoftransgenicplants:<em>Arabidopsis</em>andtobaccoplants
expressingPGfrom<em>Aspergillusniger</em>,and<em>Arabidopsis</em>,tobaccoand
tomatoplantsexpressingPGIPfrom<em>Phaseolusvulgaris</em>.Thesealterationschangedthe
pectinsinthecellswalls.
Althoughtheyexpectedthatthemorphogenesisoftransformedplantswouldnotbethesameas
normalones,theyweresurprisedwhenthealteredplantsgrewmuchlargerandmorevigorously
thanthewild-type,akindofplantversionofsupermouse.Anotherunexpectedfindingwasthat
pectinsfromtomatoeswiththe<em>pgip</em>transgeneexhibitedahigherdegreeof
methylationandacetylationthanthoseisolatedfromnon-transformedplants.Thisisconsistent
withearlierfindingsthatPGIP<em>invitro</em>interactswithmethylatedpectinsbetterthan
withnon-methylatedhomogalacturonans,aperferencethatprobablyprotectspectinsfrom
demethylation.Althoughmuchmoreresearchisneeded,evenatthisearlystageitisclearthatplant
transformationwithPGsandPGIPsisavaluabletoolforexploringhowchangesinpectinstructure
affectplantdevelopment,physiology,anddefense.
<em>Pseudomonasaeruginosapathogenesisindiversehosts
</em>LaurenceRahme,AssistantProfessor
HarvardMedicalSchoolandMassachusettsGeneralHospital
Email:<ahref=""mailto:[email protected]"">LaurenceRahme</a>
Dr.Rahme'slaboratorystudiesthemolecularmechanismsunderlying<em>Pseudomonas
aeruginosa</em>pathogenesisinmammals.Thisisamedicallyimportantopportunisticbacterium
thatinfectsburnandtraumapatients,aswellasotherimmunocompromisedindividuals,anditis
theleadingcauseofdeathinpeoplewithcysticfibrosis.
Sheandhercollaboratorshaveshownthatanovelstrainof<em>P.aeruginosa</em>usesthe
samesubsetofvirulencefactorstocausediseasebothinplantsandinawiderangeofanimals,
includinghumans.Thisimportantfindinggavethemtheopportunitytouseplantsasascreening
systemforbacterialvirulencefactors,whichoncefoundcouldpointthewaytotheidentificationof
newantibiotics.Thisscreeningalgorithmmarkedlydecreasestheuseoflaboratoryanimals,yetit
generatesdatarelevanttopathogenesisinmammals.Inplants,Dr.Rahme'sgrouphasidentified
severalnovel<em>P.aeruginosa</em>virulence-relatedfactors,themajorityofthemaffectingthe
persistenceandseverityofinfection.Whenthesefactorsweretestedinamousemodelthat
involvesinfectionafteranon-lethalburn,theymadeinfectionsworseandacceleratedsepsis
development.
Morerecently,Dr.Rahmehasbeenusing<em>C.elegans</em>toidentifyavirulencemutations
thatenablethewormtofeedonpathogenic<em>P.aeruginosa</em>andsurvive.Shehasfound
severalmutationsthatappeartoincreasethehost'sabilitytolimitdiseasedevelopment,notjustin
wormsbutinmammalsaswell.
<em>Moleculargeneticsofplantbacterialdiseaseresistance
</em>BrianStaskawicz,Professor
DepartmentofPlantandMicrobialBiology,UniversityofCalifornia,Berkeley
Email:<ahref=""mailto:[email protected]"">BrianStaskawicz</a>
Scientistshavelonganticipatedatimewhentransgenicplantswillberesistanttodiseasesthat
farmersnowcombatwithchemicalpesticides.Dr.Staskawiczandhiscoworkerswillsoondiscover
whetherthismomenthasarrivedforatypeofbacterialspotdiseasecausedby<em>Xanthomonas
campestrispvvesicatorio(XCV)</em>,whichafflictsbothpeppersandtomatoes.Agenethat
producesdurableresistanceinpeppersisbeingexperimentallyintroducedintotomatoplants,
whichhavenonaturalresistancetothispathogen.IttookDr.Staskawicz'labmorethan8yearsto
naildownthefunctionofthisgene,isolateit,andprepareitforuseasatransgene.
Thisworkgrewoutofabroaderinquiryintothemoleculargeneticsofdiseaseresistance,which
showedthatplantsareprotectedagainstXCV-causedspotdiseasewhenthebacteriumcarriesthe
avirulencegene(actuallyaneffectorprotein)avrBs2andthehostplanthastheresistancegeneBs2.
AseriesofgeneticandbiochemicalexperimentsdemonstratedthattheBs2geneproduct
recognizesthebusinessendofXCV-aneffectorproteinthatthepathogenneedstobeatitsmost
virulent.HavingdecidedthatBs2wasagoodcandidateforinsertionintothetomatogenome,Dr.
Staskawiczandhiscolleaguessetouttomapandclonethegene-notrivialtaskinagenomefour
timesaslargeasthehumangeneticendowment.Thenextstepistoshowprotectioninfieldtrials,
whichareexpectedtobeginthisyear.
<strong>Session4:NeurobiologyII</strong>
<strong>Overview</strong>
Inthissession,newscientifictoolsbegintopickapartsomeofneurobiology'svenerableknots.For
example,Dr.ElioRaviolasaidinhisintroduction,circadianbiologyusedtomeanwatchinghowrats
behavedwhenthelightwasswitchedonandoff.Thefirstpaperdescribedhowmolecularbiology
canpryopenthemammaliancircadianclock,sothatsomeofitsgearsandspringscanbespread
outforinspection.Thesecondpresentationexaminedcalciumpumps,certainlyamongthebiggest
andmostimportantmembranetransporters,andaskedhowtheymightbeexpressedduring
neuronaldevelopment.Thenextspeakertackledaclassicproblem,the""inside-out""migrationof
newlyhatchedneuronstotheirproperplacesinthecerebralcortex,andidentifiedtwomajor
actorsinthisjourney.Finally,theconcludingspeakerbegantolifttheveilonwhatcouldproveto
bethemasterswitchforneuronalexocytosis.
<strong>Presentations</strong>
<em>Studiesonthemolecularmechanismofthevertebratecircadianclock
</em>CharlesJ.Weitz,AssistantProfessor
LaboratoryforGeneTransfer&amp;Therapy,InstituteforCancerResearch,Departmentof
Neurobiology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">CharlesJ.Weitz</a>
Circadianclocksareendogenousoscillatorsthatdrivedailyrhythmsandphysiology,andassuch
theyprobablyrepresentanancientandfundamentalmechanism.Inmammals,thecentralclockis
locatedinthesuperchiasmaticnucleusofthebrain,withindependentclocksineachretina.Two
yearsago,investigatorsatNorthwesternUniversityidentifiedthefirstmammaliancircadiangene,
whichtheycalled<em>Clock</em>.Furtherresearchshowedthatthisgeneencodesa
presumptivetranscriptionfactorthatiscloselyrelatedtoafamilyofproteinsthatmediate
dimerizationandbindDNA.
ThesefindingsimmediatelyremindedDr.Weitzofwhathislabhadlearnedabouttheworkingsof
theDrosophilaclock,where<em>per</em>genesmakeproteinsthatdimerizewiththeproductof
<em>tim</em>(timeless).Thisproteinpairistransportedtothenucleuswhereitsomehowshuts
downthe<em>per</em>and<em>tim</em>genesuntiltheproteinsdisappearandthegenes
turnonagain.Thesefindingsmotivatedhimtolookforasimilarfeedbackloopinmice.Thesearch
forapartnerforCLOCKproteinturnedupBMAL1,whichisco-expressedwithCLOCKandPER1at
knowncircadianclocksitesinbrainandretina.
AdditionalexperimentsshowedthatCLOCK-BMAL1heterodimersactivatetranscriptionfromEboxelements,atypeoftranscriptionfactorbindingsite,locatedadjacenttothemouse
<em>per1</em>gene,andfromanidenticalE-boxknowntobeimportantforexpressionof
<em>per</em>genesinDrosophila.IftheCLOCKproteinwasmutated,however,itjoinedwith
BMAL1toformheterodimersthatboundDNAbutfailedtoactivatetranscription.AccordingtoDr.
Weitz,CLOCK-BMAL1heterodimersdrivethepositivecomponentof<em>per</em>
transcriptionaloscillations,whichappeartounderliecircadianrhythmicity.Thisisthefirsttime
thatbindinghasbeenprovedtoactivatetranscription.
Morerecently,transfectionstudiesinmicehaveprovideddirectevidencethatexpressionofthe
PERproteininhibits<em>Per1</em>geneactivationbyCLOCK-BMAL1.Proteininteraction
experimentsandfurtheranalysissuggestthatPERbindingsequesterstheCLOCK-BMAL1
heterodimerinamannerthatkeepsthetranscriptionfactorfrombindingtoitsE-boxtargetsite.
<em>Calciumcontrolsthetranscriptionofitsowntransportersindevelopingcerebellarneurons
</em>ErnestoCarafoli,Professor
DepartmentofBiochemistry,UniversityofPadova
Email:<ahref=""mailto:[email protected]"">ErnestoCarafoli</a>
EucaryoticcellsmaintainalowintracellularconcentrationoffreeCa<sup>2+</sup>mainlyby
relyingonamembrane-boundATPase,PMCA,thatservesasahigh-affinitypump.Calciumisalso
exportedfromcellsbyalow-affinityNa+/Ca<sup>2+</sup>exchanger(NCX).ThePMCAprotein
hasfourisoformsthatvaryslightlyinaminoacidsequence,withPMCA4beingthebeststudiedof
these.Inordertolearnmoreabouttheothers,Dr.CarafoliÕslaboratorymademonoclonal
antibodiestoisoforms1-3.Usingthesetools,theyfoundPMCA2and3onlyinbraintissue,whereas
isoforms1and4appearubiquitous.
InordertoexploretheroleofPMCAisoforms1-3inneurondevelopment,Dr.Carafoliandhis
colleaguesusedantibodiestotrackchangesinthesekeycarrierproteinsinculturesofrat
cerebellargranulecells(CGC).TheyfoundthatPMCAisoforms2and3,andasplicingvariantof
PMCA1(designatedasPMCA1CII)arestronglyupregulatedinthe3to5daysrequiredforfull
maturationofthegranulecells,whereasPMCA4ismuchmorerapidlydownregulated.Theseeffects
occuratboththetranscriptionalandtranslationallevels.Maturationofcerebellargranulecells
requiresthesustainedinfluxofCa<sup>2+</sup>throughL-typechannels;whenthiswasblocked
withnifedipinetheup-and-downregulationofPMCAswasabolished.Atvariancewiththe
upregulationofPMCA2,3,and1CII,thedown-regulationofPMCA4dependsonincreasedlevelsof
calcineurin,arelationshipthatwasdisruptedbyimmunosuppressivedrugs.Dr.Carafolialso
identifiedthreeisoformsofNCXatworkintheseculturedcells.NCXIandNCXIIIbecomeslightly
upregulatedasthegranulecellsmature,whereasNCXIIisstronglyandrapidlydown-regulatedina
calcineurin-dependentway.ThesplicingvariantsofNCX1alsoundergoaswitchduringmaturation.
ExpressionofCa<sup>2+</sup>transportersmaychangebecausethecellsneedtogainbetter
controlovertheincreasedCa<sup>2+</sup>influxrequiredfortheincreasedgenetranscription
thatisintegraltotheirmaturation.Futureresearchwillexplorethespecificpropertiesofthe
individualCa<sup>2+</sup>carrierproteins.
<em>Functionanddysfunctionofcyclin-dependentkinase5indevelopmentanddegeneration
</em>Li-HueiTsai,AssistantProfessor
DepartmentofPathology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">Li-HueiTsai</a>
Neuronsarebornclosetotheinnersurfaceoftheneuraltubeandmigrateoutwardtoformthe
layersofthemammaliancerebralcortex.Intheselayers,neuronsaregroupedaccordingto
morphology.Duringcorticaldevelopment,successivegenerationsofneuronsmigrantinan
""inside-out""fashion,withthefirst-bornbraincellssettlingclosesttohomeandlatercohorts
travelingjustpastthemtoformthenextlayer.Althoughthispatterniswell-known,thefactorsthat
guidenewbornneuronsintoplacehavebeenamystery.
Overthepastseveralyears,Dr.Tsaihasnotonlyfiguredouthowneuronsmigratebutalsohas
uncoveredconnectionsbetweenthisphenomenonandAlzheimer'sdisease.Herexperiments
suggestthatnormal""inside-out""migrationrequiresthatcyclin-dependentkinase5workclosely
withp35,aregulatoryprotein.Whensheandhercolleaguesknockedoutthep35geneinmice,and
whenanotherlabindependentlyknockedoutthegeneforcdk5,eachproducedmicewithneurons
layered""outside-in.""Dr.Tsai'smiceweredefectivebutviable;theanimalswithoutcdk5werenot
viable.Shehassincefoundevidencesuggestingthatthep35/cdk5kinasecomplexfacilitates
""inside-out""migrationbyregulatingactincytoskeletondynamicsandreducingcell-celladhesion,
makingiteasierforfreshlymintedneuronstoslippasttheonesthathavealreadysettledintheir
appropriatelayers.
Whenischemia,hydrogenperoxide,orothermeanswereusedtostressthebrainsofmice,the
animalsconvertedp35top25,aproteinthatDr.Tsaibelievesderegulatescdk5andhasnonormal
developmentalfunction.Herteamfoundmassiveaccumulationsofp25andcdk5inpost-mortem
brainsamplesfromAlzheimer'sdiseasepatients,especiallyintheneurofibrillarytanglesthatare
onemajorhallmarkofthedisease.Abnormallyphosphorylatedtauproteinalsoaboundsinthese
tangles.Futureresearchfocusesontheroleofp25/cdk5inapoptosis,andthepossibilitythatthis
mightbeusedasatargetfortreatingneurodegenerativedisorders.
<em>Regulatedsecretionexpressioncompetenceandmultiplicityinneurosecretorycells
</em>JacopoMeldolesi,Professor
DepartmentofNeuroscience,DIBIT,SanRaffaeleInstitute
Email:<ahref=""mailto:[email protected]"">JacopoMeldolesi</a>
Neurosecretionistheprocessbywhichcellsexpressandreleasebyexocytosisboththesmall
vesicles,whichcontainclassicalneurotransmitters,andthelargedensevesiclescontaining
mixturesofamines,ATP,proteinsandpeptides.Becauseneuronsandendocrinecellsacquire
secretorycapacityduringdevelopmentandretainit,thisisgenerallyregardedasastabletraitthat
canbelostonlyincaseofcellde-differentiation.Someyearsback,Dr.Meldolesi'slaboratory
developedwhathecharacterizesas""aneurosecretorycellthatisincompetentforsecretion.""
DefectiveclonesofpheochromocytomaPC12cellsappearphenotypicallynormalexceptthatthey
lackthedensegranulestypicalofneurosecretorycells;functionallytheyhavelosttheabilityto
secrete.Theseratcellslacknotonlysecretionproductsbutalsovesiclemembraneproteins,
includingthevSNAREVAMP2,theplasmalemmatSNAREs,whicharenecessaryforexocytosis,and
varioussolubleregulatoryproteins.Themechanism(s)sustainingthedefectappear(s)tobeatleast
inpartpost-transcriptional.WhendefectivecellsarefusedwithnormalratPC12orwithsecretory
humancells,orwhentheyaretransfectedwithoneormorenormalgenes,neurosecretionreturns
tonormallevels.Thisimpliestheexistenceofgeneticcontrolsforexocytosis,andthenatureof
thesemechanismsisnowbeinginvestigated
<strong>Session5:Proteolysis/Apoptosis/CellCycle</strong>
<strong>Overview</strong>
Thetopicscoveredinthissession-proteindegradation,cellcycleregulation,andapoptosis-may
holdthekeystothedevelopmentofbettercancertherapeutics,Dr.GiulioDraettasaidinhis
openingremarks.Unliketreatmentsforinfectiousdiseases,whichcanbedirectedagainstfeatures
ofthepathogenthataren'tfoundinthehumanhost,mostcancertreatmentsmustaimatmolecules
thatarenormallypresent.Andthis,ofcourse,explainswhysomanyanti-cancertreatmentsareso
toxictopatients.Inthefuture,molecularoncologistshopetohavetreatmentsthatselectivelykill
cancercellswhileleavingnormalonesalone.Somedayitmaybepossibleforphysicianstoobtaina
molecularfingerprintofthepatient'scancer,thentoselectinhibitorsthatwillmoderateresponses
oftheindividualpatientandthespecifictumor.Thepresentationsinsessionfocusonsignaling
cascadesthatcouldultimatelyproverelevanttotheultimategoaloffindinglesstoxictreatments
forcancer,Dr.Draettasaid.Hepredictedthatthenextstepwillbelookingforcross-talkamong
signalingpathways,inadditiontoexploringindividualpathways,andthatthiswillleadtoeven
moreideasforhigh-specificity,low-toxicitytreatments.
<strong>Presentations</strong>
<em>Theregulatoryparticleoftheproteasome
</em>DanielFinley,AssociateProfessor
LaboratoryforGeneTransfer&amp;Therapy,InstituteforCancerResearch,DepartmentofCell
Biology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">DanielFinley</a>
Inhispresentationonthefirstdayofthesymposium,Harvard'sDr.AlfredGoldbergdescribedhow
cellsusetheubiquitin-proteasomepathwaytodegradeproteinsthatwouldcausetroubleifthey
wereallowedtoaccumulate.Dr.Finley'sworkshedsmorelightonthiscrucialpathway.Heuseda
yeastmodeltounderstandtheopeningandclosingofthedoorwaythroughwhichubiquitin-protein
conjugatesenterthelumenofthegiganticproteasome's28-subunitcoreparticle(CP).Onceinside
thischamber,taggedproteinsarereducedtoconfetti.Althoughitwouldbeeasytoviewthe
proteasomeasamonolith,itisactuallyformedbytheassociationoftheCPwiththe19Sregulatory
particle(RP),whichsitsovertheCPchannelandselectsubiquitin-conjugatesfordegradation.In
addition,Dr.Finley'sresearchindicatesthattheRPisacomplexstructurethatfullyunfolds
substratessotheywillfitthroughthe13 -wideopeningthatleadstotheCPlumen.
TheyeastRPcontains17subunits,6ofthemATPases,andwhenviewedwithanelectron
microscopethisstructurelookslikeasetofjawsthatopenandclosetoadmitselectedproteins.The
lidisan8-subunitsubcomplexwhichcanbedissociatedfromyeastproteasomesinvitro.Thebase
isalsoan8-subunitcomplexbutitcannotbeseparatedfromtheCP.Thebasecontainsall6
proteasomalATPases,whichmaybothjointheRPtotheCPandpropelsomeoftheproteasome's
targetsintotheCPfordestruction,Dr.Finleysaid.ByexperimentingwithvariousRPmutations,he
andhiscolleagueshavedeterminedthatrpt2,oneoftheATPasesfoundinthebase,isneededto
openthegatedchannelintotheCP.ThebaseissufficienttoactivatetheCPfordegradationof
peptides,perhapsindicatingthatitiscompetenttoopenthechannelintotheCP.However,the
proteasomeneedsthelidtorecognizeubiquitin-conjugates.
Todeterminewhetherthebaseofthisassemblyunfoldedproteinsinadditiontoopeningthedoor,
theresearchersusedcitratesynthase(CS)asamodelsubstrate.Astheypredicted,baseATPases
actedasmolecularchaperonesforCS.OnlyafteritwasunfoldedcouldCSbeboundbythebase,Dr.
Finleysaid,andthisreactionwasindependentofubiquitintagging.Thesedatasuggestthat
ubiquitin-proteinconjugatesareinitiallytetheredtotheproteasomeviaspecificrecognitionof
theirubiquitinchains,followedbyanonspecificinteractionbetweenthebaseandthetarget
protein,whichiscoupledtounfoldingandtranslocationofthetargetproteinintotheCP.
<em>Mitochondriaincelldeath:the(w)holestory
</em>PaoloBernardi
DepartmentsofBiomedicalSciencesandBiologicalChemistry,UniversityofPadova
Email:PaoloBernardi
Mitochondriaareoftenreferredtoasthe""powerplants""ofthecellbecausetheyspecializein
synthesizingATP.Themoststrikingfeatureoftheseorganellesisthattheyhaveadouble
membrane,whichdividesthemintotwocompartments:theintermembranespaceandthematrix
spaceinthecenterofthestructure.Mitochondriamakefewproteinsin-house,andforthemost
parttheyimportproteinsfromthecytosolwhichformcomplexeswithmitochondria-made
proteins.Sincetheearly1990s,mitochondriahavebeenunderclosescrutinyasregulatorsof
apoptosis,andaspotentialtargetsfortherapeuticinterventionsdirectedataccidentalor
programmedcelldeath.
Someresearcherssuggestthatthepermeabilitytransitionpore(PTP)isamajorplayerin
mitochondrialapoptoticsignalling.Theypostulatethatwhenthishigh-conductanceinner
membranechannelexpandstoadmitsolutes,leadingtotremendousswellingofthemitochondria,
thismaytriggerreleaseofintermembraneapoptosis-inducingfactorandpossiblyofcytochromec.
Inmechanisticterms,however,itisdifficulttounderstandhowthisporemightbelinkedtothe
releaseofdeathfactorsbytheorganelle'sinnermembrane,Dr.Bernardisaid.Onebarrierto
understandingthisprocessisthat<em>invitro</em>studiesofcell-freemitochondriamaynot
correspondwellwithinvivoevents.Workingwithpopulationsofmitochondriainsuspension,Dr.
Bernardiandhiscolleaguesmanipulatedavarietyoffactorstotryandmimicthe<em>in
vivo</em>openingandclosingofthePTP.Theyfoundthatdepolarizationalwaysleadstoopening
ofthepore,butthatthereverseisnottrue.Further,theydeterminedthataclosedporedidnot
necessarilymeanthatthepumpthatdrivesATPsynthesiswasalsooutofcommission.More
recently,theresearchersdevisedanovelmethodforstudyingmitochondriainintactcells,which
involveschemicallyblottingoutbackgroundactivityinthecellsothatmitochondrialeventsstand
out.SofaritappearsthatdepolarizationcanresultnotonlyfromopeningofthePTP,butalsofrom
increasedATPdemandorcalciuminflux.ItalsoappearsthatGD3gangliosideopenstheporeand
increasesthelikelihoodofapoptosis,Dr.Bernardisaid,andthisisbeingstudiedfurther.
<em>Structuralcommunicationinapoptoticpathways
</em>GerhardWagner,Professor
DepartmentofBiologicalChemistryandMolecularPharmacology,HarvardMedicalSchool
Email:<ahref=""mailto:[email protected]"">GerhardWagner</a>
Inorderforacelltodieorcommitsuicide,itsmitochondrial""powerplants""mustbeshutdown.
Butwhichofthemanyproteinsinvolvedinintracellularapoptosispathwaysdeliversthefatalblow
totheseorganelles?TheanswerappearstobeBID,anewproteinthatDr.Wagnerandhis
colleagueshaveidentifiedinthecomplicatedFassignaltransductionpathway.BIDappearstolink
intracellulardeathsignalstothemitochondria,whereitsetsinmotionachainofeventsthat
culminateswiththeactivationoffatalcaspaceenzymes.
WhentheresearchersusedNMRspectroscopytodeterminethestructureofBID,theywere
surprisedtofindthatthispro-apoptoticproteinlooksmuchlikeBcl-xL,aproteinknowntoinhibit
apoptosis.ModelsofBIDandBcl-<sub>xL</sub>bindingindicatethatthetwojoineasilyinthe
presenceofcaspace-8,adeathenzymethatloosensBcl-<sub>xL</sub>'sordinarilytightstructure.
ThecomplexofBIDandBcl-<sub>xL</sub>mayinterferewiththeanti-apoptoticeffectsofAPAF1,whichordinarilybindswithBcl-<sub>xL</sub>.Thissetsthestageforlethalcaspaceactivation
thatknocksoutthemitochondria.
<em>RegulationofcellcycleprogressionbytheE2Ftranscriptionfactors
</em>KristianHelin
DepartmentofExperimentalOncology,EuropeanInstituteofOncology
Email:<ahref=""mailto:[email protected]"">KristianHelin</a>
Inmammaliancells,theretinoblastomaproteins(pRB)arekeyregulatorsofthecellcycle,serving
asoneofthemainbrakesonprogressaroundthecell-divisioncycle.Theseproteinsareessential
forfundamentaldecisionsaboutwhetheracellshouldproliferate,differentiate,orundergo
apoptosis.OfthenumerouscellularproteinsthatinteractwithmembersofthepRBfamily,thebest
characterizedaretheE2Ftranscriptionfactors.ItiswidelybelievedthattheabilityofthepRB
familyproteinstorestrictcellproliferationdependsontheirabilitytoinhibitE2Ftranscriptional
activity.
E2Fsareimportantfornormalcellfunction,anddysregulationoftheseproteinshasmany
consequences.Dr.HelinandhiscolleagueshavegeneratedcelllinesexpressingE2F-1,E2F-2,and
E2F-3,eachfusedtotheestrogenreceptorligandbindingdomain(ER),aninnovationthatmakesit
possibletomanipulateERE2Flevelswithhydroxytamoxifen.Usingthissystem,theresearchers
havefoundthatactivationofallthreeE2FscanrelievethemitogenrequirementforentryintoS
phase,andactivationoftheE2FsleadstoashorteningoftheG0-G1phaseofthecellcycleby6-7
hours.E2Fcanalsoinduceapoptosisevenincellsfedgrowthfactorsthatwouldordinarilysustain
them,Dr.Helinreported.Theresearchershavealsodemonstratedthatseveralgenescontaining
E2FDNAbindingsitesareefficientlyinducedbytheE2Fsintheabsenceofproteinsynthesis.More
recently,Dr.Helin'slaboratoryhasidentifiedtwonoveltargetsforE2Ftranscriptionfactors,both
cell-division-cyclegenes.One,cdc25A,isatyrosinephosphataseessentialfortheactivationof
certaincyclin-dependentkinasesandS-phaseinitiation;itisalsooverexpressedinmanytumors.
Thesecond,cdc6,isnotonlyneededforthecellcycletoadvancebutalsoisaverysensitivemarker
forcellproliferation;insomecancersitmaybeamarkerfortheaggressivenessoftumorcells.In
thefuture,E2Fscanbeusedtoscanthehumangenomeforadditionalgenesimportantfor
apoptosis,cellproliferation,orDNAreplication,Dr.Helinsaid.