Research Article The Aryl-Hydrocarbon Receptor Protein … · 2019. 5. 13. ·...

Hindawi Publishing CorporationJournal of ToxicologyVolume 2013 Article ID 279829 12 pageshttpdxdoiorg1011552013279829

Research ArticleThe Aryl-Hydrocarbon Receptor Protein InteractionNetwork (AHR-PIN) as Identified by Tandem AffinityPurification (TAP) and Mass Spectrometry

Dorothy M Tappenden12 Hye Jin Hwang13 Longlong Yang4

Russell S Thomas4 and John J LaPres123

1 Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI 48824-1319 USA2Center for Integrative Toxicology Michigan State University East Lansing MI 48824-1319 USA3 Center for Mitochondrial Science and Medicine Michigan State University East Lansing MI 48824-1319 USA4The Hamner Institutes for Health Sciences Research Triangle Park NC 27709 USA

Correspondence should be addressed to John J LaPres lapresmsuedu

Received 2 August 2013 Revised 9 October 2013 Accepted 14 October 2013

Academic Editor Robert Tanguay

Copyright copy 2013 Dorothy M Tappenden et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aryl-hydrocarbon receptor (AHR) a ligand activated PAS superfamily transcription factor mediates most if not all of thetoxicity induced upon exposure to various dioxins dibenzofurans and planar polyhalogenated biphenyls While AHR-mediatedgene regulation plays a central role in the toxic response to dioxin exposure a comprehensive understanding of AHR biologyremains elusive AHR-mediated signaling starts in the cytoplasm where the receptor can be found in a complex with the heatshock protein of 90 kDa (Hsp90) and the immunophilin-like protein aryl-hydrocarbon receptor-interacting protein (AIP) Therole these chaperones and other putative interactors of the AHR play in the toxic response is not known To more comprehensivelydefine the AHR-protein interaction network (AHR-PIN) and identify other potential pathways involved in the toxic response aproteomic approach was undertaken Using tandem affinity purification (TAP) and mass spectrometry we have identified severalnovel protein interactionswith theAHRThese interactions physically link theAHR to proteins involved in the immune and cellularstress responses gene regulation not mediated directly via the traditional AHRARNT heterodimer and mitochondrial functionThis new insight into the AHR signaling network identifies possible secondary signaling pathways involved in xenobiotic-inducedtoxicity

1 Introduction

Dioxins a family of toxic chemical compounds that are highlystable and readily bioaccumulate have been the focus ofextensive research for several decades [1 2] Investigationshave shown that these compounds can arise naturally fromforest fires and cooking however industrial processes suchas paper bleaching and pesticide manufacturing produceddioxins in large quantities This increased production haslead to pervasive environmental contamination In fact over500000 tons of soil and sediment are contaminated with2378 tetrachlorodibenzo-p-dioxin (TCDD) in the US alone[3]

TCDD and other dioxins induce a host of toxic responsesin mammals such as thymic involution immunosuppres-sion wasting syndrome and chloracne The aryl-hydrocar-bon receptor (AHR) mediates virtually all of these dioxin-induced pathologies [4 5] Studies revealed that dioxinsserve as ligands which activate the AHR [6] a mem-ber of the basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS)superfamily of transcription factors [7] In the absence ofligand the AHR can be found in the cytosol bound to adimer of the heat shock protein of 90 kDa Hsp90 and theimmunophilin-like protein AIP (also known as XAP2 andARA9) [8 9] Upon ligand binding the AHR translocates to

2 Journal of Toxicology

the nucleus and binds its heterodimeric partner the aryl-hydrocarbon receptor nuclear translocator (ARNT) proteinThe AHRARNT dimer regulates expression of a battery ofgenes involved in metabolism and elimination includingxenobiotic metabolizing enzymes CYP1A1 and CYP1B1 [1011] The translocation and subsequent DNA binding of theAHR is necessary for TCDD-induced toxicity however thetoxicity associated with dioxin exposure does not directlycorrelate with the levels of the AHR found in the varioustissues [12ndash16] In fact the liver an organ with relatively highAHR expression is fairly resistant to TCDD-induced toxicity[17] This tissue-specific response raises the possibility thataccessory factors and secondary signaling are involved inmodulating the receptorrsquos ability to promote toxicity

Besides Hsp90 and AIP the AHR also has been shownto interact with p23 Cdk4 the retinoblastoma protein (Rb)and RelA of the NF120581B complex [16 18ndash20] Early reportsdemonstrated that p23 was a core AHR complex proteinThe p23 protein has been reported to function in numerouscellular roles including organelle function DNA repair andcell mobility [21] Recently p23 was shown to specificallyinteract with the N-terminus of Hsp90 [22] Other reportshave shown its interaction with the AHR to be transientand nonessential for the receptorrsquos function [23 24] Theinteractions with Cdk4 Rb and RelA link the receptor to cellcycle kinase signaling and gene regulation not exclusivelymediated by the AHR Moreover the AHR has been impli-cated in estrogen and glucocorticoid receptor signaling anddevelopmental processes [25ndash27] Taken together this mul-tifaceted interaction network suggests that AHR biology iscomplex and capable of impacting several cellular responses

The extent of the AHR protein interaction network(AHR-PIN) has not been addressed on a proteomic level ina mammalian system To investigate the role that protein-protein interactions have in AHR-mediated dioxin toxicitywe have established the AHR-PIN using tandem affinitypurification (TAP) and mass spectrometry (MS) Here wereport the AHR-PIN as assembled fromMS data collected inthe presence and absence of TCDD ligand using a Hepa1c1c7cell line with stable overexpression of an AHR-TAP-taggedconstruct [28] The Hepa1c1c7 cell line is extensively usedin AHR research and well characterized in its responses toTCDD exposure Although the liver is not considered highlysensitive to TCDD-induced toxicity the tissue does experi-encemarked changes in gene expression hyperlipidemia andhepatosteatosis [29] Defining the AHR-PIN in this cell lineoffers insights to how TCDD influences AHR biology andinduces toxicity in this tissue type IdentifiedAHR interactorsinclude mitochondrial cell cycle immune response andtranscription factor proteins The new AHR-PIN has identi-fied different secondary pathways that could potentially influ-ence TCDD-induced AHR-mediated toxicity Previously wereportedAHRrsquos influence onmitochondrial function throughits interaction with the ATP51205721 subunit of the ATP synthasecomplex [28]Herewe focus on the receptorrsquos interactionwithanother mitochondrial protein mitochondrial ribosomalprotein L40 (Mrpl40)

2 Materials and Methods

21 Materials Oligonucleotides were synthesized at theMacromolecule Synthesis Facility at Michigan State Uni-versity The pTarget and pGEM-T Easy vectors were pur-chased from Promega (Madison WI) and pZome1C vectorwas obtained from Cellzome (Cambridge UK) Antibodiesused in these experiments were obtained from the follow-ing resources rabbit polyclonal anti-AHR antibodies werea generous gift from Dr Christopher Bradfield (Univer-sity of Wisconsin-Madison) rabbit anti-Mrpl40 (cat noHPA006181) was purchased from Sigma (St Louis MO)mouse anti-COX4 (cat no A21348) was purchased from LifeTechnologies (Grand Island NY) goat anti-rabbit (cat nosc2004) goat anti-mouse (cat no sc2005) normal rabbit IgG(cat no sc2027) and proteinG resinwere obtained from SantaCruz (Santa Cruz CA) All other chemicals used in theseexperiments were reagent grade and purchased from SigmaAldrich (St Louis MO)

22 Plasmid Constructs and Cell Culture The cDNAs for themurine AHR and the green fluorescent protein (GFP) wereamplified using the following primers

AHR 51015840-ggatccccaccatgagcagcggcgccaacatcacc-31015840 and 51015840-ggatcctgcactctgcaccttgcttagg-31015840 GFP 51015840-gggggatccaccatggtg-agcaagggcgac-31015840 and 51015840-gtggatccccgggcccgcggtaccgtcgactgc-31015840 The amplicons were subcloned into pZome1C vectorplacing the TAP-tag at the C terminal of both the AHRand GFP genes Ampicillin resistance was used for clonalselection and each positive clone was sequence verifiedPhoenix-eco and Hepa1c1c7 cells were cultured in DMEMhigh glucose media with L-glutamate and supplementedwith 10 cosmic calf serum (CCS) 100 unitsmL peni-cillin 100 120583gmL streptomycin and 1mM sodium pyruvateHepaC12 cellswere cultured inDMEMhigh glucosemediawL-glutamate and supplemented with 10 cosmic calf serum(CCS) and 1mM sodium pyruvate Tissue culture media andsupplements were obtained from Life Technologies and CCSwas obtained fromHyClone (Waltman MA) Hepa1c17 TAP-AHRandTAP-GFPwere grown in themedia described aboveand puromycin (2120583gmL US Biological Swampscott MA)was used for selection

23 TransfectionStable Cell Line Infection The retroviralvectors AHR-TAP and GFP-TAP were transfected intoPhoenix-eco cells with Lipofectamine 2000 (Life Technolo-gies) via manufacturerrsquos instructions After incubation (5 to8 hours 37∘C) in the presence of DNA the media werechanged to Phoenix cell growth media containing chloro-quine (25 120583M) Cells were then incubated for 24 hours (37∘C)After incubation cells were given fresh Phoenix cell growthmedia and incubated for an additional 24 hours (32∘C)The media were collected after incubation centrifugationwas used to remove cellular debris (3mins 45timesg in RT7Sorvall Rockford IL) and the media were purified using a045 120583mmembrane filter (Millipore) Virus containingmediawas placed on Hepa1c1c7 wild type target cell lines andincubated for 3 hrs at 32∘C and 5 CO

2 Media containing

15 120583gmL polybrene were then added to target cells and

Journal of Toxicology 3

incubated for 24 hours (32∘C) After incubation fresh mediawere added to target cells and incubated (37∘C) until platesreached 80 confluence Cells were passaged and selectedusing puromycin (2120583gmL)

24 Western Blot Analysis Tissue culture samples were har-vested and total protein concentration was determined aspreviously described [30] Proteins samples were separatedonNu-Page Bis-Tris 4ndash12 gradient gels transferred to nitro-cellulose membranes and probed with assorted antibodies aspreviously described [31]Western blots were visualized usingPierce (Rockford IL) ECL western blotting substrate

25 Tandem Affinity Purification An estimated 25 times 109Hepa1c1c7 TAP-AH and TAP-GFP cell were challenged withDMSO (vehicle control 001) or TCDD (10 nM) for 30 120or 240 minutes in 3 independent experiments (Figure 1(a))Media were removed and cells were washed three times incold phosphate buffered saline (PBS) Cells were harvested inTAP-tag lysis buffer (TTLB 5 glycerol 50mM Tris pH 7550mM MgCl

2100mM NaCl 01 NP40 1mM DTT 1mM

Na3VO4 25mM NaF and protease inhibitor tablets) and

lysed by two cycles of freezethaw and insoluble material wasremoved by centrifugation (7000timesg 20mins) Next sampleswere incubated with 200 120583L of IgG Sepharose beads (GEHealthcare Waukesha WI) for 4 hrs at 4∘C and rotationBeads were collected by centrifugation (45timesg 30 sec) andtransferred to Poly-Prep Chromatography columns (BioRadHercules CA) Beads were extensively washed with TAP-tag lysis buffer and then with TEV cleavage buffer (10mMTris pH 75 150mM NaCl 01 NP40 05mM EDTA and1mMDTT) IgG beads were incubated (18 hrs 4∘C) in 15mLTEV cleavage buffer containing AcTEV protease (450 units)Eluates were collected by gravity flow Beads were washedwith 15mL of TEV buffer and 9mL of calmodulin bindingbuffer (CBB) (10mM 120573-mercaptoethanol 10mM Tris pH75 150mM NaCl 1mM MgOAc 1mM imidazole 01NP40 and 2mM DTT) Eluate and washes were combinedSamples were then incubated with 200120583L calmodulin affinityresin (Stratagene La Jolla CA) (4 hrs 4∘C) After incuba-tion calmodulin resin was transferred to new Ploy-PrepChromatography columns and resin was washed extensivelywith CBB Warm 3x SDS buffer (48 SDS 100mM TrispH 68 16 glycerol 8 120573-mercaptoethanol and 04bromophenol blue) was used to elute protein complexes fromresin (Figure 1(b))

26 Gel Electrophoresis andMass Spectrometry Samples wereseparated on a 4ndash12 Bis-Tris gradient gel (NuPage Invit-rogen) by electrophoresis SilverSNAP staining kit (PierceRockford IL) was used to visualize proteins accordingto manufacturerrsquos protocol and the gel was photographed(Figure 1(c)) Specific protein bands were excised from thegel matrix destained with SilverSNAP destaining kit (PierceRockford IL) and subjected to in-gel tryptic digestion as pre-viously described [32] (Figure 1(d)) The extracted peptideswere then automatically injected by aWaters nanoACQUITYSample Manager (Milford MA) loaded for 5 minutes onto aWaters Symmetry C18 peptide trap (5 120583m 180 120583m times 20mm)

at 4 120583Lmin in 5 acetonitrile01 formic acid The boundpeptides were eluted onto aWaters BEHC18 nanoACQUITYcolumn (17 120583m 100 120583m times 100mm) over 35 minutes witha gradient of 2 B to 35 B in 21min 90 B from 21ndash24min and back to constant 5 B at 241min using a WatersnanoACQUITY UPLC (buffer A = 999 water01 formicacid buffer B = 999 acetonitrile01 formic acid) withan initial flow rate of 600 nLmin ramping to 700 nLmin at80min and back to 600 nLmin at 86min Eluted peptideswere sprayed into a Thermo Fisher LTQ Linear Ion trapmass spectrometer outfitted with a MICHROM BioresourcesADVANCE nanospray source The top five ions in eachsurvey scan are then subjected to data-dependent zoomscans followed by low energy collision induced dissociation(CID) and the resulting MSMS spectra are converted topeak lists using BioWorks Browser v 331 (Thermo FisherRockford IL) using the default LTQ instrument parametersPeak lists were searched against all mouse sequences availablein the NCBI nr database downloaded on November 162008 fromNCBI using theMascot searching algorithm v22(httpwwwmatrixsciencecom) The Mascot output wasthen analyzed using Scaffold (httpwwwproteomesoftwarecom) to probabilistically validate protein identificationsusing the ProteinProphet computer algorithm (Figure 1(e))

27 Coimmunoprecipitation Wild type Hepa1c1c7 cells weregrown to 80 confluenceMediawere removed and cells werewashed with ice cold PBS and then harvested with TTLBCellular supernatants (500 120583g) were incubated with normalrabbit IgG (2 120583g120583L) or an anti-AHR antibody (2 120583g120583L) for90 minutes Following incubation protein G beads (30 120583L)were added to the samples and incubated for an additional 90minutes Beads were collected by centrifugation The super-natant was removed and the beads were washed extensivelyin TTLB Samples were eluted from the beads with warm3X SDS buffer and separated on a Nu-PAGE Bis-Tris 4ndash12gradient gel matrix transferred to nitrocellulose membraneand probed with primary AHR or Mrpl40 antibodies

28 Cellular Fractionation Wild type Hepa1c1c7 and AHRnull HepaC12 cell lines were treated with vehicle (001DMSO) or TCDD (10 nM) for 6 hrs After treatment cellswere harvested using fractionation buffer (25mM sucrose20mM Tris-HCl 1mM EDTA pH74) The cells underwentone round of freezethaw and then dounce homogenized (100stokes) Insoluble materials were removed by centrifugation(400timesg 10min) and the supernatant was transferred to a newtube An aliquot of this supernatant was taken as the wholecell lysate (WLC) fraction Separation of the supernatantand organelle fractions was performed by centrifugation(4500timesg 10min) The supernatant was removed and analiquot was taken as the cytosolic fraction (Cyto) Theorganelle pellet was resuspended in 1mL of fractionationbuffer and dounce homogenized (14ndash20 strokes) An aliquotof the pellet after resuspension and homogenization wastaken as the impure mitochondrial fraction (MF1) Largedebris was removed from the MF1 sample by centrifuga-tion (400timesg 10min) The supernatant was transferred to anew tube and mitochondria were isolated by centrifugation


DM

SO

Treatment Sample

DM

SO GFP-Hepa 1c1c7 cells

AHR-Hepa 1c1c7 cells




30min

120min

240minTC

DD10

nM40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

(a) Tissue culture experiment

IgG sepharosepurification IgG

IgG

Protein A

Protein A

TEV

TEV

CBD

CBD

CBD

CBD

AHR

AHR

AHR

AHR

TEV cleavage

CalmodulinCalmodulin sepharose

purification

AHR-interactingproteins

Identify AHR associated proteins using gel electrophoresis and mass spectrometry

(b) TAP-tagging method

AHRTCDD

MW GFP D 30 120 240

(c) Protein sample separation

Gel band excision and destaining

Reduction

Alkylation

Trypsin digestion

(d) In gel trypsin digestion

Probability legend Over 95

80 to 94

50 to 79

20 to 490 to 19

Bio viewIdentified proteins (8)

1 3

3

33

132

77

22

14

1 1

1

1

2 8

21

16

32

1

1

11

38kDa62kDa42kDa25kDa85kDa83kDa22kDa24kDaMitochondrial ribosomal protein L gi|6754862

Cardiotrophin-like cytokine factor gi|9910314

Hippocalcin-like 1 (Mus musculus) gi|7949055Heat shock protein 1 beta (Mus gi|40556608

Heat shock protein 1 alpha (Mus gi|6754254

Beta actin (Homo sapiens) gi|66 gi|4501885 Bcl2-associated athanogene 3 (M gi|1152709

Aryl-hydrocarbon receptor-intera gi|7709982

Acce

ssio

n nu

mbe

r

Mol

ecul

ar w

eigh

t

Prot

ein

grou

ping

ambi

guity

Dor

othy

A3

x

Dor

othy

A1

xx

Dor

othy

A2

xx

Dor

othy

ACx

(e)

Figure 1 Experimentation overview (a) Schematic outline of TCDD exposure experiments in Hepa1c1c7 cell lines GFP and AHR sampleswere treated with vehicle (DMSO (D) 001) the remaining three AHR samples were treated with TCDD (10 nM) for the labeled time (30120 and 240mins) (b) Schematic of the tandem affinity purification (TAP) protocol After TCDD exposure cell lysate samples underwenttwo rounds of purification utilizing the TAP methodology AHR and GFP TAP-tagged proteins and their associated complexes were isolatedand then separated using gel electrophoresis (c) Representative image of gel replicates for final TAP eluate samples separated in 4ndash12 Nu-page gel matrix Boxes around the areas of the AHR-TAP 240min TCDD dose sample lane denote the areas of the gel that were excised fromeach of the sample lanes (d) Schematic of the in-gel trypsin digestion protocol Excised gel samples were destained and proteins underwentreduction alkylation and trypsin digestion (e) Sample of MS analysis data set Lastly the protein fragment samples then underwent MSanalysis as described in Section 2


(4500timesg 10min)Themitochondrial pellet was resuspendedin 100 120583L fractionation buffer and is the pure mitochondrialfraction (MF2)

3 Results

31 Workflow for Proteomic Screen Each replicate of theproteomic screen was processed following the workflowdescribed in Figure 1 and detailed in the materials andmethods section

32 Identification of AHR Interactors TAPwas performed onDMSO treated GFP-TAP and AHR-TAP cells and AHR-TAPHepa1c1c7 samples treated with TCDD (10 nM) for differenttimes (30 120 240 minutes) followed by MS analysis of theTAP final eluatesThe cytosolic partners of the AHRHsp90aHsp90b and AIP were identified in the AHR-TAP sampleat a greater than 95 confidence The identification of thecore AHR cytosolic complex proteins demonstrates that thismethod is a valid means to investigate novel AHR proteininteractions

TCDD and vehicle treated AHR-TAP Hepa1c1c7 samplesfrom three separate experiments per treatment and timepoint were analyzed for AHR protein interactors The GFP-TAP vehicle treated sample data sets were used as a negativecontrol The TAP samples were separated into discreet bandsin a gel matrix and excised for MS analysis (Figure 1(c)) Thebreakdown of proteins identified per treatment group is asfollows There are 74 proteins identified in the vehicle treat-ment group of which 13 were identified in 2 or more replicatesamples 4 novel proteins 4 known interactors and 5 nonspe-cific interactors (Figure 2) The 30-minute TCDD treatmentgroups contained 66 proteins including 12 that were identi-fied in 2 or more of the replicates 6 novel proteins 2 knowninteractors and 4 nonspecific interactors (Figure 3) Thesmallest protein set a total of 51 proteins was identified in the120-minute TCDD treatment groupsThis group contained 10proteins that were identified in 2 or more replicates 6 novelinteractors 1 known interactor and 3 nonspecific interactors(Figure 4) Lastly the 240 minute TCDD treatment data setcontains 59 proteins of which 14 were identified in multiplereplicates 10 novel interactors no known interactors and 4nonspecific interactors (Figure 5) Here novel proteins areconsidered previously unreported AHR interactions some ofthese novel proteins were found in more than one data setandwill be discussed belowThe group of previously reportedproteins includedHsp90 isoforms Hsp90120572 andHsp90120573 AIPand the ATP synthase F1 complex alpha subunit (ATP51205721)Nonspecific interactorswere found in both theAHR-TAPandGFP-TAP sample data sets and will not be discussed furtherThe unique proteins of these data sets are detailed below

In the DMSO treated AHR-TAP samples two proteins areexclusive to this data set (Figure 2) One is the previouslyreported ATP51205721 subunit of the ATP synthase complex [28]The other is Tesp4 a ubiquitously expressed homolog of pan-creatic trypsin [33] One protein Mrpl40 was also identifiedin the 30- and 240-minute TCDD treatment samples In yeastthis protein has been shown to play a role in growth ratemitochondrial protein folding and mitochondrial function

[34 35] In humans the Mrpl40 gene is part of a chro-mosomal deletion of 22q11 in velo-cardio-facial syndrome(VCFS) and DiGeorge syndrome [36 37]The remaining twoproteins cardiotrophin-like cytokine factor 1 (Clcf1) andHIVTAT specific factor 1 (Htatsf1) were identified in this and allthree (30 120 240minute) TCDD treatment data sets Clcf1 isa cytokine highly expressed in tissues of the immune systemand is an activator of kinase pathways [38 39] Htatsf1 is atranscriptional cofactor that regulates expression of theHIV-1LTR [40]

The 30-minute TCDD treated AHR-TAP samples con-tained one protein exclusive to this treatment set thecAMP responsive element binding protein 3-like 3 (Creb3l3)(Figure 3) Creb3l3 which is also known as CrebH functionsas an endoplasmic reticulum bound transcription factorresponsible for regulating hepatic gluconeogenesis [41] Twoof these proteins Enhancer-trap-locus-1 (Smarcad SWISNF-related) and Eukaryotic translation elongation factor 1 alpha 1(Eef1a1) were identified in all three of the TCDD treatedsample sets Smarcad functions as a DNA helicase associatedwith SWISNF complexes [42] Eef1a1 facilitates proteinsynthesis through tRNA transfer to ribosomal machinery[43]The remaining three proteins in this sample set Mrpl40Clcf1 and HIV TAT were introduced above

There were no unique proteins to the 120min TCDDtreated AHR-TAP sample data sets There are two proteinsin common between this and the 240min TCDD treateddata sets an uncharacterized transcript hypothetical pro-tein LOC56279 and the activated leukocyte cell adhesionmolecule CD166 (Alcam) (Figure 4) Alcam has becomewidely recognized as a cellular marker for several formsof cancer including melanoma squamous cell carcinomaprostate breast colorectal bladder esophageal and ovariancancers [44ndash46] The four remaining proteins of these datasets Smarcad Eef1a1 Clcf1 and HIV TAT have been dis-cussed above

There were three unique proteins to the 240 min-utes TCDD treatment AHR-TAP samples Bcl2-associatedathanogene 3 (Bag3) ArfGAP with SH3 domain ankyrinrepeat and PH domain 2 (Asap2) and ubiquitin associ-ated protein 2-like (Ubap2l) (Figure 5) Bag3 the secondmitochondrial associated protein identified is a cochaperoneprotein involved in the stress response and disease [4748] Bag3 is considered a prooncoprotein by inhibiting theapoptotic response through its influence on Bcl-2 familymembers [49 50] Asap2 also known as development anddifferentiation enhancing factor 1 (Ddef1) is an Arf-GTPaseactivating protein [51 52] Ubap2l is associated with protea-some mediated protein degradation and has been implicatedin infertility [53] The seven remaining proteins identified inthese data sets were introduced in the previous data sets

33 Coimmunoprecipitation Verification of Identified Interac-tors Independent verification of the interactions betweenthe AHR and select proteins identified by mass spectrome-try was performed by coimmunoprecipitation in wild typeHepa1c1c7 cells Similar to previously reported verificationof the AHRATP51205721 interaction [28] this method was usedto investigate the interaction between the AHR and Mrpl40


Trial 1 Trial 2

2

2613 5

42

22

Trial 3

DMSO control

(a)

Aip 11632Hsp90aa1 15519

11946Cardiotrophin-like cytokine factor 1 Clcf1 56708

Hsp90ab1 15516HIV TAT specific factor 1 Htatsf1 72459Mitochondrial ribosomal protein L40 Mrpl40 18100Tesp4 protein 1810049H19Rik 435889

ACTB 60Il18 16173

Transferrin receptor Tfrc 22042Tubulin 5 Tubb5 22154Unnamed protein product A430108E01Rik 384382

Protein Entreznumber

Officialsymbol

Atp51205721ATP synthase F1 complex alpha subunit

Aryl-hydrocarbon receptor-interacting protein 9lowast

Interferon gamma inducing factorBeta actin

Heat shock protein 1 beta

Heat shock protein 1 alphalowast

(b)

Figure 2 DMSO control data set (a) Venn diagram of proteins identified by mass spectrometry in 3 data sets of AHR-TAP DMSO (001)control (b) Table listing of proteins identified in two out of three data sets Bold type with lowast denotes a protein represented in all three datasets Italicized type denotes nonspecific proteins identified in both AHR-TAP and GFP-TAP samples

Trial 1 Trial 2

1

2318 1

73

13

Trial 3

30min exposure

(a)

116321551656708

cAMP responsive element binding protein 3-like 3 208677Enhancer-trap-locus-1 Smarcad1 13990

Eef1a1 136277245918100

Hippocalcin-like 1 Hpcal1 536021617322154

384382


Officialsymbol

AipHsp90ab1

Clcf1Creb3l3

Htatsf1Mrpl40

Il18Tubb5

A430108E01Rik

Cardiotrophin-like cytokine factor 1

HIV TAT specific factor 1 Mitochondrial ribosomal protein L40

TubulinUnnamed protein product

Eukaryotic translation elongation factor 1 alpha 1

Interferon gamma inducing factor

Heat shock protein 1 betaAryl-hydrocarbon receptor-interacting protein 9lowast

(b)

Figure 3 TCDD 30-minute treatment data set (a) Venn diagram of proteins identified by mass spectrometry in 3 data sets of AHR-TAP30min TCDD (10 nM) dosing (b) Table listing of proteins identified in two out of three data sets Bold type with lowast denotes a proteinrepresented in all three data sets Italicized type denotes nonspecific proteins identified in both AHR-TAP and GFP-TAP samples

(Figure 6) Mrpl40 was chosen to further investigate thedirect link between theAHRandmitochondrial function thatwas recently reported [28] Mrpl40 showed marked enrich-ment in the AHR Co-IP samples over the normal mouse IgGcontrol These interactions were reproducible in at least twoof the three trials performed The functional consequence ofthese interactions is currently under investigation

34 TCDD Influence on MRPL40 Cellular Localization Wepreviously reported that TCDD exposure decreased the poolof AHR in the mitochondrial but did not alter ATP51205721 levels

[28] Given AHRrsquos interaction with MRPL40 we wantedto determine if TCDD-induced depletion of mitochondriallocalized AHR impacted the cellular localization ofMRPL40To investigate this cellular fractionation was performed onwild type Hepa1c1c7 cells treated with TCDD (10 nM) andvehicle (DMSO) for 6 hrs (Figure 7(a)) A marked decreasein mitochondrial MRPL40 levels was observed in the 6 hrTCDD treatment samples Next we examined the localizationof Mrpl40 in the AHR null cell line HepaC12 The AHRnull cell line does not experience a decrease in mitochondrialMrpl40 levels in the presence of TCDD (Figure 7(b))


Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708

Smarcad1 SWISNF-related Smarcad1 13990

Eef1a1 13627

15516

72459

Hypothetical protein LOC56279 56279

5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik

HIV TAT specific factor 1

Unnamed proteinlowast

Hippocalcin-like 1lowast

Eef1a1 eukaryotic translation elongation factor 1 alpha 1

Cardiotrophin-like cytokine factor 1lowastActivated leukocyte cell adhesion molecule CD166lowast

Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658

56708Bcl2-associated athanogene 3

Smarcad1 13990Arf-GAP with SH3 domain ankyrin repeat and PH domain containing protein 2 Asap2 211914

Eef1a1 13627724595627918100

Ubiquitin associated protein 2-like Ubap2l 74383

Tuba 1b 22143Hpcal1 53602

16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik



Mitochondrial ribosomal protein L40

Unnamed protein product

Smarcad1 SWISNF-related

Hypothetical protein LOC56279

Activated leukocyte cell adhesion molecule CD166lowast


Alpha-tubulin isotype M-alpha-2Hippocalcin-like 1


(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR

Figure 6 Independent protein interaction verification Coimmunoprecipitations (Co-IP) using anti-AHR or normal rabbit IgG as negativecontrol were performed in wild type Hepa1c1c7 cells (a) Western blot probed with anti-MRPL40 (Invitrogen) (b) Positive control westernblot probed with anti-AHR MRPL40 and AHR control all show marked enrichment in the AHR pulldown lanes over the IgG control lane


Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)

Figure 7 TCDD treatment influences Mrpl40 mitochondrial localization (a) Cellular fractionation was performed on AHR in wild typeHepa1c1c7 cells Fractions from cells exposed to TCDD for 6 hours were probed with antibodies specific to MRPL40 lactate dehydrogenase(LDH) and cytochrome c oxidase subunit 4 (COXIV)The results demonstrate a decreased level of mitochondrial localizedMrpl40 followingTCDD exposure (b) Similar cellular fractionations were next performed on AHR null Hepac12 cells No decrease in Mrpl40 was observedafter exposure toTCDD for 6 hours LDHwas used as the cytosolic fraction control protein andCOXIVwas used as the purifiedmitochondrialfraction control protein

4 Discussion

The AHRrsquos functional relevance is centered on sensingenvironmental pollutants and regulating developmental pro-cesses Identifying interacting partners for the AHR there-fore is important to our understanding of the receptor-mediated changes in signaling pathways involved in theseprocesses The research detailed here represents the firstcomprehensive analysis of the AHR-PIN in a mammaliansystem using proteomic technology The AHR-PIN revealsphysical interactionswith proteins involved in several cellularprocesses and disease states with which the receptor hasbeen previously linked These include cell cycle apoptosisimmune response mitochondrial function and cancer [1654ndash58] In addition to AHR mediated gene regulation thisdata suggests the receptorrsquos potential influence on cellularbiology through proteinprotein interactions

A 2004 study conducted using S cerevisiae yeast asa model system reported 54 genes which influence AHRbiology [59] Interestingly we have identified proteins thatdemonstrate some correlation with this data In yeast SNF12and SWI3 were shown to influence the AHR and here wereport a protein interaction between the AHR and SMAD-CAD a helicase linked to the SWISNF complex In additionsimilar to the identified yeast genes encoding kinases andGTPases we identified several proteins involved in kinasesignaling including Asap2 and Clcf1While these similaritiesare not direct correlations they do provide another layer ofevidence to the complexity of AHR biology and the possiblemechanisms involved in AHR mediate toxicity and cellularfunction

There are previously reported AHR protein interactionsthat were not observed here that are of note The cytosoliccomplex protein p23 was not identified in any of our MS dataas stated earlier Given the transient nature of its interactionwith the AHR its absence is not wholly unexpected We alsodid not identify Rb or RelA in any of the data sets Theseinteractions are also of a transient nature making identi-fication difficult and we cannot rule out cell type-specificinteractions [19 60] Moreover it is possible that the levelof these proteinsrsquo interaction with the AHR is below thedetection limit of this methodology as well Interestingly wedid identify Cdk4 in some of ourMSdata however this resultwas not consistent across multiple data sets but the findingscorrelate with earlier reports of AHRCDK4 crosstalk [16]Finally the AHRrsquos nuclear partner ARNT was detected invarious TCDD samples across the dosing time course TheARNT detected however did not meet our threshold cutoff(gt80 confidence in two or more samples) in a given timepoint to be included in the tables This is most likely due tothe inherent variability in the assay and the transient natureof the interaction

The absence of these known interactors and the variabilityin the data sets were also noted This type of variability isnot unexpected given that many of the proteins were nearthe limit of detection of the methodology It is difficult todetermine significant differences between samples as oneoperates closer to the limit of detection [61] This increasedvariability might also be involved in the transient identifi-cation of some of the identified interactors For exampleMrpl40 was identified in every sample except the 120-minuteone This might be biologically important or it might be


due to the inherent variability in the methodology and thelimitations in terms of sample size and ability to detect lowabundant proteins This experimental variability might alsobe confounded by the overexpression system used in thisscreen It is difficult however to determine the extent towhich overexpression has influenced the observations Inaddition the transient nature and complex sample handlingprotocol will also induce significant amounts of variabilitybetween samples For data analysis of novel AHR proteininteractions we relaxed the confidence level to 80 cutoffThis was done to include proteins that were identified ata greater than 95 confidence in at least one replicateexperiment but not all replicates It should be noted thatthe variability seen in this screen is consistent with previouspublished reports for 14-3-3 and c-Myc TAP experiments[62 63] Finally given the inherent variability in this typeof analysis it is as important to investigate the biologicalimportance of those interactions that occurred in a singlesample with high significance as it is to investigate those thatoccurred in multiple replicates

Previous studies have linked TCDD exposure to mito-chondrial dysfunction including hyperpolarization of theinner membrane influence on ATP levels and ROS produc-tion [57 64 65] The AHRATP51205721 interaction and an AHR-dependent hyperpolarization of the mitochondrial innermembrane that was independent of transcription indicatea role for the AHR in mitochondrial function and cellularenergetic [28] Taken together the novel interaction betweentheAHR and two othermitochondrial associated proteins areof great interestThe first Bag3 a prooncoprotein is involvedin the mitochondrial stress response and disease [47 48] Ithas also been shown to inhibit the apoptotic response throughinteractions with Bcl-2 family members [49 50] Moreoverthere is evidence that it influences theNF120581B pathway throughits interaction with p65 The interaction between Bag3 andp65 plays a role in the human immunodeficiency virustype 1 (HIV-1) LTR response to NF120581B signaling [66] Thephysical interactions between Bag3 and the AHR providean interesting connection between the AHR and cell cycleregulation apoptosis and the NF120581B pathway [20] AHRrsquosinteraction with Bag3 observed after the longest TCDD treat-ment could indicate amechanismbywhich activated receptorinfluences apoptotic signaling cascadesThe second Mrpl40influences growth rate mitochondrial protein folding andmitochondrial function in yeast [34 35] It is linked totwo diseases in humans velo-cardio-facial and DiGeorgesyndromes [36 37] Interestingly these diseases present withheart defects and immune deficiencies AHR null mice havebeen shown to have abnormal heart development Further-more the immune system is one of the more sensitive toTCDD-induced toxicity [67ndash69] We have currently focusedour attentions on the AHRMrpl40 interactions

These AHR protein interactions could indicate otherroles for the receptor in cellular energy homeostasis andgene regulation not directly mediated by DNA bindingThe temporal differences in the AHR interactions with themitochondrial proteins reported here and the ATP51205721 afterTCDD exposure are of significant interest The AHRMrpl40interaction appears transiently across TCDD treatments in

our MS studies Cellular fractionation experiments demon-strated a decreased mitochondrial pool of Mrpl40 afterTCDD treatment in AHR expressing hepatoma cells Thisdecrease in mitochondrial Mrpl40 was not observed in theAHR null hepatoma cell line It is noteworthy that thedecreased mitochondrial pool of Mrpl40 is observed at thesame TCDD exposure time point 6 hrs as we observed theincrease in membrane polarization previously reported [28]

The novel interactions reported here demonstrate poten-tial roles for the AHR in signaling pathways which mayfunction in concert with AHR-mediated gene regulationin dioxin induced toxicity While the physical interactionsreported here provide another layer of evidence for the AHRrsquospotential roles in several signaling pathways disease statesand gene regulation events the functional relevance of theseinteractions remains in question This AHR-PIN presentsnew avenues of investigation to better define a comprehensiveunderstanding of AHR biology

Acknowledgments

The authors would like to thank Drs Christopher Bradfieldand Garry Nolan for their willingness to share cell linesand reagents This work was supported by NIH Grant P42ES04911

References

[1] A Poland and E Glover ldquoChlorinated dibenzo p dioxinspotent inducers of 120575 aminolevulinic acid synthetase and arylhydrocarbon hydroxylase II A study of the structure activityrelationshiprdquoMolecular Pharmacology vol 9 no 6 pp 736ndash7471973

[2] J C Knutson and A Poland ldquo2378-tetrachlorodibenzo-p-dioxin toxicity in vivo and in vitrordquo in Toxicity of HalogenatedHydrocarbons M A Q Khan and R H Stanton Eds pp 187ndash201 Pergamon Press New York NY USA 1981

[3] PHaglund ldquoMethods for treating soils contaminatedwith poly-chlorinated dibenzo-p-dioxins dibenzofurans and other poly-chlorinated aromatic compoundsrdquo Ambio vol 36 no 6 pp467ndash474 2007

[4] J V Schmidt G H T Su J K Reddy M C Simon and C ABradfield ldquoCharacterization of amurineAhr null allele involve-ment of the Ah receptor in hepatic growth and developmentrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 93 no 13 pp 6731ndash6736 1996

[5] A Poland and E Glover ldquoCharacterization and strain distribu-tion pattern of themurine Ah receptor specified by the119860ℎd and119860ℎ

bminus3 allelesrdquoMolecular Pharmacology vol 38 no 3 pp 306ndash312 1990

[6] R S Pollenz C A Sattler and A Poland ldquoCharacterization ofthe Ah receptor for 2378-tetrachlrodibenzo-p-dioxin use ofchemical crosslinking and a monoclonal antibody directedagainst a 59-kDa protein associated with sterod receptorsrdquoMolecular Pharmacology vol 45 no 3 pp 428ndash438 1994

[7] R Robles Y Morita K K Mann et al ldquoThe aryl hydrocarbonreceptor a basic helix-loop-helix transcription factor of the PASgene family is required for normal ovarian germ cell dynamicsin the mouserdquo Endocrinology vol 141 no 1 pp 450ndash453 2000

[8] L A Carver V Jackiw and C A Bradfield ldquoThe 90-kDa heatshock protein is essential for Ah receptor signaling in a yeast


expression systemrdquoThe Journal of Biological Chemistry vol 269no 48 pp 30109ndash30112 1994

[9] L A Carver J J LaPres S Jain E E Dunham and C A Brad-field ldquoCharacterization of the Ah receptor-associated proteinARA9rdquoThe Journal of Biological Chemistry vol 273 no 50 pp33580ndash33587 1998

[10] DWNebert S A Atlas TMGuenthner andR E Kouri ldquoTheAh locus genetic regulation of the enzymes which metabolizepolycyclic hydrocarbons and the risk for cancerrdquo in PolycyclicHydrocarbons and Cancer Chemistry Molecular Biology andEnvironment P O P Tsrsquoo and H V Gelboin Eds p 345Academic Press New York NY USA 1978

[11] DWNebert A L RoeM Z DieterW A Solis Y Yang and TP Dalton ldquoRole of the aromatic hydrocarbon receptor and [Ah]gene battery in the oxidative stress response cell cycle controland apoptosisrdquo Biochemical Pharmacology vol 59 no 1 pp 65ndash85 2000

[12] M K Bunger E Glover S M Moran et al ldquoAbnormal liverdevelopment and resistance to 2378-tetrachlorodibenzo-p-dioxin toxicity inmice carrying amutation in theDNA-Bindingdomain of the aryl hydrocarbon receptorrdquo Toxicological Scien-ces vol 106 no 1 pp 83ndash92 2008

[13] M K Bunger S M Moran E Glover et al ldquoResistance to2378-tetrachlorodibenzo-p-dioxin toxicity and abnormal liverdevelopment in mice carrying a mutation in the nuclear locali-zation sequence of the aryl hydrocarbon receptorrdquo The Journalof Biological Chemistry vol 278 no 20 pp 17767ndash17774 2003

[14] L A Carver J B Hogenesch and C A Bradfield ldquoTissue spe-cific expression of the rat Ah-receptor and ARNT mRNAsrdquoNucleic Acids Research vol 22 no 15 pp 3038ndash3044 1994

[15] A B Okey ldquoAn aryl hydrocarbon receptor odyssey to the shoresof toxicology the deichmann lecture international congress oftoxicology-XIrdquo Toxicological Sciences vol 98 no 1 pp 5ndash382007

[16] M A Barhoover J M Hall W F Greenlee and R S ThomasldquoAryl hydrocarbon receptor regulates cell cycle progression inhuman breast cancer cells via a functional interaction withcyclin-dependent kinase 4rdquo Molecular Pharmacology vol 77no 2 pp 195ndash201 2010

[17] H I Swanson and C A Bradfield ldquoThe AH-receptor geneticsstructure and functionrdquo Pharmacogenetics vol 3 no 5 pp 213ndash230 1993

[18] K A Hutchison L F Stancato J K Owens-Grillo et al ldquoThe23-kDa acidic protein in reticulocyte lysate is the weakly boundcomponent of the hsp foldosome that is required for assemblyof the glucocorticoid receptor into a functional heterocomplexwith hsp90rdquoThe Journal of Biological Chemistry vol 270 no 32pp 18841ndash18847 1995

[19] N L Ge and C J Elferink ldquoA direct interaction between thearyl hydrocarbon receptor and retinoblastoma protein linkingdioxin signaling to the cell cyclerdquo The Journal of BiologicalChemistry vol 273 no 35 pp 22708ndash22713 1998

[20] Y Tian S Ke M S Denison A B Rabson and M A GalloldquoAh receptor andNF-120581B interactions a potentialmechanism fordioxin toxicityrdquoThe Journal of Biological Chemistry vol 274 no1 pp 510ndash515 1999

[21] F J Echtenkamp E Zelin E Oxelmark et al ldquoGlobal functionalmap of the p23 molecular chaperone reveals an extensivecellular networkrdquo Molecular Cell vol 43 no 2 pp 229ndash2412011

[22] G E Karagoz A M S Duarte H Ippel et al ldquoN-terminaldomain of human Hsp90 triggers binding to the cochaperone

p23rdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 2 pp 580ndash585 2011

[23] M B Cox and C A Miller III ldquoCooperation of heat shockprotein 90 and p23 in aryl hydrocarbon receptor signalingrdquoCellStress and Chaperones vol 9 no 1 pp 4ndash20 2004

[24] B D Hollingshead J R Petrulis and G H Perdew ldquoThe arylhydrocarbon (Ah) receptor transcriptional regulator hepatitisB virus X-associated protein 2 antagonizes p23 binding toAh receptor-Hsp90 complexes and is dispensable for receptorfunctionrdquo The Journal of Biological Chemistry vol 279 no 44pp 45652ndash45661 2004

[25] B D Abbott M R Probst G H Perdew and A R BuckalewldquoAH receptor ARNT glucocorticoid receptor EGF receptorEGF TGF 120572 TGF 120573 1 TGF 120573 2 and TGF 120573 3 expression inhuman embryonic palate and effects of 2378-tetrachlorodi-benzo-p-dioxin (TCDD)rdquo Teratology vol 58 no 2 pp 30ndash431998

[26] T V Beischlag and G H Perdew ldquoER120572-AHR-ARNT protein-protein interactions mediate estradiol-dependent transrepres-sion of dioxin-inducible gene transcriptionrdquo The Journal ofBiological Chemistry vol 280 no 22 pp 21607ndash21611 2005

[27] L Biegel and S Safe ldquoEffects of 2378-tetrachlorodibenzo-p-dioxin (TCDD)on cell growth and the secretion of the estrogen-induced 34- 52- and 160-kDa proteins in human breast cancercellsrdquo Journal of Steroid Biochemistry andMolecular Biology vol37 no 5 pp 725ndash732 1990

[28] D M Tappenden S G Lynn R B Crawford et al ldquoThe arylhydrocarbon receptor interacts with ATP51205721 a subunit of theATP synthase complex and modulates mitochondrial func-tionrdquo Toxicology and Applied Pharmacology vol 254 no 3 pp299ndash310 2011

[29] J V Schmidt and C A Bradfield ldquoAH receptor signalingpathwaysrdquo Annual Review of Cell and Developmental Biologyvol 12 pp 55ndash89 1996

[30] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo The TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[31] A Vengellur and J J LaPres ldquoThe role of hypoxia inducible fac-tor 1120572 in cobalt chloride induced cell death inmouse embryonicfibroblastsrdquo Toxicological Sciences vol 82 no 2 pp 638ndash6462004

[32] A Shevchenko M Wilm O Vorm and M Mann ldquoMass spec-trometric sequencing of proteins from silver-stained polyacryl-amide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash8581996

[33] K Ohmura N Kohno Y Kobayashi et al ldquoA homologue ofpancreatic trypsin is localized in the acrosome of mammaliansperm and is released during acrosome reactionrdquoThe Journal ofBiological Chemistry vol 274 no 41 pp 29426ndash29432 1999

[34] L Jia J Kaur and R A Stuart ldquoMapping of the Saccharomycescerevisiae oxa1-mitochondrial ribosome interface and identifi-cation ofMrpL40 a ribosomal protein in close proximity to oxaland critical for oxidative phosphorylation complex assemblyrdquoEukaryotic Cell vol 8 no 11 pp 1792ndash1802 2009

[35] R Accardi E Oxelmark N Jauniaux V de Pinto A Marchiniand M Tommasino ldquoHigh levels of the mitochondrial largeribosomal subunit protein 40 prevent loss of mitochondrialDNA in null mmfl Saccharomyces cerevisiae cellsrdquo Yeast vol 21no 7 pp 539ndash548 2004

[36] T M Maynard D W Meechan M L Dudevoir et al ldquoMito-chondrial localization and function of a subset of 22q11 deletion


syndrome candidate genesrdquo Molecular and Cellular Neuro-science vol 39 no 3 pp 439ndash451 2008

[37] B Funke A Puech B Saint-Jore R Pandita A Skoultchi andB Morrow ldquoIsolation and characterization of a human genecontaining a nuclear localization signal from the critical regionfor velo-cardio-facial syndrome on 22q11rdquoGenomics vol 53 no2 pp 146ndash154 1998

[38] C J Auernhammer N B Isele F B Kopp et al ldquoNovelneurotrophin-1B cell-stimulating factor-3 (cardiotrophin-likecytokine) stimulates corticotroph function via a signal trans-ducer and activator of transcription-dependent mechanismnegatively regulated by suppressor of cytokine signaling-3rdquoEndocrinology vol 144 no 4 pp 1202ndash1210 2003

[39] E Lelievre H Plun-Favreau S Chevalier et al ldquoSignaling path-ways recruited by the cardiotrophin-like cytokinecytokine-like factor-1 composite cytokine Specific requirement of themembrane-bound form of ciliary neurotrophic factor receptor120572 componentrdquoThe Journal of Biological Chemistry vol 276 no25 pp 22476ndash22484 2001

[40] G Blot S Lopez-Verges C Treand et al ldquoLuman a newpartnerof HIV-1 TMgp41 interferes with Tat-mediated transcription ofthe HIV-1 LTRrdquo Journal of Molecular Biology vol 364 no 5 pp1034ndash1047 2006

[41] M W Lee D Chanda J Yang et al ldquoRegulation of hepaticgluconeogenesis by an ER-bound transcription factor CREBHrdquoCell Metabolism vol 11 no 4 pp 331ndash339 2010

[42] C N Adra J Donato R Badovinac et al ldquoSMARCAD1 anovel human helicase family-defining member associated withgenetic instability cloning expression and mapping to 4q22-q23 a band rich in breakpoints and deletion mutants involvedin several human diseasesrdquoGenomics vol 69 no 2 pp 162ndash1732000

[43] K Moldave ldquoEukaryotic protein synthesisrdquo Annual Review ofBiochemistry vol 54 pp 1109ndash1149 1985

[44] C Kahlert H Weber C Mogler et al ldquoIncreased expressionof ALCAMCD166 in pancreatic cancer is an independentprognostic marker for poor survival and early tumour relapserdquoThe British Journal of Cancer vol 101 no 3 pp 457ndash464 2009

[45] V Kulasingam Y Zheng A Soosaipillai A E Leon MGion and E P Diamandis ldquoActivated leukocyte cell adhesionmolecule a novel biomarker for breast cancerrdquo InternationalJournal of Cancer vol 125 no 1 pp 9ndash14 2009

[46] S F Ofori-Acquah and J A King ldquoActivated leukocyte celladhesion molecule a new paradox in cancerrdquo TranslationalResearch vol 151 no 3 pp 122ndash128 2008

[47] S Song S Kole P Precht M J Pazin and M BernierldquoActivation of heat shock factor 1 plays a role in pyrroli-dine dithiocarbamate-mediated expression of the co-chaperoneBAG3rdquo International Journal of Biochemistry and Cell Biologyvol 42 no 11 pp 1856ndash1863 2010

[48] A K McCollum G Casagrande and E C Kohn ldquoCaught inthemiddle the role of Bag3 in diseaserdquo Biochemical Journal vol425 no 1 pp e1ndashe3 2010

[49] M Iwasaki S Homma A Hishiya S J Dolezal J C Reed andS Takayama ldquoBAG3 regulates motility and adhesion of epithe-lial cancer cellsrdquo Cancer Research vol 67 no 21 pp 10252ndash10259 2007

[50] A Rosati E Di Salle L Luberto et al ldquoIdentification of a Btk-BAG3 complex induced by oxidative stressrdquo Leukemia vol 23no 4 pp 823ndash824 2009

[51] S Hashimoto A Hashimoto A Yamada Y Onodera and HSabe ldquoAssays and properties of the ArfGAPs AMAP1 and

AMAP2 in Arf6 functionrdquo Methods in Enzymology vol 404pp 216ndash231 2006

[52] CMatsui S Kaieda T Ikegami and YMimori-Kiyosue ldquoIden-tification of a link between the SAMP repeats of adenomatouspolyposis coli tumor suppressor and the Src homology 3domain of DDEFrdquoThe Journal of Biological Chemistry vol 283no 47 pp 33006ndash33020 2008

[53] R K Naz and L Dhandapani ldquoIdentification of human spermproteins that interact with human zona pellucida3 (ZP3) usingyeast two-hybrid systemrdquo Journal of Reproductive Immunologyvol 84 no 1 pp 24ndash31 2010

[54] N I Kerkvliet ldquoImmunological effects of chlorinated dibenzo-p-dioxinsrdquoEnvironmental Health Perspectives vol 103 no 9 pp47ndash53 1995

[55] J G Vos andH van Loveren ldquoMarkers for immunotoxic effectsin rodents andmanrdquo Toxicology Letters vol 82-83 pp 385ndash3941995

[56] A Puga CMa and J LMarlowe ldquoThe aryl hydrocarbon recep-tor cross-talks with multiple signal transduction pathwaysrdquoBiochemical Pharmacology vol 77 no 4 pp 713ndash722 2009

[57] H A Aly and O Domenech ldquoCytotoxicity and mitochondrialdysfunction of 2378-tetrachlorodibenzo-p-dioxin (TCDD) inisolated rat hepatocytesrdquo Toxicology Letters vol 191 no 1 pp79ndash87 2009

[58] D L Alexander S E Eltom and C R Jefcoate ldquoAh receptorregulation of CYP1B1 expression in primary mouse embryo-derived cellsrdquo Cancer Research vol 57 no 20 pp 4498ndash45061997

[59] G YaoM Craven NDrinkwater andC A Bradfield ldquoInterac-tion networks in yeast define and enumerate the signaling stepsof the vertebrate aryl hydrocarbon receptorrdquo PLoS Biology vol2 no 3 article E65 2004

[60] Y Tian A B Rabson and M A Gallo ldquoAh receptor and NF-120581B interactions mechanisms and physiological implicationsrdquoChemico-Biological Interactions vol 141 no 1-2 pp 97ndash1152002

[61] P L Roulhac J M Ward J W Thompson et al ldquoMicropro-teomics quantitative proteomic profiling of small numbers oflaser-captured cellsrdquo Cold Spring Harbor Protocols no 2 2011

[62] P Puri A Acker-Palmer R Stahler Y Chen D Kline and SVijayaraghavan ldquoIdentification of testis 14-3-3 binding proteinsby tandem affinity purificationrdquo Spermatogenesis vol 1 no 4pp 354ndash365 2011

[63] P Agrawal K Yu A R Salomon and J M Sedivy ldquoProteomicprofiling of Myc-associated proteinsrdquo Cell Cycle vol 9 no 24pp 4908ndash4921 2010

[64] S J Stohs ldquoOxidative stress induced by 2378-tetrachlorodi-benzo-p-dioxin (TCDD)rdquo Free Radical Biology and Medicinevol 9 no 1 pp 79ndash90 1990

[65] H G Shertzer M B Genter D Shen D W Nebert Y Chenand T P Dalton ldquoTCDD decreases ATP levels and increasesreactive oxygen production through changes in mitochondrialF0F1-ATP synthase and ubiquinonerdquo Toxicology and AppliedPharmacology vol 217 no 3 pp 363ndash374 2006

[66] A Rosati A Leone L Del Valle S Amini K Khalili and MC Turco ldquoEvidence for BAG3 modulation of HIV-1 gene tran-scriptionrdquo Journal of Cellular Physiology vol 210 no 3 pp 676ndash683 2007

[67] A K Lund M B Goens N L Kanagy and M K WalkerldquoCardiac hypertrophy in aryl hydrocarbon receptor null mice iscorrelated with elevated angiotensin II endothelin-1 and mean


arterial blood pressurerdquo Toxicology and Applied Pharmacologyvol 193 no 2 pp 177ndash187 2003

[68] D L Morris H G Jeong S D Jordan N E Kaminski andM P Holsapple ldquoCharacterization of the effects of 2378-tetra-chloredibenzo-p-dioxin in B6C3F1 and DBA2 mice followingsingle and repeated exposuresrdquo Archives of Toxicology vol 72no 3 pp 157ndash168 1998

[69] C E W Sulentic M P Holsapple and N E Kaminski ldquoArylhydrocarbon receptor-dependent suppression by 2378-tetra-chlorodibenzo-p-dioxin of IgM secretion in activated B cellsrdquoMolecular Pharmacology vol 53 no 4 pp 623ndash629 1998

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MEDIATORSINFLAMMATION

of


the nucleus and binds its heterodimeric partner the aryl-hydrocarbon receptor nuclear translocator (ARNT) proteinThe AHRARNT dimer regulates expression of a battery ofgenes involved in metabolism and elimination includingxenobiotic metabolizing enzymes CYP1A1 and CYP1B1 [1011] The translocation and subsequent DNA binding of theAHR is necessary for TCDD-induced toxicity however thetoxicity associated with dioxin exposure does not directlycorrelate with the levels of the AHR found in the varioustissues [12ndash16] In fact the liver an organ with relatively highAHR expression is fairly resistant to TCDD-induced toxicity[17] This tissue-specific response raises the possibility thataccessory factors and secondary signaling are involved inmodulating the receptorrsquos ability to promote toxicity

Besides Hsp90 and AIP the AHR also has been shownto interact with p23 Cdk4 the retinoblastoma protein (Rb)and RelA of the NF120581B complex [16 18ndash20] Early reportsdemonstrated that p23 was a core AHR complex proteinThe p23 protein has been reported to function in numerouscellular roles including organelle function DNA repair andcell mobility [21] Recently p23 was shown to specificallyinteract with the N-terminus of Hsp90 [22] Other reportshave shown its interaction with the AHR to be transientand nonessential for the receptorrsquos function [23 24] Theinteractions with Cdk4 Rb and RelA link the receptor to cellcycle kinase signaling and gene regulation not exclusivelymediated by the AHR Moreover the AHR has been impli-cated in estrogen and glucocorticoid receptor signaling anddevelopmental processes [25ndash27] Taken together this mul-tifaceted interaction network suggests that AHR biology iscomplex and capable of impacting several cellular responses

The extent of the AHR protein interaction network(AHR-PIN) has not been addressed on a proteomic level ina mammalian system To investigate the role that protein-protein interactions have in AHR-mediated dioxin toxicitywe have established the AHR-PIN using tandem affinitypurification (TAP) and mass spectrometry (MS) Here wereport the AHR-PIN as assembled fromMS data collected inthe presence and absence of TCDD ligand using a Hepa1c1c7cell line with stable overexpression of an AHR-TAP-taggedconstruct [28] The Hepa1c1c7 cell line is extensively usedin AHR research and well characterized in its responses toTCDD exposure Although the liver is not considered highlysensitive to TCDD-induced toxicity the tissue does experi-encemarked changes in gene expression hyperlipidemia andhepatosteatosis [29] Defining the AHR-PIN in this cell lineoffers insights to how TCDD influences AHR biology andinduces toxicity in this tissue type IdentifiedAHR interactorsinclude mitochondrial cell cycle immune response andtranscription factor proteins The new AHR-PIN has identi-fied different secondary pathways that could potentially influ-ence TCDD-induced AHR-mediated toxicity Previously wereportedAHRrsquos influence onmitochondrial function throughits interaction with the ATP51205721 subunit of the ATP synthasecomplex [28]Herewe focus on the receptorrsquos interactionwithanother mitochondrial protein mitochondrial ribosomalprotein L40 (Mrpl40)

2 Materials and Methods

21 Materials Oligonucleotides were synthesized at theMacromolecule Synthesis Facility at Michigan State Uni-versity The pTarget and pGEM-T Easy vectors were pur-chased from Promega (Madison WI) and pZome1C vectorwas obtained from Cellzome (Cambridge UK) Antibodiesused in these experiments were obtained from the follow-ing resources rabbit polyclonal anti-AHR antibodies werea generous gift from Dr Christopher Bradfield (Univer-sity of Wisconsin-Madison) rabbit anti-Mrpl40 (cat noHPA006181) was purchased from Sigma (St Louis MO)mouse anti-COX4 (cat no A21348) was purchased from LifeTechnologies (Grand Island NY) goat anti-rabbit (cat nosc2004) goat anti-mouse (cat no sc2005) normal rabbit IgG(cat no sc2027) and proteinG resinwere obtained from SantaCruz (Santa Cruz CA) All other chemicals used in theseexperiments were reagent grade and purchased from SigmaAldrich (St Louis MO)

22 Plasmid Constructs and Cell Culture The cDNAs for themurine AHR and the green fluorescent protein (GFP) wereamplified using the following primers

AHR 51015840-ggatccccaccatgagcagcggcgccaacatcacc-31015840 and 51015840-ggatcctgcactctgcaccttgcttagg-31015840 GFP 51015840-gggggatccaccatggtg-agcaagggcgac-31015840 and 51015840-gtggatccccgggcccgcggtaccgtcgactgc-31015840 The amplicons were subcloned into pZome1C vectorplacing the TAP-tag at the C terminal of both the AHRand GFP genes Ampicillin resistance was used for clonalselection and each positive clone was sequence verifiedPhoenix-eco and Hepa1c1c7 cells were cultured in DMEMhigh glucose media with L-glutamate and supplementedwith 10 cosmic calf serum (CCS) 100 unitsmL peni-cillin 100 120583gmL streptomycin and 1mM sodium pyruvateHepaC12 cellswere cultured inDMEMhigh glucosemediawL-glutamate and supplemented with 10 cosmic calf serum(CCS) and 1mM sodium pyruvate Tissue culture media andsupplements were obtained from Life Technologies and CCSwas obtained fromHyClone (Waltman MA) Hepa1c17 TAP-AHRandTAP-GFPwere grown in themedia described aboveand puromycin (2120583gmL US Biological Swampscott MA)was used for selection

23 TransfectionStable Cell Line Infection The retroviralvectors AHR-TAP and GFP-TAP were transfected intoPhoenix-eco cells with Lipofectamine 2000 (Life Technolo-gies) via manufacturerrsquos instructions After incubation (5 to8 hours 37∘C) in the presence of DNA the media werechanged to Phoenix cell growth media containing chloro-quine (25 120583M) Cells were then incubated for 24 hours (37∘C)After incubation cells were given fresh Phoenix cell growthmedia and incubated for an additional 24 hours (32∘C)The media were collected after incubation centrifugationwas used to remove cellular debris (3mins 45timesg in RT7Sorvall Rockford IL) and the media were purified using a045 120583mmembrane filter (Millipore) Virus containingmediawas placed on Hepa1c1c7 wild type target cell lines andincubated for 3 hrs at 32∘C and 5 CO

2 Media containing

15 120583gmL polybrene were then added to target cells and













DM

SO

Treatment Sample

DM






30min

120min

240minTC

DD10

nM40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates



IgG

Protein A

Protein A

TEV

TEV

CBD

CBD

CBD

CBD

AHR

AHR

AHR

AHR

TEV cleavage


purification




AHRTCDD

MW GFP D 30 120 240



Reduction

Alkylation

Trypsin digestion



80 to 94

50 to 79

20 to 490 to 19


1 3

3

33

132

77

22

14

1 1

1

1

2 8

21

16

32

1

1

11







Acce

ssio

n nu

mbe

r

Mol

ecul

ar w

eigh

t

Prot

ein

grou

ping

ambi

guity

Dor

othy

A3

x

Dor

othy

A1

xx

Dor

othy

A2

xx

Dor

othy

ACx

(e)




3 Results











Trial 1 Trial 2

2

2613 5

42

22

Trial 3

DMSO control

(a)

Aip 11632Hsp90aa1 15519



ACTB 60Il18 16173



Officialsymbol






(b)


Trial 1 Trial 2

1

2318 1

73

13

Trial 3

30min exposure

(a)

116321551656708


Eef1a1 136277245918100


384382


Officialsymbol

AipHsp90ab1

Clcf1Creb3l3

Htatsf1Mrpl40

Il18Tubb5

A430108E01Rik







(b)






Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708


Eef1a1 13627

15516

72459


5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik






Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658



Eef1a1 13627724595627918100



16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik











(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR



Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













DM

SO

Treatment Sample

DM






30min

120min

240minTC

DD10

nM40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates

40x 15 cm plates



IgG

Protein A

Protein A

TEV

TEV

CBD

CBD

CBD

CBD

AHR

AHR

AHR

AHR

TEV cleavage


purification




AHRTCDD

MW GFP D 30 120 240



Reduction

Alkylation

Trypsin digestion



80 to 94

50 to 79

20 to 490 to 19


1 3

3

33

132

77

22

14

1 1

1

1

2 8

21

16

32

1

1

11







Acce

ssio

n nu

mbe

r

Mol

ecul

ar w

eigh

t

Prot

ein

grou

ping

ambi

guity

Dor

othy

A3

x

Dor

othy

A1

xx

Dor

othy

A2

xx

Dor

othy

ACx

(e)




3 Results











Trial 1 Trial 2

2

2613 5

42

22

Trial 3

DMSO control

(a)

Aip 11632Hsp90aa1 15519



ACTB 60Il18 16173



Officialsymbol






(b)


Trial 1 Trial 2

1

2318 1

73

13

Trial 3

30min exposure

(a)

116321551656708


Eef1a1 136277245918100


384382


Officialsymbol

AipHsp90ab1

Clcf1Creb3l3

Htatsf1Mrpl40

Il18Tubb5

A430108E01Rik







(b)






Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708


Eef1a1 13627

15516

72459


5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik






Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658



Eef1a1 13627724595627918100



16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik











(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR



Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



3 Results











Trial 1 Trial 2

2

2613 5

42

22

Trial 3

DMSO control

(a)

Aip 11632Hsp90aa1 15519



ACTB 60Il18 16173



Officialsymbol






(b)


Trial 1 Trial 2

1

2318 1

73

13

Trial 3

30min exposure

(a)

116321551656708


Eef1a1 136277245918100


384382


Officialsymbol

AipHsp90ab1

Clcf1Creb3l3

Htatsf1Mrpl40

Il18Tubb5

A430108E01Rik







(b)






Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708


Eef1a1 13627

15516

72459


5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik






Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658



Eef1a1 13627724595627918100



16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik











(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR



Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Trial 1 Trial 2

2

2613 5

42

22

Trial 3

DMSO control

(a)

Aip 11632Hsp90aa1 15519



ACTB 60Il18 16173



Officialsymbol






(b)


Trial 1 Trial 2

1

2318 1

73

13

Trial 3

30min exposure

(a)

116321551656708


Eef1a1 136277245918100


384382


Officialsymbol

AipHsp90ab1

Clcf1Creb3l3

Htatsf1Mrpl40

Il18Tubb5

A430108E01Rik







(b)






Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708


Eef1a1 13627

15516

72459


5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik






Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658



Eef1a1 13627724595627918100



16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik











(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR



Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Trial 1 Trial 2

4

216 1

32

14

Trial 3

120min exposure

(a)

Alcam 1165856708


Eef1a1 13627

15516

72459


5360216173

384382


Officialsymbol

Clcf1

Hsp90ab1

Htatsf1

Il18

A430108E01Rik






Hpcal1



(b)


Trial 1 Trial 2

3

1712 4

34

11

Trial 3

240min exposure

(a)

Alcam 11658



Eef1a1 13627724595627918100



16173384382


Officialsymbol

Clcf1

Htatsf1

Mrpl40

Il18A430108E01Rik











(b)


IgG

(a)

(b)

AHR

120572 MRPL40

120572 AHR



Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Hepa1c1c7

Cytosol

Purified

mitochondria

LDH

MRPL40

COX IV

MRPL40

DMSO TCDD

6hr treatment

(a)

HepaC12

LDH

MRPL40

Cytosol

COX IV

MRPL40

Purified

mitochondria

DMSO TCDD

6hr treatment

(b)


4 Discussion











Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







Acknowledgments


References



















































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article The Aryl-Hydrocarbon Receptor Protein … · 2019. 5. 13. ·...

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Transcript of Research Article The Aryl-Hydrocarbon Receptor Protein … · 2019. 5. 13. ·...