Nuclear receptors - Jagiellonian...

Nuclear receptorsNuclear receptors

structure structure and functionand function

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LetLet’’s start from a small fragment..... (?)s start from a small fragment..... (?)

Types of Types of leukemiasleukemias

A.A.

-- LymphoLymphoidid

-- MyeloMyeloidid

B.B.

-- acuteacute(results from a transformation relatively undifferentiated proge(results from a transformation relatively undifferentiated progenitor cells, with only limited nitor cells, with only limited capabilities of differentiation)capabilities of differentiation)

-- chronicchronic(results from a transformation of more differentiated cells, whi(results from a transformation of more differentiated cells, which have capability to mature)ch have capability to mature)

AcuteAcute myelocyticmyelocytic leukemialeukemia

ChronicChronic myelocyticmyelocytic leukemialeukemia

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)-- sustains about sustains about 3% of cancers3% of cancers in humans (15in humans (15--20% all 20% all leukemiasleukemias in adults), in adults), approximately approximately 11--2 cases per 100 000 people2 cases per 100 000 people

-- average ageaverage age of diagnosed patients: of diagnosed patients: 53 years53 years (30% patients is older than 60 years, (30% patients is older than 60 years, less than 10% less than 10% underunder 20 years20 years of of ageage))

-- in about half of patients the disease is in about half of patients the disease is asympthomaticasympthomatic and is detected during and is detected during routine blood controlroutine blood control

-- untreated is fataluntreated is fatal

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)-- First cancer for which the First cancer for which the geneticalgenetical mutation causing the disease has been identified mutation causing the disease has been identified (1960, Philadelphia)(1960, Philadelphia)

-- Mutated chromosome Philadelphia forms after translocation Mutated chromosome Philadelphia forms after translocation of of fragment of long arm of fragment of long arm of chromosome 9 (coding chromosome 9 (coding for for AblAbl kinasekinase) to long arm ) to long arm of chromosome 22 (coding of chromosome 22 (coding for for BcrBcr....) ....) (9 22)(9 22)

-- This mutation is present in 95% patients with This mutation is present in 95% patients with CML; it can also be found in CML; it can also be found in patintspatints with other with other leukemiasleukemias (e.g. in 15(e.g. in 15--30% cases of acute 30% cases of acute lymphoid leukemia)lymphoid leukemia)

-- Translocation leads to formation of hybrid geneTranslocation leads to formation of hybrid geneBcr/AblBcr/Abl and fusion protein of constitutive, and fusion protein of constitutive, unregulated unregulated kinasekinase activityactivity

Chromosome 9 Chromosome 9’

Chromosome 22 Philadelphia

AblBcr

Bcr/Abl

Normal Normal AblAbl tyrosine tyrosine kinasekinase in leukocytes is associated with regulation of cell proliferatioin leukocytes is associated with regulation of cell proliferation n and differentiationand differentiation

BcrBcr--AblAbl kinasekinase, which is constitutively active leads to:, which is constitutively active leads to:-- increased proliferationincreased proliferation-- decreased rate of apoptosisdecreased rate of apoptosis-- decreased expression of decreased expression of adhesinsadhesins (attenuation of adhesion and disturbed signal (attenuation of adhesion and disturbed signal transduction from a bone morrow transduction from a bone morrow microenviromentmicroenviroment))

Chronic Chronic myelomyeloidid leukemia (CML)leukemia (CML)- The most common symptoms at presentation in the chronic phase of CML include:fatigue, weight loss, abdominal fullness, bleeding, and sweating.

- As the disease progresses, bone pain and pain from splenic infarction may be seen, and end stage CML may present with signs and symptoms of acute leukaemia.

Chronic Chronic myelomyeloidid leukemialeukemia(CML)(CML)Therapy:Therapy:

-- Bone mBone maarrowrrow transplantation transplantation (achievable for (achievable for less than 20% of new diagnosed patients)less than 20% of new diagnosed patients)

-- Treatment with interferonTreatment with interferon--IFNIFN

-- chemotherapychemotherapy (e.g. (e.g. hydroxyureahydroxyurea))

-- GlGleeveevecec

At advanced phase no treatment is effectiveAt advanced phase no treatment is effective

KinaKinasesses inhibitedinhibited byby glgleeeevecvec

GlGleeeevecvec ((imatinibimatinib))

-- first commercially available inhibitorfirst commercially available inhibitorof tyrosine of tyrosine kinasekinase accepted for a accepted for a clinical useclinical use

-- specifically inhibits specifically inhibits kinaseskinases BcrBcr--AblAbl, c, c--Kit (CD117) and PDGFKit (CD117) and PDGF--RR

-- this way it inhibits proliferation and this way it inhibits proliferation and increasesincreases apoptosis of cells expressing those apoptosis of cells expressing those kinaseskinases

-- causes relatively mild sidecauses relatively mild side--effecteffect

-- significant improvement is observed in 49% significant improvement is observed in 49% patientspatients in the chronic phase of CML, who in the chronic phase of CML, who did not respond to treatment with did not respond to treatment with IFNIFN

gleevec

22--phenylaminopyrimidine derivativephenylaminopyrimidine derivative

HIFHIF--11

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Power of Power of nuclearnuclear receptorsreceptors……....

AmbystomaAmbystoma mexicanummexicanum

Prof. Laura KProf. Laura KaaufmanufmanUniwerytetUniwerytet JagielloJagiellońńskiski

19171917

Willson TM and Moore JT

19021902 –– wordword ‘‘hormonehormone’’ coinedcoined leadingleading to to conceptconcept of chemical of chemical messengersmessengers19111911 –– mammalianmammalian thyroidthyroid extractextract inducedinduced amphibianamphibian metamorphosismetamorphosis1919 1919 –– thyroxinthyroxin and and cortisonecortisone purifiedpurified19351935--19451945 –– synthesissynthesis of of severalseveral hormonshormons and and theirtheir analoguesanalogues19601960 –– ecdysoneecdysone inducedinduced chromosomalchromosomal puffspuffs indicatedindicated genegene activationactivation19611961 –– estrogen estrogen shownshown to bind to to bind to cytoplasmaticcytoplasmatic protein and to protein and to translocatetranslocate to to thethe nucleusnucleus19761976--19791979 –– estrogen receptor estrogen receptor clonedcloned19811981 –– hormon hormon receptorsreceptors shownshown to to interactinteract withwith promoterspromoters of target of target genesgenes

1988 1988 –– methodmethod for for cloningcloning receptorsreceptors withwith no no knownknown ligandsligands defineddefined((orphanorphan receptorsreceptors))19901990 –– heterodimerizationheterodimerization of of somesome receptorsreceptors withwith RXRRXR19961996 –– interactioninteraction withwith coco--activatorsactivators and and coco--repressorsrepressors19981998 –– phosphorylationphosphorylation cancan regulateregulate thethe activationactivation of of nuclearnuclear receptorsreceptors20002000 –– many many receptorsreceptors existexist as as multiplemultiple subtypessubtypes and and splicingsplicing formsforms......20022002 –– interplayinterplay betweenbetween membranemembrane and and nuclearnuclear signalingsignaling pathwayspathwaysdeterminesdetermines genegene expressionexpression

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-- -- Nuclear receptors exist in the all Nuclear receptors exist in the all MetazoaMetazoa sstudiedtudied, , but have not been found in the other organismsbut have not been found in the other organisms

-- Most of receptors are very old and Most of receptors are very old and conservconserveded: mammalian proteins : mammalian proteins have their counterparts in insectshave their counterparts in insects ((exceptionexception: steroid : steroid hormonehormone receptorsreceptors))

-- NNuclear receptors sustain of one uclear receptors sustain of one superfamilysuperfamily, originating perhaps from , originating perhaps from one ancestorone ancestor

-- Primary nuclear receptor acted possibly as a constitutive, Primary nuclear receptor acted possibly as a constitutive, homodimerichomodimerictranscription factortranscription factor

Evolution of nuclear receptorsEvolution of nuclear receptors

-- inin CCaaenorhabditisenorhabditis eleganselegans more than 2more than 25500 different different nucleanuclear r receptorreceptor genesgenes have been have been found, but in Drosophila found, but in Drosophila melanogastermelanogaster only only 2222 (they mediates e.g. action of e(they mediates e.g. action of eccdysodysonne)e)

-- in human there are in human there are 4499 nuclear receptor genesnuclear receptor genes

-- atat the protein level the number of receptors is higher the protein level the number of receptors is higher ((alternative alternative splicing,splicing, different promoters,different promoters,postranslationalpostranslational modificationsmodifications))

ArandaAranda A. & A. & PascualPascual A. A. PhysiolPhysiol Rev 2001.Rev 2001.

Modular structure of nuclear receptorsModular structure of nuclear receptors

A typical nuclear receptor is composed of A typical nuclear receptor is composed of several domainsseveral domains The variable NH2The variable NH2--terminal region (terminal region (region region A/BA/B) contains the ligand) contains the ligand--independent independent AFAF--11

transactivationtransactivation domain.domain.

The conserved DNAThe conserved DNA--binding domain binding domain ((DBD, region CDBD, region C) is responsible for the ) is responsible for the recognition of specific DNA sequencesrecognition of specific DNA sequences

A variable linker A variable linker region Dregion D connects DBD connects DBD to the E/F regionto the E/F region

The conserved The conserved E/FE/F region contains ligand region contains ligand binding domain (binding domain (LBDLBD), ), dimerizationdimerizationstructure and structure and ligandligand--dependenddependend AF2 AF2 transactivationtransactivation domain domain

RRegion A/Begion A/B

The most variable in respect of size and sequence. Very often iThe most variable in respect of size and sequence. Very often it includes AFt includes AF--1. 1.

The alternative splicing occurs usually within this domainThe alternative splicing occurs usually within this domain

Perhaps this domain is responsible for cellPerhaps this domain is responsible for cell--specific action of the nuclear receptorsspecific action of the nuclear receptors

It can be It can be phosphorylatedphosphorylated by different by different kinaseskinases involved in signal transduction involved in signal transduction (MAPK, (MAPK, cyclinecycline--dependent dependent kinaseskinases)), which regulates the activity of the receptor, which regulates the activity of the receptor

RRegion DBDegion DBD (domain C)(domain C)

The most The most conservconserveded domaindomain,, confers the ability to recognize specific targetconfers the ability to recognize specific target sequences sequences and activate genesand activate genes

There are There are 2 2 zinc fingers divided by the fragment of zinc fingers divided by the fragment of 6060--70 70 aminoacidsaminoacids –– in each finger in each finger four four cysteinescysteines coordinates one zinc ioncoordinates one zinc ion

The sequence at the base of the first finger The sequence at the base of the first finger (P box) (P box) recognizes DNA, whereas the recognizes DNA, whereas the sequence at the base of the second finger sequence at the base of the second finger (D box) (D box) is involved in the receptor is involved in the receptor dimerizationdimerization


DBD of nuclear receptorDBD of nuclear receptor DBDDBD consists of two zinc fingersconsists of two zinc fingers (60(60--70 70 aminoacidsaminoacids)). In each zinc finger, four of the. In each zinc finger, four of theinvariable invariable cysteinescysteines coordinate one zinccoordinate one zinc ion, ion, and both zinc finger modules fold together to and both zinc finger modules fold together to form aform a compact, interdependent structurecompact, interdependent structure

First finger contains First finger contains P boxP box residues, involved residues, involved in discrimination of response elementin discrimination of response element inin DNADNA

Second finger contains Second finger contains D boxD box, which form a , which form a dimerizationdimerization interfaceinterface

CTE CTE (COOH(COOH--terminal extension) is critical for terminal extension) is critical for monomericmonomeric DNA bindingDNA binding

Fingers form the DBDFingers form the DBDthat recognizes a hemithat recognizes a hemi--sitesiteof the response elementof the response element

HingHingee rregion egion (domain D)(domain D)

Moderately Moderately conservconserveded,, servesserves as a as a hingehinge betweenbetween thethe DBD DBD andand thethe LBD, LBD, allowingallowingrotationrotation of of thethe DBDDBD

VeryVery oftenoften itit includesincludes a a nuclearnuclear localizationlocalization signalssignals

MutationsMutations withinwithin thisthis region region abolishesabolishes interactioninteraction withwith nuclearnuclear receptor receptor coco--repressorsrepressors

RRegion LBDegion LBD ((domaindomain E)E) ItIt bindsbinds ligand and ligand and mediatesmediates homohomo-- oror heterohetero--dimerdimerizationization and and interactioninteraction withwith heatheatshockshock proteinsproteins

The The LBDsLBDs are formed by 12 conservedare formed by 12 conserved --helical regionshelical regions. On . On thethe 1212thth helisehelise itit containscontains an an AFAF--22 domaindomain, , responsibleresponsible for a ligandfor a ligand--dependent dependent transactivationtransactivation. . MutationsMutations withinwithin AF2 AF2 maymay leadlead to e.g. to e.g. androgenandrogen--insensitivityinsensitivity syndromesyndrome, , oror thyroidthyroid hormoneshormones--insensitivityinsensitivitysyndomesyndome..

ThisThis domaindomain includesincludes alsoalso otherother sequencessequences necessarynecessary for for transactivationtransactivation, , disperseddispersed inin thetherestingresting receptor, but receptor, but locatedlocated closelyclosely eacheach otherother upon ligand upon ligand bindingbinding..

A. LazarA. Lazar

Domain structure of nuclear receptorsDomain structure of nuclear receptors

N-terminal DBD LBD

NNuclearuclear receptorsreceptors regulateregulate transcriptiontranscription throughthrough bindingbinding to consensus to consensus sequencesequence inintarget target genesgenes. . TheseThese sequencessequences areare usuallyusually locatedlocated withinwithin promoterpromoter, but , but sometimessometimes theytheyareare eveneven severalseveral thousandthousand nucleotidesnucleotides upup-- oror downdown--streamstream of of thethe start of start of transcriptiontranscriptionsitesite..

TATA box

TATA box

TATA box

TATA box

consensus consensus sequensesequense

DNADNA

DNADNA

Early primary response

Delayed secondaryresponse

ligandligand

nuclearnuclear receptorsreceptors activateactivateprimaryprimary responseresponse genesgenes

primaryprimary responseresponse proteinsproteinsturnturn on on secondarysecondary responseresponse genesgenes

secondarysecondary responseresponse proteinsproteins

adopted from Deanna Kroetz, University of California, San Francisco

Consensus Consensus sequencesequence consistsconsists of of 6 nu6 nucleotidescleotides: :

AGAGAAAACACA ((recognizedrecognized by by thethe steroid steroid hormonehormone receptorsreceptors))

AGAGGGTCATCA ((recognizedrecognized by by thethe otherother receptorsreceptors))

AGAGTTTCATCA ((recognizedrecognized by by thethe otherother receptorsreceptors))

Consensus Consensus sequencessequences of of nuclearnuclear receptorsreceptors

fragment of fragment of acylCoAacylCoA oxidaseoxidasepromoterpromoter

TypTypee II ((homodimerhomodimericic receptorreceptorss of steroid of steroid hormoneshormones)) –– e.g.e.g. ER,ER, AR, GR, MR.AR, GR, MR.

TypTypee IIII ((hodimerichodimeric orphansorphans) ) –– e.g. e.g. RXRRXR

TTypypee IIIIII ((heterodimersheterodimers) ) –– e.g. e.g. TRTR, , RAR, VDR, PPAR.RAR, VDR, PPAR.

TypTypee IVIV ((monomericmonomeric orphansorphans) ) –– e.g. Nurre.g. Nurr--11

-- on on thethe basisbasis of of primaryprimary structurestructure nuclearnuclear receptorsreceptors theythey cancan be be classifiedclassified to to 7 7 familiesfamilies, , but one but one familyfamily maymay containcontain receptorsreceptors for for veryvery distinctdistinct ligandsligands, , whereaswhereas thethe same ligand same ligand cancan be be boudboud by by membersmembers of of differentdifferent familiesfamilies..

ClassificationClassification of of nuclearnuclear receptorsreceptors

On On thethe basisbasis of of ligand ligand bindingbinding and and dimerizationdimerization nuclearnuclearreceptorsreceptors cancan be be classifiedclassified to to 4 4 typtypeses::

SomeSome nuclearnuclear receptorsreceptors actact as as monomermonomerssbindingbinding to to hexamerhexamer of of nucleotidesnucleotides..

Most Most receptorsreceptors actact as as dimersdimers, , bindingbinding to to thethe sequencesequence of of twotwo hexamershexamers

DimersDimers cancan be be bothboth homohomo-- oror heterodimersheterodimers. In . In thethe latterlatter casecase thethe partner for partner for heterodimerizationheterodimerization isis alwaysalways a a nuclearnuclear receptor receptor RXRRXR

e.g.e.g. NurrNurr--11

homodimershomodimers RXR RXR heterodimersheterodimers

ERERGRGR

RARRARTRTRVDRVDRPPARPPAR

RXRRXR

consensus consensus sequencessequences recognizedrecognized by a by a dimersdimers cancan be:be:

palindrompalindromss ((onlyonly suchsuch sequencessequences cancan be be recognizedrecognized by steroid by steroid hormonehormone receptorsreceptors))

invertedinverted palindromspalindroms

directdirect repetitionsrepetitions (DR1, DR2, DR3 etc.) (DR1, DR2, DR3 etc.)

AGGTCA...TGACCTAGGTCA...TGACCT

AGGTCA...AGGTCA...AGGTCAAGGTCA

TCCAGT...ACTGGATCCAGT...ACTGGA

TCCAGT...TCCAGT...TCCAGTTCCAGT

ACTGGA...TCCAGTACTGGA...TCCAGTTGACCT...AGGTCATGACCT...AGGTCA

Consensus Consensus sequencessequences of of nuclearnuclear receptorsreceptors


Binding of receptors toBinding of receptors to theirtheir consensus consensus sequencessequences

Receptors can bind as monomers, Receptors can bind as monomers, homodimershomodimers or RXR or RXR heterodimersheterodimers..

Steroid receptors bind as Steroid receptors bind as homodimershomodimers to to palindromicpalindromicelements spaced by three elements spaced by three nucleotides.nucleotides.

MonomericMonomeric binding requires the binding requires the halfhalf--core motif precede by a 5core motif precede by a 5’’--flanking A/T reach sequence.flanking A/T reach sequence.

HeterodimersHeterodimers can can recognmizerecognmizediverse diverse HREsHREs in which halfin which half--core core motifs can be arranged as motifs can be arranged as polindromespolindromes, direct repeats or , direct repeats or inverted inverted polindromespolindromes

Permissive Permissive heterodimersheterodimers, such as PPAR/RXR, such as PPAR/RXR, FXR/RXR , FXR/RXR oror LXR/RXRLXR/RXR, can be , can be activated by activated by ligandsligands of either RXR or its partner receptor and are synergistically of either RXR or its partner receptor and are synergistically activated in the presence of both activated in the presence of both ligandsligands..


Permissive and nonPermissive and non--permissive permissive heterodimersheterodimers


In In nonnon--permissive permissive heterodimersheterodimers, such as RXR/RAR,, such as RXR/RAR, RXR/RT RXR/RT oror RXR/VDRRXR/VDRheterodimerizationheterodimerization precludes binding of the RXR ligand. precludes binding of the RXR ligand.

Binding of ligand to the RAR moiety causes receptor activation aBinding of ligand to the RAR moiety causes receptor activation and allows binding of nd allows binding of the RXR ligand resulting in synergism. the RXR ligand resulting in synergism.

Permissive and nonPermissive and non--permissive permissive heterodimersheterodimers


Ligand can be generated in three different ways: Ligand can be generated in three different ways:

11) an active ligand or hormone is synthesized in a ) an active ligand or hormone is synthesized in a classical endocrine organ and enters the cell, classical endocrine organ and enters the cell,

22) the ligand may be generated from a precursor or ) the ligand may be generated from a precursor or prohormoneprohormone within the target cell, and within the target cell, and

33) the ligand may be a metabolite synthesized within the ) the ligand may be a metabolite synthesized within the target cell.target cell.

The The unligandedunliganded receptor may have a nuclear location. receptor may have a nuclear location. However, some steroid receptors are However, some steroid receptors are cytoplasmiccytoplasmic in the in the absence of ligand due to their association with a large absence of ligand due to their association with a large multiproteinmultiprotein complex of chaperones, including complex of chaperones, including Hsp90Hsp90andand Hsp56Hsp56. . Ligand binding induces dissociation of the Ligand binding induces dissociation of the complex and nuclear translocation. Once in the nucleus, complex and nuclear translocation. Once in the nucleus, the receptors regulate transcription by binding, generally the receptors regulate transcription by binding, generally as as dimersdimers, to hormone response elements (, to hormone response elements (HREsHREs) ) normally located in regulatory regions of target genes.normally located in regulatory regions of target genes.

Nuclear receptors are activated after ligand Nuclear receptors are activated after ligand binding. binding.

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Mechanism of action of nuclear receptorsMechanism of action of nuclear receptors

AlternativeAlternative, , ligandligand--independent pathways for independent pathways for activation of nuclear receptorsactivation of nuclear receptors existexist::

•• Some receptors may be constitutively active, Some receptors may be constitutively active,

•• the activity of others is modulated by other the activity of others is modulated by other means, for instance,means, for instance, phosphorylationphosphorylation mediated by mediated by hormones and growth factors that stimulate diverse hormones and growth factors that stimulate diverse signal transduction pathways.signal transduction pathways.

Nuclear receptors Nuclear receptors cancan actactindependentlyindependently of of ligandsligands

•• TheThe hormonehormone transducestransduces itsits signalsignal throughthrough a a heterodimericheterodimericcomplexcomplex of of twotwo nulearnulear receptorsreceptors EcREcR ((ecdysoneecdysone receptor) and USP receptor) and USP ((ultraspiracleultraspiracle). ). TheThe EcREcR/USP /USP heterodimersheterodimers bind to bind to thethe ecdysteroidecdysteroidresponseresponse element (element (EcREEcRE) ) presentpresent inin thethe promoterpromoter of target of target genesgenes..

•• EcREcR/USP /USP directlydirectly inducesinduces genesgenes codingcoding for for transcriptiontranscription factorsfactors, , thatthat inin turnturn, , regulateregulate cascadecascade of of genesgenes essentialessential for for appropriateappropriatebiologicalbiological responsesresponses to 20to 20--hydroxyecdysone.hydroxyecdysone.

•• EcREcR mutantsmutants diedie duringduring earlyearly stagesstages of development. of development.

EcdysoneEcdysone and and EcREcR/USP /USP receptorsreceptors

•• EcdysteroidsEcdysteroids regulateregulate insectinsect development, development, reproductionreproduction, and , and severalseveral otherother physiologicalphysiologicalprocessesprocesses. . TheThe most most activeactive form form isis 2020--hydroxyecdysone (20E).hydroxyecdysone (20E).

• Production of ecdysone in prothoracic gland is induced by brain neuropeptide –prothoracicotropic hormone. Ecdysone is then converted in peripheral tissues to active molting hormone – 20-hydroxyecdysone (20E).

• The increase in 20E initiates the molt. It causes:- division of epidermis- its separation from the old cuticle- synthesis and secretion of new cuticle components

• 20E directs also much more complicated than ”simple” molts – metamorphic molts

EcdysoneEcdysone and and EcREcR/USP /USP receptorsreceptors

Moltingdragonfly

brain

prothoracic gland

Pulses of 20-hydroxyecdysone act as critical signal that direct each of themajor developmental transitions in the Drosophila life cycle, includingmolting and metamorfosis.

RegulatedRegulated by by EcREcR--likelike nuclearnuclear receptorsreceptors?.....?.....

MoltingMolting trilobitestrilobites……..

FruitFruit flyfly EcREcR cancan induceinduce genesgenes inin humanhuman cellscells

The transfected cells in medium containing DMSO or EcR activator. At 48 h after addition of ligand, cells were assayed for luciferase activity

Homo sapiens Locusta migratoria

LmUSPHsRXR

•• TheThe pERV3 pERV3 vectorvector isis engineeredengineered suchsuch thatthatbothboth receptorsreceptors areare expressedexpressed fromfrom a single a single mRNAmRNA transcribedtranscribed fromfrom thethe CMV CMV promoterpromoter..

•• ItIt isis accomplishedaccomplished by by placingplacing thethe internalinternalribosomeribosome entryentry sitesite (IRES) (IRES) upstreamupstream of of thethesecondsecond (RXR) (RXR) openopen readingreading frameframe (ORF), (ORF), whichwhich allowsallows highhigh--levellevel internalinternal (cap(cap--independent) independent) initiationinitiation of of translationtranslation. .

•• TheThe plasmidplasmid alsoalso containscontains a a neomycinneomycin--resistanceresistance genegene, , whichwhich isis expressedexpressed inin bothboth E. E. colicoli ((kanamycinkanamycin--resistanceresistance) and ) and mammalianmammaliancellscells (G418(G418--resistance).resistance).

EcREcR derivativesderivatives as as thethe transgenetransgene switchersswitchers

EcR and 20E are not found in mammaliancells, thus they are attractive target for development of gene switches.

In mammalian cells, EcR heterodimerizeswith RXR, the homologue of USP.

The EcR-RXR heterodimer is capable of binding to and activating reporters thatcontain multiple copies of the ecdysone-responsive element (EcRE).

EcR protein and EcRE recognitionsequence were modified to create both a synthetic ecdysone-inducible receptor that would not bind to and trans-activateany endogenous host genes, as well as a synthetic recognition site that would not be recognized by host transcriptionfactors.

WhatWhat wouldwould be be profitableprofitable to to rememberremember inin JuneJune::

-- structurestructure of of thethe nuclearnuclear receptorsreceptors

-- thethe wayway of action of of action of thethe nuclearnuclear receptorsreceptors

SSlideslides cancan be be foundfound inin thethe librarylibrary and and atat thethe HemeHeme OxygenaseOxygenase Fan Fan ClubClubpagepage::

http://http://biotka.mol.uj.edu.pl/~hemeoxygenasebiotka.mol.uj.edu.pl/~hemeoxygenase

ThankThank youyou and and seesee youyou nextnext weekweek......

Molting cicada

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