Exogenous Expressions of FTO Wild-Type and R316Q Mutant...

Research ArticleExogenous Expressions of FTO Wild-Type and R316Q MutantProteins Caused an Increase in HNRPK Levels in 3T3-L1 Cells asDemonstrated by DIGE Analysis

Nil Guzel1 Murat Kasap1 Aylin Kanli1 Gurler Akpinar1

M Dogan Gulkac1 and Kubra Karaosmanoglu2

1Department of Medical Biology Kocaeli University Medical School Umuttepe 41380 Kocaeli Turkey2Department of Biomedical Engineering Technology Faculty Kocaeli University Umuttepe 41380 Kocaeli Turkey

Correspondence should be addressed to Murat Kasap mkasap2008gmailcom

Received 23 December 2016 Revised 8 March 2017 Accepted 27 March 2017 Published 7 May 2017

Academic Editor Rita Casadio

Copyright copy 2017 Nil Guzel et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fat mass and obesity-associated protein is an enzyme that oxidatively demethylates DNA Although there are numerous studiesregarding the catalytic function of FTO the overall existence or absence of FTO on cellular proteome has not been investigatedThis study investigated the changes in the soluble proteome of 3T3-L1 cells upon expression of the WT and the mutant (R316Q)FTO proteins Protein extracts prepared from 3T3-L1 cells expressing either the WT or the mutant FTO proteins were used inDIGE experiments Analysis of the data revealed the number of spots matched to every member and there were 350 plusmn 20 spots with305 overall mean coefficient of variation Eleven regulated protein spots were excised from the gels and identified by MALDI-TOFTOF One of the identified proteins was heterogeneous nuclear ribonucleoprotein K which displayed more than 26- and37-fold increases in its abundance in the WT and the mutant FTO expressing cells respectively Western blot analysis validatedthese observationsThis is the first study revealing the presence of a parallel increase in expressions of FTO andHNRNPK proteinsThis increase may codictate the metabolic changes occurring in the cell and may attribute a significance to HNRNPK in FTO-associated transformations

1 Introduction

Fat mass and obesity-associated protein (FTO) is anenzyme known to catalyze Fe (II)- 2-oxoglutarate-dependentdemethylation in both ssDNA and RNA [1ndash3] Since thediscovery of the FTO gene in a genome wide associationstudy (GWAS) in 2007 numerous efforts have been placedon understanding the physiological function of the FTOFTO is able to catalyze 3-methyl thymine to thymine and3-methyl uracil to uracil in ssDNA and RNA respectively[3] This reversible activity is interpreted as part of a nucleicacid repair mechanism [4] However in a recent study N6-methyladenosine is shown to be a better suited substrate forthe FTO indicating that FTO may possess a physiologicalfunction different than the predicted one [2] FTO canoxidatively demethylate the methyl group and change it to

short lived mRNAs including N6-hydroxymethyladenosineand N6-formylmethyladenosine in the nucleus [5]

The clinical significance of FTO was recognized in earlierstudies when researches deleted a FTO containing regionfrom the mouse genome and studied its phenotypic effectsWhat they observed was that there were a partial syndactylythymic hyperplasia growth retardation and serious malfor-mations in craniofacial structure Even death was an outcomeduring the early embryonic stage [6 7] Later detailed studiesestablished a direct link betweenFTOandobesity which endsup producing a devastating disease diabetes [8 9]

Several of the mutations occurring on the FTO geneand their effects on the FTO protein were characterized indetail [10 11] The mutations generally affect the activityof the enzyme For example the R316Q mutation causescomplete loss of the enzyme activity [1 12] The phenotypic

HindawiBioMed Research InternationalVolume 2017 Article ID 8216180 11 pageshttpsdoiorg10115520178216180

2 BioMed Research International

effects of R316Q mutation were studied using fibroblast cellsisolated from a patient with an autosomal-recessive lethalsyndrome [12] In comparison to the control cultures theR316Q mutant FTO expressing cells displayed altered cellmorphology enlarged cell size decreased cell viability andincreased senescence [12] However the molecular detailsoccurring in fibroblast cells carrying the mutant FTO proteinare not known in detail In this study we sought to searchfor the changes occurring at the molecular level using aproteomics approach in 3T3-L1 cells expressing the mutantFTOprotein 3T3-L1 cells are adipose tissue-derived cells dis-playing fibroblast-like morphology As a control we used theWT FTO-expressing 3T3-L1 cells and performed a detailedcomparative study Our hypothesis was that exogenouslyexpressed mutant FTO protein might cause changes at theproteome level in 3T3-L1 cells that would shed some lightonto the observed phenotype Recent studies suggested thatm6A is a key molecule and its modification by FTO creates acode for the regulation of gene expression and in turn proteintranslation [2 13]

The proteome of 3T3-L1 cells did not display pronouncedchanges following either the WT or the mutant FTO expres-sionsWedetectedminor changes in abundance of 11 proteinsHowever after implementation of strict twofold regulationcriteria there were two proteins left that were significantlyregulated Those were heterogeneous nuclear ribonucleopro-tein K (HNRNPK) and Vimentin HNRNPK is a multifunc-tional mRNA-binding protein and Vimentin is one of theclass-III intermediate filaments found in various cell typesVimentin which is listed as one of the most frequentlyidentified proteins in proteomics studies was disregardedin our further analysis More of the focus was placed onHNRNPK to elucidate its association with FTO

2 Materials and Methods

21 Creation of Plasmid Constructs The full-length cDNAof the human FTO gene was cloned into pcDNA4TO (LifeTech USA) by reverse transcription The pcDNA4TO clonefor the mutant FTO (R316Q) was created in our laboratoryby Site-Directed Mutagenesis using a commercial kit (Invit-rogen USA) The constructs were sequenced and in-frameFTO sequences were verified (Eurofins DE)

22 Expression of the WT and the Mutant FTO Proteins3T3-L1 cells were grown in DMEM supplemented with10 (VolVol) fetal bovine serum 100Uml penicillin-streptomycin and 2mML-glutamine at 37∘C in a humidified5 CO

2atmosphere Cells were grown in Petri dishes to 60

confluence To express FTO in 3T3-L1 cells pcDNA4TOharboring FTO gene sequences was transfected using Lipo-fectamine 3000 (LifeTech USA) Empty pcDNA4TO trans-fected cells were used as a negative control

23 Fluorescence Microscopy 3T3-L1-WT and mutant FTOexpressing cells were cultured under standard tissue cultureconditions Culture plates contained glass cover slips that

allowed fluorescence imaging After 48 hrs of FTO expres-sion cells were fixed with formaldehyde and stained for FTOwith an anti-FTO antibody (Clone C-3 (sc-271713) SantaCruz USA) The nuclei of the cells were stained with DAPI(Lifetech USA) Coverslips were mounted in Mowiol beforethe analysis [14] Imaging was performed with an invertedmicroscope (Olympus CKX41) using appropriate filter sets

24 Western Blot Analysis Cell-free extracts were preparedas described by Akpinar et al (2014) [15] Western blot anal-ysis was performed using anti-FTO (Clone C-3 (sc-271713)Santa Cruz USA) anti-HNRNP K (D-6 (sc-28380) SantaCruz USA) and anti-beta actin (Santa Cruz USA ACTBDsc-81178) antibodies as described by Kasap et al (2015) [16]Untransfected 3T3-L1 cells were used to determine the levelof endogenous FTO expression

For the purpose of band analysis ImageJ a freely availablesoftware was used as described by Kasap et al [17] The soft-ware is available at the National Institutes of Health and canbe obtained from httprsbinfonihgovijdownloadhtmlImageJ measures the integrated density of each band byoutlining it and using the AnalyzeMeasure command Thevalues obtained were plotted with Microsoft Excel software

25 Minimal Protein Labeling for DIGE Cells from threebiologic replicates were used for protein extraction Theextracts were prepared in minimal DIGE lysis buffer (7MUrea 2MThiourea 30mM Tris and 5mMMg(CH

3COO)

2

pH9) and equal amounts of proteins were pooled to createthree different pools which were labeled with Cy2 Cy3 orCy5 according to the instructions provided by the supplier(Life Tech USA) The whole labeling experiments werecarried out in the dark

In short 50 120583g of protein sample was used in eachlabeling experiment After adjusting the pH of the extractsto 85 CyDye DIGE minimal dyes were added directly to thesamples and incubated at 4∘C for 30min The reactions werestopped by adding 10mM lysine Labeled samples were thenpooled and used in 2D experiments

26 2D Gel Electrophoresis Experiments For DIGE experi-ments three sets of gels were produced The labeled proteinsamples were loaded onto immobilized 11 cm pH gradientstrips (IPG) (pH 3ndash10) by passive rehydration Separationbased on isoelectric points was performed by a ProteanIsoelectric Focusing Cell (Bio-Rad USA) The followingconditions were used for IEF twenty min at 250V with arapid ramp followed by 2 hrs at 4000V with a slow ramp and25 hrs for 4000Vwith a rapid ramp until a total of 32000Vhwas reached at 20∘CAfter isoelectric focusing the stripswerewashed with buffer I (6M Urea 375mM Tris-HCl pH 882 SDS 20 glycerol 2 (wv) DTT) for 30min and thenwith buffer II (6M Urea 375mM Tris-HCl pH 88 2 SDS20 glycerol 25 iodoacetamide (wv)) for 30min at roomtemperature in the dark and subjected to SDS-PAGE usingTGX precast gels in a Dodeca gel running system (Bio-RadUSA) In parallel to the DIGE experiment a preparative 2Dgel experiment was run to cut protein spots for identification

BioMed Research International 3

A total of 240 120583g of protein (80 120583g from each sample) wasloaded for each separation

27 ImageAnalysis DIGE imageswere captured using appro-priate filter sets with VersaDoc4000 MP (Bio-Rad USA)PDQuest Advance 2D-analysis software (BioRad USA) wasused for comparative analysis of protein spots Quantity ofeach spot was normalized by linear regression model Thestatistical significance of image analysis was determined byStudentrsquos t-test (statistical level of 119901 lt 005 is significant) Gelspots significantly differed in expression (more than 2-fold)and were selected and excised from gels using ExQuest Spotcutter (Bio-Rad USA) for protein identification A manualediting tool was used to inspect the determined protein spotsdetected by the softwareThe 3D view of the selected spots foreach group was created to perform visual comparison usingPDQuest Advance 2D-analysis software The software is runwith default settings

28 Protein Identification Protein identification experi-ments were performed at Kocaeli University DEKART pro-teomics laboratory (httpkabiproteomicskocaeliedutr)using ABSCIEX MALDI-TOFTOF 5800 system In-geltryptic digestions of the proteins were performed using anIn-gel Digestion Kit following the recommended protocolby the manufacturer (Pierce USA) Before deposition ontoa MALDI plate all samples were desalted and concentratedwith a 10 120583L ZipTipC18 following the recommendedprotocol (Millipore USA) Peptides were eluted in avolume of 1 120583L using a concentrated solution of 120572-cyano-4-hydroxycinnamic acid (120572-CHCA) in 50 acetonitrileand 01 trifluoroacetic acid in water and spotted onto theMALDI target plate The TOF spectra were recorded in thepositive ion reflector mode with a mass range from 400 to2000Da Each spectrum was the cumulative average of 200laser shots The spectra were calibrated with the trypsinautodigestion ion peaks 119898119911 (842510 and 22111046) asinternal standards Ten of the strongest peaks of the TOFspectra per sample were chosen for MSMS analysis All ofthe Peptide Mass Fingerprints (PMFs) were searched in theMASCOT version 25 (Matrix Science) using a streamlinesoftware ProteinPilot (ABSCIEX USA) with the followingcriteria Swissprot species restriction to M musculus andR norvegicus enzyme of trypsin at least five independentpeptides matched at most one missed cleavage site MStolerance set toplusmn50 ppmandMSMS tolerance set toplusmn04Dafixed modification being International carbamidomethyl(Cys) and variable modification being oxidation (Met)peptide charge of 1+ and being monoisotopic Onlysignificant hits as defined by the MASCOT probabilityanalysis (119901 lt 005) were accepted Protein score is minus10 lowastlog(119901) where 119901 is the probability that the observed match isa random event Protein score which has a value of 119901 lt 005is considered significant hit Protein scores are derived fromion scores as a nonprobabilistic basis for ranking protein hits

29 STRING Analysis The STRING database (httpstring-dborg) aims to provide a critical assessment and integration

1 2 3 4 5 6 7 8

FTO

5min 10min 20min 40min

120573-Actin

Figure 1 Western blot analysis of endogenously expressed FTOprotein in 3T3-L1 (Lanes 1 3 5 and 7) and the WT FTOtransfected SH-SY5Y control cells (Lanes 2 4 6 and 8) Fordetection of FTO and actin bands mouse monoclonal anti-FTOand anti-actin antibodies were used respectivelyThe FTObandwasonly observed in the WT FTO transfected SH-SY5Y cells The lackof FTO band in 3T3-L1 cells implies the absence or low level of FTOexpression

of protein-protein interactions including direct (physical) aswell as indirect (functional) associations STRING analysiswas performed at httpstring-dborg

3 Results

31 Endogenous FTO Expression in 3T3-L1 Cells Before theexpression of exogenous FTO proteins in 3T3-L1 cells wemeasured the expression of endogenous FTO using westernblot analysis A western blot was created using consecutiveprotein loads of 5120583glane from a cell-free extract of 3T3-L1cells The blot was cut into four pieces and each piece wasprobed with an anti-FTO antibody To visualize endogenousFTO expression X-ray films were exposed to the blotsand different exposure times ranging from 5min to 40minwere used There was no distinctive endogenous FTO bandappearing on the blots indicating that 3T3-L1 cells did notexpress FTOprotein (Figure 1)The control band appeared onthe blots came fromSH-SY5Y cells expressing FTOHoweverif more than 15120583g total protein was loaded to the gels a veryfaint FTO band can be visualized within 20min of exposureindicating that a low level of FTO expression was present in3T3-L1 cells

32 Exogenous FTO Expression in 3T3-L1 Cells 3T3-L1 cellswere transfected with pcDNA4TO itself or pcDNA4TOharboring the WT and the mutant FTO genes After 48 hrsof expression FTO levels were monitored using SDS-PAGE(Figure 2(a)) There was no overexpressed band indicatingthat FTO proteins were expressed at a moderate level whichwould not cause toxic effects on physiological events due tooverexpression To demonstrate FTO expressions westernblot analysis was also performed with the same cell-freeextracts used in SDS-PAGE analysis There was an increasein FTO levels indicating the success of the transfection(Figure 2(b))

33 Fluorescence Imaging of FTO To observe the localizationof both the WT and the mutant FTO in 3T3-L1 cells


250

75

150100

50

37

2520

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

pcD

NA4

TO

-FTO

WT

Mar

ker

(a)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

(kD

a)

250150

7055

35

25

15

120573-Act (41kDa)FTO (58kDa)

Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

(b)

Figure 2 SDS-PAGE analysis of the cell-free extracts prepared from the pcDNA4TO pcDNA4TO-WT FTO and pcDNA4TO-R316Q FTOtransfected cells to demonstrate the moderate expression level of the FTO (a) Western blot analysis of the WT FTO and the mutant (R316Q)expressions in 3T3-L1 cells The positive control was from the WT FTO transfected SH-SY5Y cell-free extract The bar graph was createdusing the intensity values of the bands obtained from Image J

immunofluorescence staining was performed Superposedimages were generated using ImageJ software A similarnuclear localization was observed for the WT FTO and theR316Q mutant proteins in the cells (Figure 3)

34 2D-DIGE Analysis of the WT and the Mutant FTOExpressing 3T3-L1 Cells To monitor the changes observedat the proteome level well-resolved DIGE gels were gener-ated from the soluble protein extracts prepared from FTO


Texas Red-WT FTO DAPI-WT FTO Merge-WT FTO

Merge-R316Q FTODAPI-R316Q FTOTexas Red-R316Q FTO

Figure 3 Immunofluorescence analysis of the WT and the mutant FTO-expressing 3T3-L1 cells The images clearly demonstrated nuclearlocalizations of the FTO proteins

expressing and nonexpressing cells (Figure 4(a)) Analysis ofDIGE images using spot detection tool revealed 350 (plusmn20)spots per gel Superimposition of the gel images over eachother using PDQuest Advance revealed the presence of 11differentially expressed proteins among the protein extractsThe regulated spots were excised from the preparative gelsvia ExQuest Spot cutter into a 96-well plate and successfullyidentified with MALDI-TOFTOF analysis Spots positionson the preparative gel are shown in Figure 4(b) with theircorresponding SSP numbers The identified proteins listedin Table 1 included Vimentin Keratin Type II Cytoskeletal1 Acetyl-CoA Acetyltransferase Parathymosin FTO het-erogeneous nuclear ribonucleoprotein K PhosphoglycerateKinase 1 and 120572-2-macroglobulin Receptor-associated Pro-tein To determine the level of regulation we generated3D images for each protein spot and the calculated spotintensities were used for comparisons (Figure 5) Statisticalanalysis with Studentrsquos t-test as implemented in PDQuestAdvanced software yielded ratios for the identified proteinsover the control (Table 2) Except two proteins HNRNPKand Vimentin the regulation ratios did not meet the 2-foldcriteria Therefore to validate the differential expression ofHNRNPK western blot analysis was performed The resultsindicated that the observed regulation represented the actualchanges that occurred at the proteome level (Figure 6)

4 Discussion

In the present study we investigated the possible effects ofexogenously expressed FTO on the soluble proteome of 3T3-L1 cells This cell line is one of the most robust cell culture

models for adipogenesis and its relevance to FTO researchhas been confirmed [18] Our purpose was to substantiatethe existing theories on the FTO pathogenesis by way ofexpressing the WT and the mutant FTO proteins We choseto study the R316Q mutant because it is the loss of functionmutant of the FTO protein [12] In FTO-deficient mice thismutation causes severe malformations including postnatalgrowth retardation significant reduction in adipose tissueand lean body mass [19] The purpose of expressing themutant FTO protein was to repress the existing activity ofendogenous FTO protein via increasing the quantity of themutant FTO protein in the cells In addition to the mutantprotein we expressed theWT FTO to enhance the activity ofthe existing endogenous FTO via increasing the quantity ofthe WT FTO proteins in the cell The experimental approach(DIGE) allowed us to make comparisons among the WTFTO and the mutant FTO overexpressing cells over thecontrol (the pcDNA4TO transfected cells) Furthermore wewere able to mutually compare the protein profiles of theWT- and the mutant- overexpressing cells Overall therewas a superior match among the protein profiles When 2-fold regulation criteria were applied to the matching spotsthree proteins came into view One of those proteins wasFTO itself indicated that any regulated protein spot couldbe caught by the experimental approach that was used inthis study This finding was also served as an internal controlfor the validity of the observed regulated protein spots Theother two proteins were Vimentin and HNRNPK Petrak etal (2008) have revealed in their meta-analysis that someproteins and protein families which are identified frequentlyas regulated in proteomic studies are regulated due to the


Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)

Figure 4 Representative 2D images of the DIGE gels (a) The green color represents Cy3 labeled protein extracts prepared from the WTFTO expressing cellsThe red color represents Cy2 labeled protein extracts prepared from the mutant (R316Q) FTO expressing cellsThe bluecolor represents Cy5 labeled protein extracts prepared from the pcDNA4TO expressing cells The Image for the 2D preparative gel (b) Thespots labeled with SSP numbers were excised and identified

stress caused by experimental conditions [20] Interpretationof this suspicious regulation can cause misunderstanding ofthe results Vimentin which is listed as one of the frequentlyidentified proteins was thus disregarded in our furtheranalysis We focused our attention to HNRNPK which wasupregulated by 3-fold over the control What was interestingto see was that in comparison to the control HNRNPK wasupregulated both in the WT and the mutant expressing cellsThis has led us to propose that the regulation of HNRNPKwas independent of the activity displayed or repressed bythe FTO In other words the increase in HNRNPK levelwas irrespective of the activity enhanced by the WT FTO

or repressed by the mutant FTO The observed change inHNRNPK level may be reflected onto the FTO interactomeFor example if the increase in FTO level causes a saturationin FTO interaction partners such saturation may indirectlyor directly cause an increase in the levels of proteins thatare part of that interactome We believe that HNRNPK ispart of this interactome since its function is relevant to thephysiological function of FTO in the cell Indeed both FTOand HNRNPK are part of the hnRNA metabolism FTOoxidatively demethylates the adenine nucleotide on hnRNAwhile HNRNP binds this methylated adenine nucleotides[2 21 22]


Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe

rasecytosolic250119864minus21

1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin

receptor-associatedprotein25119890minus019

1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT


Table 2 Respective ratios for the regulated proteins over the control

Protein name R316Qcontrol 119901 values WTcontrol 119901 valuesVimentin 575 00021 507 0002Acetyl-CoA acetyltransferase 0865 003 0675 0025Parathymosin 0655 002 076 0032FTO 584 00019 573 0002Heterogeneous nuclear ribonucleoprotein factor K 3775 0002 26 0003Phosphoglycerate kinase 1 064 0031 051 004Α-2-macroglobulin receptor-associated protein 1545 0035 076 003Keratin type II cytoskeletal 1 087 003 0885 004

FTO-WT FTO-R316Q Control

SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities

Figure 5 Close-up images of the regulated protein spots and their corresponding spot intensities The arrows point to the exact position ofthe spots Relative quantification of the spots was performed using PD Quest Advanced software (BioRad USA)

250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Figure 6 Western blot analysis of HNRNPK expression in pcDNA4TO the WT FTO and the mutant (R316Q) FTO expressing cells Thebar graph was created using the intensity values of the bands obtained from ImageJ


YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH

Figure 7 STRING analysis of HNRNPK and FTO interactionsTheabbreviations stand for the following FTO fat mass and obesity-associated protein HNRNPK heterogeneous nuclear ribonucle-oprotein K UBC ubiquitin C YBX1 Y box binding protein 1RBMX RNA binding motif protein X-linked MAGOH mago-nashi homolog HNRNPF heterogeneous nuclear ribonucleopro-tein F

HNRNPK is a subunit of heterogeneous ribonucleopro-tein complex which is a conserved RNA binding proteinthat is involved in multiple processes of gene expressionincluding processing of pre-mRNAs mRNA export fromthe nucleus RNA splicing mRNA stability and translation[23ndash25] m6A is the most prevalent internal modification inhnRNAs in higher eukaryotes and widely conserved amongeukaryotic species [26 27] This methylation of primertranscripts is catalyzed by METTL3-METTL14-WTAP het-erotrimer structured methyltransferase complex [28] Recentstudies indicated that the methylation of hnRNA is a keycode of hnRNA splicing and themRNA translationThere arethree different protein groups that control the translationalregulation writers erasers and readers One of the erasersis FTO which mediates oxidative demethylation of m6A onRNA [28 29] Within this perspective HNRNPK may beas the one of the translational regulator (trans-factor) andplays function as a reader on modified hnRNA Like otherreaders HNRNPK may be a part of the decision-makingmechanism determining whether the transcript is going to betranslated and at what level of quantity Here we suggestedthat there could be an indirect relationship between FTOand HNRNPK To validate our prediction we performedSTRING analysis using FTO and HNRNPK protein namesAfter retrieval of the experimental evidence from primarynonredundant interaction databases STRING predicted thatthere is an indirect connection between FTO and HNRNPKproteins (Figure 7) This connection is achieved via ubiquitinC (UBC)

The increase of HNRNPK levels may seem very distantfrom the process typical of both FTO and UBC Howeverthe role of ubiquitin within the cell can be very complex andunique Its functions extend from the well-known protea-some dependent proteolysis to cell signaling transcriptionapoptosis and so on [30] In addition the effect of FTOexpression on translation of certain proteins appears tobe feasible Zhou et al demonstrated that a single m6Amodification site in the 51015840UTR enables translation initiationindependent of the 51015840-cap The dynamic features of 51015840UTR

methylation expand the boundaries of physiological rolesof FTO [31] The details of such physiological roles arenot understood to their full extend Therefore the parallelincrease in expressions of FTO and HNRNPK may codictatethe metabolic changes occurring at the protein level

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This project was supported by TUBITAK under the Grant noof 113S965

References

[1] T Gerken C A Girard Y-C L Tung et al ldquoThe obesity-associated FTO gene encodes a 2-oxoglutarate-dependentnucleic acid demethylaserdquo Science vol 318 no 5855 pp 1469ndash1472 2007

[2] G Jia Y Fu X Zhao et al ldquoN6-methyladenosine in nuclearRNA is amajor substrate of the obesity-associated FTOrdquoNatureChemical Biology vol 7 no 12 pp 885ndash887 2011

[3] G Jia C-G Yang S Yang et al ldquoOxidative demethylation of3-methylthymine and 3-methyluracil in single-stranded DNAand RNA by mouse and human FTOrdquo FEBS Letters vol 582no 23-24 pp 3313ndash3319 2008

[4] C Yi and C He ldquoDNA repair by reversal of dna damagerdquo ColdSpring Harbor Perspectives in Biology vol 5 no 1 2013

[5] Y Fu G Jia X Pang et al ldquoFTO-mediated formation of N6-hydroxymethyladenosine and N 6-formyladenosine in mam-malian RNArdquo Nature Communications vol 4 Article IDa012575 2013

[6] I Anselme C Laclef M Lanaud U Ruther and S Schneider-Maunoury ldquoDefects in brain patterning and head morphogen-esis in the mouse mutant Fused toesrdquo Developmental Biologyvol 304 no 1 pp 208ndash220 2007

[7] F VanDerHoeven T Schimmang A VolkmannM-GMatteiB Kyewski and U Ruther ldquoProgrammed cell death is affectedin the novel mouse mutant Fused toes (Ft)rdquo Development vol120 no 9 pp 2601ndash2607 1994

[8] C Dina D Meyre C Samson et al ldquoComment on rsquoA commongenetic variant is associated with adult and childhood obesityrsquordquoScience vol 315 no 5809 p 187 2007

[9] T M Frayling N J Timpson M NWeedon et al ldquoA commonvariant in the FTO gene is associated with body mass index andpredisposes to childhood and adult obesityrdquo Science vol 316no 5826 pp 889ndash894 2007

[10] D Meyre K Proulx H Kawagoe-Takaki et al ldquoPrevalence ofloss-of-function FTOmutations in lean and obese individualsrdquoDiabetes vol 59 no 1 pp 311ndash318 2010

[11] K A Fawcett and I Barroso ldquoThe genetics of obesity FTO leadsthe wayrdquo Trends in Genetics vol 26 no 6 pp 266ndash274 2010

[12] S Boissel O Reish K Proulx et al ldquoLoss-of-functionmutationin the dioxygenase-encoding FTO gene causes severe growthretardation and multiple malformationrdquo American Journal ofHuman Genetics vol 85 no 1 pp 106ndash111 2009


[13] M E Hess S Hess K DMeyer et al ldquoThe fat mass and obesityassociated gene (Fto) regulates activity of the dopaminergicmidbrain circuitryrdquoNatureNeuroscience vol 16 no 8 pp 1042ndash1048 2013

[14] M Kasap S Thomas E Danaher V Holton S Jiang and BStorrie ldquoDynamic nucleation of Golgi apparatus assembly fromthe endoplasmic reticulum in interphase HeLa cellsrdquo Trafficvol 5 no 8 pp 595ndash605 2004

[15] G Akpinar M Kasap A Aksoy G Duruksu G Gacar and EKaraoz ldquoPhenotypic and proteomic characteristics of humandental pulp derived mesenchymal stem cells from a natal anexfoliated deciduous and an impacted third molar toothrdquo StemCells International vol 2014 Article ID 457059 19 pages 2014

[16] M Kasap I Yegenaga G Akpinar M Tuncay A Aksoy and EKaraoz ldquoComparative proteome analysis of hAT-MSCs isolatedfrom chronic renal failure patients with differences in their boneturnover statusrdquo PLoS ONE vol 10 no 11 Article ID e01429342015

[17] M Kasap S Torol G Gacar and F Budak ldquoEthidium bromidespot test is a simple yet highly accurate method in determiningDNA concentrationrdquo Turkish Journal of Medical Sciences vol36 no 6 pp 383ndash386 2006

[18] Y Jiao J Zhang L Lu J Xu and L Qin ldquoThe Fto gene regulatesthe proliferation and differentiation of pre-adipocytes in VitrordquoNutrients vol 8 no 2 article 102 2016

[19] J Fischer L Koch C Emmerling et al ldquoInactivation of the Ftogene protects from obesityrdquoNature vol 458 pp 894ndash898 2009

[20] J Petrak R Ivanek O Toman et al ldquoDeja vu in proteomicsA hit parade of repeatedly identified differentially expressedproteinsrdquo Proteomics vol 8 no 9 pp 1744ndash1749 2008

[21] N Liu andT Pan ldquoN6-methyladenosine-encoded epitranscrip-tomicsrdquo Nature Structural and Molecular Biology vol 23 no 2pp 98ndash102 2016

[22] C R Alarcon H Goodarzi H Lee X Liu S Tavazoie andS F Tavazoie ldquoHNRNPA2B1 is a mediator of m6a-dependentnuclear rna processing eventsrdquo Cell vol 162 no 6 pp 1299ndash1308 2014

[23] T Fukuda T Naiki M Saito and K Irie ldquohnRNP K interactswith RNA binding motif protein 42 and functions in themaintenance of cellular ATP level during stress conditionsrdquoGenes to Cells vol 14 no 2 pp 113ndash128 2009

[24] G Dreyfuss V N Kim and N Kataoka ldquoMessenger-RNA-binding proteins and the messages they carryrdquoNat RevMol CellBiol vol 3 pp 195ndash205 2002

[25] C G Burd and G Dreyfuss ldquoConserved structures and diver-sity of functions of RNA-binding proteinsrdquo Science vol 265 no5172 pp 615ndash621 1994

[26] C-M Wei A Gershowitz and B Moss ldquoN6 O2rsquo-dimeth-yladenosine a novel methylated ribonucleoside next to the 5rsquoterminal of animal cell and virus mRNAsrdquo Nature vol 257 no5523 pp 251ndash253 1975

[27] R M Krug M A Morgan and A J Shatkin ldquoInfluenza viralmRNA contains internal N6methyladenosine and 5rsquo terminal 7methylguanosine in cap structuresrdquo Journal of Virology vol 20no 1 pp 45ndash53 1976

[28] Y Fu D Dominissini G Rechavi and C He ldquoGene expressionregulation mediated through reversible m6A RNA methyla-tionrdquo Nature Reviews Genetics vol 15 no 5 pp 293ndash306 2014

[29] S Smemo J J Tena K-H Kim et al ldquoObesity-associatedvariants within FTO form long-range functional connectionswith IRX3rdquo Nature vol 507 no 7492 pp 371ndash375 2014

[30] L Radici M Bianchi R Crinelli and M Magnani ldquoUbiquitinC gene structure function and transcriptional regulationrdquoAdvances in Bioscience and Biotechnology vol 4 pp 1057ndash10622013

[31] J Zhou J Wan X Gao X Zhang S R Jaffrey and S-BQian ldquoDynamic m6 AmRNAmethylation directs translationalcontrol of heat shock responserdquo Nature vol 526 no 7574 pp591ndash594 2015

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


effects of R316Q mutation were studied using fibroblast cellsisolated from a patient with an autosomal-recessive lethalsyndrome [12] In comparison to the control cultures theR316Q mutant FTO expressing cells displayed altered cellmorphology enlarged cell size decreased cell viability andincreased senescence [12] However the molecular detailsoccurring in fibroblast cells carrying the mutant FTO proteinare not known in detail In this study we sought to searchfor the changes occurring at the molecular level using aproteomics approach in 3T3-L1 cells expressing the mutantFTOprotein 3T3-L1 cells are adipose tissue-derived cells dis-playing fibroblast-like morphology As a control we used theWT FTO-expressing 3T3-L1 cells and performed a detailedcomparative study Our hypothesis was that exogenouslyexpressed mutant FTO protein might cause changes at theproteome level in 3T3-L1 cells that would shed some lightonto the observed phenotype Recent studies suggested thatm6A is a key molecule and its modification by FTO creates acode for the regulation of gene expression and in turn proteintranslation [2 13]

The proteome of 3T3-L1 cells did not display pronouncedchanges following either the WT or the mutant FTO expres-sionsWedetectedminor changes in abundance of 11 proteinsHowever after implementation of strict twofold regulationcriteria there were two proteins left that were significantlyregulated Those were heterogeneous nuclear ribonucleopro-tein K (HNRNPK) and Vimentin HNRNPK is a multifunc-tional mRNA-binding protein and Vimentin is one of theclass-III intermediate filaments found in various cell typesVimentin which is listed as one of the most frequentlyidentified proteins in proteomics studies was disregardedin our further analysis More of the focus was placed onHNRNPK to elucidate its association with FTO

2 Materials and Methods

21 Creation of Plasmid Constructs The full-length cDNAof the human FTO gene was cloned into pcDNA4TO (LifeTech USA) by reverse transcription The pcDNA4TO clonefor the mutant FTO (R316Q) was created in our laboratoryby Site-Directed Mutagenesis using a commercial kit (Invit-rogen USA) The constructs were sequenced and in-frameFTO sequences were verified (Eurofins DE)

22 Expression of the WT and the Mutant FTO Proteins3T3-L1 cells were grown in DMEM supplemented with10 (VolVol) fetal bovine serum 100Uml penicillin-streptomycin and 2mML-glutamine at 37∘C in a humidified5 CO

2atmosphere Cells were grown in Petri dishes to 60

confluence To express FTO in 3T3-L1 cells pcDNA4TOharboring FTO gene sequences was transfected using Lipo-fectamine 3000 (LifeTech USA) Empty pcDNA4TO trans-fected cells were used as a negative control

23 Fluorescence Microscopy 3T3-L1-WT and mutant FTOexpressing cells were cultured under standard tissue cultureconditions Culture plates contained glass cover slips that

allowed fluorescence imaging After 48 hrs of FTO expres-sion cells were fixed with formaldehyde and stained for FTOwith an anti-FTO antibody (Clone C-3 (sc-271713) SantaCruz USA) The nuclei of the cells were stained with DAPI(Lifetech USA) Coverslips were mounted in Mowiol beforethe analysis [14] Imaging was performed with an invertedmicroscope (Olympus CKX41) using appropriate filter sets

24 Western Blot Analysis Cell-free extracts were preparedas described by Akpinar et al (2014) [15] Western blot anal-ysis was performed using anti-FTO (Clone C-3 (sc-271713)Santa Cruz USA) anti-HNRNP K (D-6 (sc-28380) SantaCruz USA) and anti-beta actin (Santa Cruz USA ACTBDsc-81178) antibodies as described by Kasap et al (2015) [16]Untransfected 3T3-L1 cells were used to determine the levelof endogenous FTO expression

For the purpose of band analysis ImageJ a freely availablesoftware was used as described by Kasap et al [17] The soft-ware is available at the National Institutes of Health and canbe obtained from httprsbinfonihgovijdownloadhtmlImageJ measures the integrated density of each band byoutlining it and using the AnalyzeMeasure command Thevalues obtained were plotted with Microsoft Excel software

25 Minimal Protein Labeling for DIGE Cells from threebiologic replicates were used for protein extraction Theextracts were prepared in minimal DIGE lysis buffer (7MUrea 2MThiourea 30mM Tris and 5mMMg(CH

3COO)

2

pH9) and equal amounts of proteins were pooled to createthree different pools which were labeled with Cy2 Cy3 orCy5 according to the instructions provided by the supplier(Life Tech USA) The whole labeling experiments werecarried out in the dark

In short 50 120583g of protein sample was used in eachlabeling experiment After adjusting the pH of the extractsto 85 CyDye DIGE minimal dyes were added directly to thesamples and incubated at 4∘C for 30min The reactions werestopped by adding 10mM lysine Labeled samples were thenpooled and used in 2D experiments

26 2D Gel Electrophoresis Experiments For DIGE experi-ments three sets of gels were produced The labeled proteinsamples were loaded onto immobilized 11 cm pH gradientstrips (IPG) (pH 3ndash10) by passive rehydration Separationbased on isoelectric points was performed by a ProteanIsoelectric Focusing Cell (Bio-Rad USA) The followingconditions were used for IEF twenty min at 250V with arapid ramp followed by 2 hrs at 4000V with a slow ramp and25 hrs for 4000Vwith a rapid ramp until a total of 32000Vhwas reached at 20∘CAfter isoelectric focusing the stripswerewashed with buffer I (6M Urea 375mM Tris-HCl pH 882 SDS 20 glycerol 2 (wv) DTT) for 30min and thenwith buffer II (6M Urea 375mM Tris-HCl pH 88 2 SDS20 glycerol 25 iodoacetamide (wv)) for 30min at roomtemperature in the dark and subjected to SDS-PAGE usingTGX precast gels in a Dodeca gel running system (Bio-RadUSA) In parallel to the DIGE experiment a preparative 2Dgel experiment was run to cut protein spots for identification






1 2 3 4 5 6 7 8

FTO


120573-Actin



3 Results





250

75

150100

50

37

2520

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

pcD

NA4

TO

-FTO

WT

Mar

ker

(a)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

(kD

a)

250150

7055

35

25

15


Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

(b)









4 Discussion




Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)





Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






1 2 3 4 5 6 7 8

FTO


120573-Actin



3 Results





250

75

150100

50

37

2520

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

pcD

NA4

TO

-FTO

WT

Mar

ker

(a)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

(kD

a)

250150

7055

35

25

15


Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

(b)









4 Discussion




Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)





Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250

75

150100

50

37

2520

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

pcD

NA4

TO

-FTO

WT

Mar

ker

(a)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

(kD

a)

250150

7055

35

25

15


Posit

ive c

ontro

l

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

(b)









4 Discussion




Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)





Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






4 Discussion




Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)





Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cy2- FTO R316Q

pH 3 10

Cy5-pcDNA4TO

pH 3 10

250130100

70

55

35

25

1510

pH 3 10Cy3- FTO WT

(a)

7203

1602

10031004

1005

2502

4602 3603 3602

73017302

pH 3 10

250

150

100

75

50

37

25

20

15

(kD

a)

(b)





Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Listof

theidentified

regulated

proteins

andtheirrespectiveM

ALD

I-TOFTO

Fdata

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

1005

Q4F

ZU2

59213

56Ke

ratin

typeIIc

ytoskeletal

6A0059

11796

1117

8593

50KYE

ELQITAG

RH

5

1602

Q4F

ZU2

668

88Ke

ratin

typeIIc

ytoskeletal

6A42119890minus005

11796

29117

8593

80KYE

ELQITAG

RH

7

2502

P20152

53655

668

Vimentin

400119864minus63

1296635

1295599

86REE

AES

TLQSFRQ

53

1587831

158679

85RTN

EKVEL

QEL

NDRF

1734854

1733808

76RLQ

DEIQNMKE

EMARH

1254588

125356

62RLG

DLY

EEEM

RE

1115583

1114562

53KVEL

QEL

NDRF

2201048

2199974

47REM

EENFA

LEAANYQ

DTIGRL

109354

109252

40KFA

DLSEA

ANRN

1046

547

104552

328

KLQ

EEMLQ

RE

P48657

53424

192

Desmin

16119890minus015

1587831

158679

85RTN

EKVEL

QEL

NDRF

231115583

1114562

53KVEL

QEL

NDRF

7203

Q8C

AY6

41271

250

Acetyl-C

oAacetyltransfe


1753984

1752952

69KAG

HFD

KEIV

PVLV

SSRK

29266739

1266631

250

KVA

PEEV

SEVIFGHVLT

AGCG

QNPT

RQ

9445446

9435352

36KAPH

LTHLR

T1935019

1933979

35KVNID

GGAIALG

HPL

GASG

CRI

1307802

1306797

23RILVTL

LHTL

ERV

7302

P55302

42189

230

Alpha-2-m

acroglob

ulin


1563856

156283

69KIQ

EYNVLL

DTL

SRA

361823859

1822812

55KVS

HQGYG

STTE

FEEP

RV

1475751

1474716

26KHVES

IGDPE

HISRN

1951962

1950907

16RKV

SHQGYG

STTE

FEEP

RV

3602

P20152

53655

390

Vimentin

25119890minus035

1587822

158679

59RTN

EKVEL

QEL

NDRF

42125458

125356

59RLG

DLY

EEEM

RE

1296628

1295599

59REE

AES

TLQSFRQ

1444

728

1443699

51RSLYS

SSPG

GAY

VTR

S109353

4109252

47KFA

DLSEA

ANRN

1003

Q9D

0J8

11423

69Parathym

osin

0003

1389627

1388606

66RTA

EEED

EADPK

RQ

11

1004

Q9C

0B1

58245

427

ProteinFT

O100119864minus37

191494

1913891

79KQGEE

IHNEV

EFEW

LRQ

45

9274

879

9264821

52REG

LPVEQ

RN

1708823

1707779

50KMAV

SWHHDEN

LVDRS

1792839

1791792

44KANED

AVPL

CMSA

DFP

RV

1725877

172484

36RVA

ECST

GTL

DYILQ

RC

108252

51081509

28RQFW

FQGNRY


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Con

tinued

SSP

numbers

Accession

number

Best

protein

mass

Best

protein

score

Proteindescrip

tion

Expect

Observed

Mr

(calc)

Ion

score

Matched

peptides

Sequ

ence

coverage

()

3603

P61979

50944

255

Heterogeneous

nucle

arrib

onucleop

rotein

K13119890minus020

1194702

1193692

94RNLP

LPPP

PPPR

G

171549667

1548645

63KLF

QEC

CPHST

DRV

1780834

1779791

55RTD

YNASV

SVPD

SSGPE

RI

1098471

1097445

23KGSD

FDCE

LRL

4602

P61979

50944

316

Heterogeneous

nucle

arrib

onucleop

rotein

K64119890minus028

178078

1779791

82RTD

YNASV

SVPD

SSGPE

RI

25119

4677

1193692

60RNLP

LPPP

PPPR

G1098432

1097445

52KGSD

FDCE

LRL

1053622

1052634

45RVVLIGGKP

DRV

154963

1548645

32KLF

QEC

CPHST

DRV

7301

P09411

44522

277

Phosph

oglyceratekinase

1500119864minus24

2782383

2781343

84KDCV

GPE

VEN

ACANPA

AGTV

ILLE

NLR

F

341634795

1633785

66KLG

DVYV

NDAFG

TAHRA

1769008

1767988

39KALE

SPER

PFLA

ILGGAKV

2023034

2022031

28KITLP

VDFV

TADKF

DEN

AKT





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





SSP 1602

SSP 1003

SSP 1004

134518 630420 541338

1994759 1678642 2641466

5057832 50825104 9752551

Spot intensities

Spot intensities

Spot intensities

SSP 1005

SSP 2502

SSP 4602SSP 3603SSP 3602


1121296 1095691 1350939

2283035 2339141 450946

382051 570336 158176

Spot intensities

Spot intensities

Spot intensities

SSP 7301

SSP 7302

SSP 7203


61050 76288 114206

221432 4660068 3008154

108035 144774 170496

Spot intensities

Spot intensities

Spot intensities


250150

7055

35

25

15

(kD

a)

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO

Mar

ker (

1kb

)

HNRNP K (65kDa)

120573-Act (41kDa)

0

02

04

06

08

1

12

Nor

mal

ized

val

ues

pcD

NA4

TO

-FTO

R316

Q

pcD

NA4

TO

-FTO

WT

pcD

NA4

TO



YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


YBX1RBMX

UBCFTO HNRNPK

HNRNPF

MAGOH







Acknowledgments


References








































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Exogenous Expressions of FTO Wild-Type and R316Q Mutant...

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Transcript of Exogenous Expressions of FTO Wild-Type and R316Q Mutant...