Research Article Arsenic Modulates Posttranslational S...

Research ArticleArsenic Modulates Posttranslational S-Nitrosylation andTranslational Proteome in Keratinocytes

Bin Huang12 Kuo-Hao Chiang3 Hsin-Su Yu4 Ying-Lun Chen5

Huey-Ling You6 and Wei-Ting Liao3

1 Department of Biomedical Science and Environmental Biology College of Life Science Kaohsiung Medical UniversityKaohsiung 80708 Taiwan

2Department of Biological Sciences National Sun Yat-sen University Kaohsiung 80424 Taiwan3Department of Biotechnology College of Life Science No 100 Shihchuan 1st Rd Sanming District Kaohsiung Medical UniversityKaohsiung 80708 Taiwan

4Department of Dermatology Kaohsiung Medical University Hospital and Kaohsiung Medical University College of MedicineKaohsiung 80708 Taiwan

5Department of Anesthesiology Mackay Memorial Hospital Taipei 10449 Taiwan6Departments of Laboratory Medicine Chang Gung Memorial Hospital-Kaohsiung Medical Center Kaohsiung 83301 Taiwan

Correspondence should be addressed to Wei-Ting Liao wtliaokmuedutw

Received 12 June 2014 Accepted 18 June 2014 Published 8 July 2014

Academic Editor Hsueh-Wei Chang

Copyright copy 2014 Bin Huang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Arsenic is a class I human carcinogen (such as inducing skin cancer) by its prominent chemical interaction with protein thio (-SH)group Therefore arsenic may compromise protein S-nitrosylation by competing the -SH binding activity In the present study weaimed to understand the influence of arsenic on protein S-nitrosylation and the following proteomic changes By using primaryhuman skin keratinocyte we found that arsenic treatment decreased the level of protein S-nitrosylationThis was coincident to thedecent expressions of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) By using LC-MSMSaround twenty S-nitrosoproteins were detected in the biotin-switched eluent With the interest that arsenic not only regulatesposttranslational S-nitrosylation but also separately affects proteinrsquos translation expression we performed two-dimensional gelelectrophoresis and found that 8 proteins were significantly decreased during arsenic treatment Whether these decreased proteinsare the consequence of protein S-nitrosylation will be further investigated Taken together these results provide a finding thatarsenic can deplete the binding activity of NO and therefore reduce protein S-nitrosylation

1 Introduction

Protein S-nitrosylation the covalent attachment of nitricoxide (NO) to protein thio (-SH) group is an important post-translational modification that affects a wide variety of pro-teins for cellular signaling in normal physiology and a broadspectrum of human diseases [1 2] S-nitrosylation signalingcontrols a number of cellular processes such as protein-protein interactions [3] nuclear transcriptions [4] andmembrane-associated proteins activation [5 6] Pathophys-iology is correlated with hypo- or hyper-S-nitrosylation ofspecific protein targets rather than a general cellular insultdue to not only the loss of or enhanced nitric oxide synthase

activity but also the denitrosylation by a major denitrosy-lase S-nitrosoglutathione reductase (GSNOR) [1] Abnormalprotein S-nitrosylation causes many diseases such as cardio-vascular musculoskeletal and neurological dysfunction [7]Furthermore autophagy a vacuolar degradation for long-lived and aggregate-prone proteins plays an important rolein neurodegeneration Inhibition of autophagy by S-nitro-sylation results in stress-mediated protein aggregation inneurodegenerative diseases [8 9]

S-nitrosylation is also associated with cancer [10] Poten-tialmechanisms of S-nitrosylation in carcinogenesis are focu-sed on apoptosis and DNA repair [11] Survival of tumor cellcould be induced by inactivation of proapoptotic signaling

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 360153 7 pageshttpdxdoiorg1011552014360153

2 The Scientific World Journal

or activation of antiapoptotic pathways [12] Inhibition ofcaspase protease by protein nitrosylation promotes extendedsurvival of malignant cells [13] For example nitrosylationof caspase 9 inhibits the mitochondrial pathway of apop-tosis in cholangiocarcinoma cell line [14] Additionally p53induces apoptotic cell death and causes cell cycle arrest inresponse to various stresses [15] S-nitrosylation of p53 sup-presses p53-mediated apoptosis in colon carcinogenesis[16] Bcl-2 a major anti-apoptotic regulatory proteinwas regulated by S-nitrosylation in various carcinomatissues [11 17 18] S-nitrosylation is shown to modu-late the activity stability and cellular localization of keyDNA repair proteins including O(6)-alkylguanine-DNA-alkyltransferase (AGT) 8-oxoguanine glycosylase (Ogg1)apurinic-apyrimidinic endonuclease 1 (APE1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [19]Inactivation of AGT by S-nitrosylation is found in hepatocar-cinogenesis [20] Moreover S-nitrosylated APE1 export fromthe nucleus to the cytoplasm is described in colon adenomasbreast cancer and hepatocellular carcinomas [21] AlsoS-nitrosylated DNA-PKcs shows increased transcriptionalexpression and activity in HEK-293 [22]

It is well known that arsenic a human carcinogen showedits chemical carcinogenesis activity by interaction with pro-tein -SH groups By its -SH binding activity thereforearsenic may compromise protein S-nitrosylations in cells Inaddition it is reported that arsenic has a significant effecton NO production in the endothelium [23] Arsenic remainsone of the most concerned environmental toxicants whichhas been classified as a group I human carcinogen by theInternational Agency for Research on Cancer (IARC) Epi-demiological studies demonstrated that long-term exposureto inorganic arsenic through either ingestion or inhalationis associated with an increased risk of malignant cancers inthe urinary bladder lung and especially skin since arsenictends to concentrate in keratinocytes [24] However the roleof S-nitrosylation induced by arsenic in skin carcinogenesisremains unclear In the present study we hypothesize thatarsenic can alter protein S-nitrosylation and NO productionin skin keratinocyte Our contribution is identifying inter-actions between arsenic and S-nitrosylation axis in ker-atinocytes that can provide the novel molecular and pharma-cological strategies for potential clinical applications

2 Materials and Methods

21 Human Keratinocyte Culture Freshly obtained prepucespecimenswere used to cultivate the primary human culturedkeratinocytes Briefly normal human prepuce specimenswere washed with PBS then cut into small pieces and incu-bated in medium containing 025 trypsin overnight at 4∘CThe epidermal sheet was lifted from the dermis using a fineforceps The epidermal cells were pelleted by centrifugation(500timesg 10min) and dispersed into individual cells byrepeated aspiration with a pipette Isolated keratinocytesmust be cultured in commercialized keratinocyte serum freemedium (Invitrogen Carlsbad USA) at 37∘C in a humidifiedincubator with 5 CO

2atmosphere with or without sodium

arsenite (Sigma St Louis USA) treatment (10120583M) Thisprimary keratinocyte culture protocol using human skinsamples has been approved by Institutional Review Boardof Kaohsiung Medical University Hospital (KMUH-IRB-960119)

22 Cell Lysis and Proteins Extraction Keratinocytes aftertreatment were washed with cord buffer [NaCl (014M) KCl(4mM) glucose (11mM) and HEPES (10mM pH 74)] andthen lysed with 100120583L of lysis buffer [Hepes (250mMpH 77) EDTA (1mM) neocuproine (01mM) and CHAPS(04 wv)] After centrifugation protein supernatant is col-lected and protein concentrations are determined with BCAassay reagent (Thermo Fisher Scientific Inc Rockford ILUSA)

23 Biotin SwitchMethod for Purifying S-Nitrosoproteins Thebiotin switch method was used according to previous study[22 25] Cells were washed with 1 times cord buffer (10mMHEPES pH 74 014M NaCl 4mM KCl and 11mM glu-cose) Protein lysates were obtained using ultrasound andlysis buffer (250mM HEPES pH 77 1mM EDTA 01mMneocuproine and 04 (wv) CHAPS) The free thiols weremethylated with blocking buffer (225mM HEPES pH 7709mM EDTA 009mM neocuproine 25 (wv) SDS and20mM MMTS) at the ratio of 08mg1mL and were incu-bated at 50∘C for 20min with agitation To remove residualMMTS the MMTS-treated lysate was precipitated with coldacetone and the resulting pellet was resuspended in HENSbuffer (250mM HEPES pH 77 1mM EDTA 01mMneocuproine and 1 (wv) SDS) This was followed by addi-tion of one-third of the HENS suspensionrsquos final volume of4mM N-[6-(biotinamido) hexyl]-31015840-(21015840-pyridyldithio) pro-pionamide (biotin-HPDPDMF)mixedwith 1mMascorbateThe protein lysatebiotin-HPDP mixture was incubated atroom temperature for 1 hour to allow biotinylation to occurThese mixtures were precipitated with cold acetone toremove excess biotin-HPDP and then resuspended in HENSbuffer The biotinylated proteins (ie the former S-nitro-soproteins) were recovered using neutravidin-agarose beads(15 120583Lpermg of initiated protein input) in neutralizationbuffer (20mM HEPES pH 77 100mM NaCl 1mM EDTAand 05 (vv) Triton X-100) The agarose beads were rinsedwith washing buffer (20mM HEPES pH 77 600mM NaCl1mMEDTA and 05 (vv) Triton X-100) The biotinylatedproteins were eluted by elution buffer (20mMHEPES pH 77100mM NaCl 1mM EDTA and 100mM 2-ME) We added100 femtomole of BSA in the eluent as a standard in thesubsequentMSMS analysisThe salt and detergent in eluentswere removed by SpinOUT and DetergentOUT resins (GenoTechnology Inc MO USA)

24 Mass Spectrometric Assay Around 100 ng eluents werereduced alkylated and trypsin digested according to theuserrsquos guideline (In-Gel Tryptic Digestion KitThermo FisherScientific IL USA) The peptide lysates were further desalt-ing with Proteomics C18 Column (Mass solution Ltd TaipeiTaiwan) and then subjected to mass analysis by nLCQ-TOF

The Scientific World Journal 3

(1) MMTS blocking

(3) Biotin-HPDP labeling

(2) Ascorbate reduction

SH

SNO

SS

SNO

S SO

OCH

SS

SH

SS

SSBiotin CH3

CH3CH3

CH3

(a)

MW

(kD

a) 60

50

40

7090

100

30

0 10 0 10 0 10

Ponceau S stain Actin Streptavidin-HRP

20

As (1hr)

(120583M)

(b)

Figure 1 The arsenic decreases protein S-nitrosylation in primary keratinocytes (a)The scheme of biotin switch showed that the NO on thecysteine residue was replaced by biotin (b) After biotin switch 40 120583g of biotinylated lysates treated with 10120583Mof arsenic for 1 h was separatedby SDS-PAGEThe blottedmembrane was prestained by Ponceau S and western blotted with actin (1 3000) Both two were applied as loadingcontrol Streptavidin-HRP (1 3000) was applied to detect biotinylated proteinsThe decreased S-nitrosylated proteins after arsenic treatmentwere indicated as triangle

tandem mass spectrometry (Micromass Manchester UK)MS data were searched for against the NCBI database usingan in-house MASCOT search program (Matrix ScienceLondon UK) Search parameters were set as mass valuesmonoisotopic proteinmass unrestricted peptidemass toler-ance plusmn04Da fragmentmass tolerance plusmn04Damaxmissedcleavages 1 and the instrument type ESI-QUAD-TOF

25 Western Blot Analysis Forty micrograms of cell lysateswith various treatments was mixed with equal volume ofsample buffer [Tris-HCl (625mM pH68) SDS (3 wv)2-mercaptoethanol (5 vv) and glycerol (10 vv)] andthen separated by SDS-PAGE The gel was transferred toPVDF membranes (Millipore MA USA) and immunoblot-ted with antibodies eNOS (1 3000 Cell Signaling TechMA USA) iNOS (1 3000 Cell Signaling Tech MA USA)The membranes were visualized with the SuperSignal WestFemto reagent (Thermo Fisher Scientific IL USA) on X-rayfilmsThe images on X-ray films were scanned using a digitalscanner (Microtek International Inc) and the density wascalculated by the Progenesis Samespots v20 software (Non-Linear Dynamics Newcastle UK)

26 Two-Dimensional Gel Electrophoresis Extracted protein(1mg) was precipitated with 3 volumes of cold acetone atminus20∘C for at least 20min After centrifugation the proteinpellets were air-dried for 5min dissolved in sample buffer[9M urea 2 (wv) CHAPS 60mM DTT and 2 (vv pH4ndash7) IPG buffer (GEHealthcare BioSci NJ USA)] and incu-bated for at least 30min to denature proteins completely Theprotein solution was mixed with rehydration solution [8Murea 2 (wv) CHAPS and 05 (vv pH 4ndash7) IPG buffer]to reach a final volume of 340 120583L and then soaked into an18 cmDryStrip (pH 4ndash7 GEHealthcare BioSci) for up to 12 hon Ettan IPGphor system (GEHealthcare BioSci) Isoelectricfocusing (IEF) was performed with the accumulated voltage

set to 32 kVh After IEF analysis stripped gels were equili-brated with trisbuffer [50mM Tris-HCl pH 88 containing2 (wv) SDS 6M urea 30 (vv) glycerol and 60mM DL-dithiothreitol (DTT)] for 20minThe stripped gels were thenalkylated in the same buffer containing 135mM iodoaceticacid for additional 20minThe equilibrated IEF strip was laidon the top of a vertical SDS-PAGE system to perform the 2-DE

27 Image Analysis The gels were stained with VisPRO dye(Visual Protein Biotech Taipei Taiwan) and scanned with adigital scanner (Microtek International Inc) The transla-tional levels were calculated by ImageMaster software (GEHealthcare BioSci) The gel slices excised from the silver-stained gel were digested with trypsin for 4 hours at 37∘C (In-Gel Tryptic Digestion Kit Thermo Fisher Scientific Inc) andthen subjected to mass spectrometric analysis

3 Results and Discussion

31 Arsenic Reduces Protein S-Nitrosylation Biotin switch isnow the most popular methodology for identifying S-nitro-soproteins in various tissues [22] In the present study theprocedure of biotin switch was simply indicated (Figure 1(a))By treatment with 10 120583M of arsenic for 1 hour at least threegroups of protein S-nitrosylationwere significantly decreasedin keratinocytes (Figure 1(b)) These biotinylated proteinsthat is NO-bound S-nitrosylated proteins were purified fromstreptavidin conjugated agarose and then analyzed by LC-MSMS Totally 20 and 13 S-nitrosoproteins were separatelyidentified in the control and arsenic treatments This wascoincident to the decreased NO production and the reducedS-nitrosylated proteins

32 Arsenic Decreases the Expressions of eNOS and iNOS Ithas been reported that NO in keratinocytes was produced


Ctrl 05 24 05 24

eNOS

Actin

(h)As (1120583M) As (10 120583M)

(a)

1 10

iNOS

Actin

CtrlAs (48h)

120583M)(

(b)

0

05

1

15

Ctrl 1 10

As

Rela

tive f

old

05h24h

120583M)(

lowastlowast

(c)

Rela

tive f

old

0

05

1

15

Ctrl 1 10As (48h)

120583M)(

lowastlowast

lowast

(d)

Figure 2 Arsenic attenuates the expressions of eNOS and iNOS (a) Cells treated with 1 or 10 120583M of arsenic for 05 24 were used to monitoreNOS variations (b) For investigating iNOS level the cells were incubated with same concentration of arsenic for 48 hours (c d) Theexpression levels of eNOS and iNOS were statistically calculated from three repeats Relative folds of protein levels shown as means plusmn SEcompared to control Statistical significance ( lowast119875 lt 005 lowastlowast119875 lt 001) analyzed using Fisherrsquos LSD

Table 1 Identification of arsenic-modulated proteins with nLC-MSMS

Spot number Protein namea Accessionnumberb

MW (kDa)pITheroc

MW (kDa)pIExpd

Sequencecoverage ()

MOWSEscore

PeptidesMatched

287Transformation

upregulated nuclearprotein

460789 51051 54549 12 268 3

63126S proteasome

non-ATPase regulatorysubunit 13 isoform 1

157502193 42955 44352 19 380 8

720 60S acidic ribosomalprotein P0 4506667 34257 39250 18 326 7



858 Cathepsin Dpreproprotein 4503143 44561 30453 25 422 9

899 NADH-Ubiquinonereductase 4758788 30269 26458 40 575 8

905 Enoyl-CoA hydratase 1922287 31383 26461 16 180 3aFunction of the protein obtained via theMASCOT software (httpwwwmatrixsciencecom) search program by querying the NCBI databaseThe parameterswere set at peptide mass tolerance plusmn04Da and allowed missed cleavage 1bAccession number from NCSI databasecTheoretic protein molecular weight and pI annotated in NCBI databasedExperimental protein molecular weight and pI calculated from 2-DE gel


4 7 4 7

MW

(kD

a)50

40

30

631

720 735 749

858

905899

287

631

720735

749

858

905899

287

As 0mel (0 and 10)2 As 10mel (0 and 10)2As 0 As 10

(a)

287 631 720 735

749 858 899 905

As 0As 10

(b)

Figure 3 Translational proteome regulated by arsenic The cell lysates (1mg) treated with 10120583M of arsenic for 1 h were separated by 2-DE(pH4ndash7) By using ImageMaster software the proteins with decreased expression (lt07 fold) were indicated (b)The relative expression levelsof these decreased proteins were statistically calculated from three repeats

through the activation of eNOS and iNOS [23] Thereforein the present study we examined the expressions of bothenzymes in the existence of arsenic As shown in Figures 2(a)and 2(b) the significant decreases of their expressions wereobserved

33 Arsenic Modulates Translational Proteome in Ker-atinocytes In addition to elucidating those posttranslationalS-nitrosylated proteins we further investigated the proteinschanged in their expression levels By using 2-DE 8 proteinsshowed a dramatic decrease in the expression level (Figures3(a) and 3(b)) These identified proteins are annotated func-tion in (Table 1)More specifically HNRNPK is a pre-mRNA-binding protein which has been identified to be involvedin arsenic-induced apoptosis in acute myeloid leukemiacells [26] PSMD13 is proteasome protein regulating the

degradation of ubiquitinated proteins which has been iden-tified in fetal fibroblasts from glutathione deficient mouseusing arsenic-induced apoptosis model by cDNAmicroarray[27] RPLP0 is a ribosomal protein in general conditionsconstantly expressed in cells (house-keeping gene) Thefunctional interactions between arsenic and RPLP0 requiredfurther investigations CTSD is a protease active in intracel-lular protein breakdown Increased CTSD has been identifiedin arsenic-treated human lymphoblastoid cell lines correlatedwith autophagy [28] However here we found arsenic treat-ment decreased CTSD in human keratinocytes NDUFS3 is asubunit of the mitochondrial membrane respiratory chainNADH dehydrogenase (Complex I) Decreased NDUFS3 hasbeen identified in hepatoma cell line [29] Indeed our previ-ous study has identified the dose response between arsenicand NDUFS3 in human keratinocytes Low concentrations


(01ndash10 120583M) of arsenic increased NDUFS3 associated withkeratinocyte proliferation High concentration (50120583M) ofarsenic decreased NDUFS3 correlated with cell death [30]This decreased NDUFS3 was represented in our current dataECHS1 is a catalytic enzyme in mitochondrial fatty acidbetaoxidation pathway however direct evidence betweenECHS1 and arsenic is not yet clarified To summarize fromour proteomic data altered apoptosis and mitochondrialmetabolic functions were the dominant effects of arsenic onhuman keratinocytes

4 Conclusion

In the current study we concluded that arsenic can competewith nitric oxide in binding cysteine residues so that theprotein S-nitrosylation is inhibited Whether the decreasedS-nitrosylation is correlated to arsenic-induced pathologyattracts a great attention in the further study In addition thehomeostasis of cellular apoptosis andmitochondrial dysfunc-tions is worthy of further study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Hans-Uwe Dahms for critical readingof the paper This study was supported by a grant fromthe National Science Council (NSC100-2314-B-037-053 andNSC101-2320-B-037-041) They are grateful to the Center forResources Research amp Development of Kaohsiung MedicalSchool for the support of fluorescent imaging

References

[1] M W Foster D T Hess and J S Stamler ldquoProtein S-nitro-sylation in health and disease a current perspectiverdquo Trends inMolecular Medicine vol 15 no 9 pp 391ndash404 2009

[2] D T Hess A Matsumoto S O Kim H E Marshall and J SStamler ldquoProtein S-nitrosylation purview and parametersrdquoNature ReviewsMolecular Cell Biology vol 6 no 2 pp 150ndash1662005

[3] N V Marozkina and B Gaston ldquoS-Nitrosylation signalingregulates cellular protein interactionsrdquo Biochimica et BiophysicaActa General Subjects vol 1820 no 6 pp 722ndash729 2012

[4] H E Marshall and J S Stamler ldquoInhibition of NF-120581B by S-nitrosylationrdquo Biochemistry vol 40 no 6 pp 1688ndash1693 2001

[5] W Kim Y Choi P V Rayudu et al ldquoAttenuation of NMDAreceptor activity and neurotoxicity by nitroxyl anion NOminusrdquoNeuron vol 24 no 2 pp 461ndash469 1999

[6] S A Lipton Y B Choi Z H Pan et al ldquoA redox-based mech-anism for the neuroprotective and neurodestructive effects ofnitric oxide and related nitroso-compoundsrdquo Nature vol 364no 6438 pp 626ndash632 1993

[7] T Nakamura and S A Lipton ldquoRedox modulation by S-nitro-sylation contributes to protein misfolding mitochondrialdynamics and neuronal synaptic damage in neurodegenerative

diseasesrdquo Cell Death and Differentiation vol 18 no 9 pp 1478ndash1486 2011

[8] S Sarkar V I Korolchuk M Renna et al ldquoComplex inhibitoryeffects of nitric oxide on autophagyrdquoMolecular Cell vol 43 no1 pp 19ndash32 2011

[9] S M Haldar and J S Stamler ldquoS-Nitrosylation at the interfaceof autophagy and diseaserdquoMolecular Cell vol 43 no 1 pp 1ndash32011

[10] E Aranda C Lopez-Pedrera J R de laHaba-Rodrıguez andARodrıguez-Ariza ldquoNitric oxide and cancer the emerging role ofS-nitrosylationrdquo Current Molecular Medicine vol 12 no 1 pp50ndash67 2011

[11] A K Iyer N Azad L Wang and Y Rojanasakul ldquoRole of S-nitrosylation in apoptosis resistance and carcinogenesisrdquo NitricOxide vol 19 no 2 pp 146ndash151 2008

[12] S Ghavami M Hashemi S R Ande et al ldquoApoptosis and can-cer mutations within caspase genesrdquo Journal of Medical Genet-ics vol 46 no 8 pp 497ndash510 2009

[13] J B Mannick A Hausladen L Liu et al ldquoFas-induced caspasedenitrosylationrdquo Science vol 284 no 5414 pp 651ndash654 1999

[14] N J Torok H Higuchi S Bronk and G J Gores ldquoNitricoxide inhibits apoptosis downstream of cytochrome c release bynitrosylating caspase 9rdquo Cancer Research vol 62 no 6 pp1648ndash1653 2002

[15] A Vazquez E E Bond A J Levine andG L Bond ldquoThe genet-ics of the p53 pathway apoptosis and cancer therapyrdquo NatureReviews Drug Discovery vol 7 no 12 pp 979ndash987 2008

[16] J L Williams P Ji N Ouyang L Kopelovich and B RigasldquoProtein nitration and nitrosylation by NO-donating aspirin incolon cancer cells Relevance to itsmechanismof actionrdquoExper-imental Cell Research vol 317 no 10 pp 1359ndash1367 2011

[17] N Azad V Vallyathan L Wang et al ldquoS-nitrosylation of Bcl-2 inhibits its ubiquitin-proteasomal degradation a novel anti-apoptotic mechanism that suppresses apoptosisrdquoThe Journal ofBiological Chemistry vol 281 no 45 pp 34124ndash34134 2006

[18] P Chanvorachote U Nimmannit C Stehlik et al ldquoNitric oxideregulates cell sensitivity to cisplatin-induced apoptosis throughS-nitrosylation and inhibition of Bcl-2 ubiquitinationrdquo CancerResearch vol 66 no 12 pp 6353ndash6360 2006

[19] W Xu L Liu G C M Smith and L G Charles ldquoNitricoxide upregulates expression of DNA-PKcs to protect cells fromDNA-damaging anti-tumour agentsrdquoNature Cell Biology vol 2no 6 pp 339ndash345 2000

[20] W Wei B Li M A Hanes S Kakar X Chen and L LiuldquoS-nitrosylation from GSNOR deficiency impairs DNA repairand promotes hepatocarcinogenesisrdquo Science TranslationalMedicine vol 2 no 19 Article ID 19ra13 2010

[21] J Jones Larry E L Ying A BHofseth et al ldquoDifferential effectsof reactive nitrogen species on DNA base excision repair initi-ated by the alkyladenine DNA glycosylaserdquo Carcinogenesis vol30 no 12 pp 2123ndash2129 2009

[22] M T Forrester J W Thompson M W Foster L Nogueira MA Moseley and J S Stamler ldquoProteomic analysis of S-nitro-sylation and denitrosylation by resin-assisted capturerdquo NatureBiotechnology vol 27 no 6 pp 557ndash559 2009

[23] Y Kumagai and J Pi ldquoMolecular basis for arsenic-inducedalteration in nitric oxide production and oxidative stress impli-cation of endothelial dysfunctionrdquo Toxicology and AppliedPharmacology vol 198 no 3 pp 450ndash457 2004

[24] C J Chen Y C Chuang T M Lin and H Y Wu ldquoMalignantneoplasms among residents of a blackfoot disease-endemic area


in Taiwan high-arsenic artesianwell water and cancersrdquoCancerResearch vol 45 no 11 pp 5895ndash5899 1985

[25] B Huang C L Liao Y P Lin S C Chen and D L WangldquoS-nitrosoproteome in endothelial cells revealed by a modifiedbiotin switch approach coupled with western blot-based two-dimensional gel electrophoresisrdquo Journal of Proteome Researchvol 8 no 10 pp 4835ndash4843 2009

[26] F Gao Y Wu M Zhao C Liu L Wang and G Chen ldquoProteinKinase C-delta mediates down-regulation of heterogeneousnuclear ribonucleoprotein K protein involvement in apoptosisinductionrdquoExperimental Cell Research vol 315 no 19 pp 3250ndash3258 2009

[27] S Kann C Estes J F Reichard et al ldquoButylhydroquinone pro-tects cells genetically deficient in glutathione biosynthesis fromarsenite-induced apoptosis without significantly changing theirprooxidant statusrdquo Toxicological Sciences vol 87 no 2 pp 365ndash384 2005

[28] A M Bolt R M Douglas and W T Klimecki ldquoArsenite expo-sure in human lymphoblastoid cell lines induces autophagy andcoordinated induction of lysosomal genesrdquo Toxicology Lettersvol 199 no 2 pp 153ndash159 2010

[29] D R Yoo S A Chong and M J Nam ldquoProteome profiling ofarsenic trioxide-treated human hepatic cancer cellsrdquo CancerGenomics and Proteomics vol 6 no 5 pp 269ndash274 2009

[30] C Lee S Wu C Hong et al ldquoAberrant cell proliferation byenhancedmitochondrial biogenesis viamtTFA in arsenical skincancersrdquoThe American Journal of Pathology vol 178 no 5 pp2066ndash2076 2011

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MEDIATORSINFLAMMATION

of


or activation of antiapoptotic pathways [12] Inhibition ofcaspase protease by protein nitrosylation promotes extendedsurvival of malignant cells [13] For example nitrosylationof caspase 9 inhibits the mitochondrial pathway of apop-tosis in cholangiocarcinoma cell line [14] Additionally p53induces apoptotic cell death and causes cell cycle arrest inresponse to various stresses [15] S-nitrosylation of p53 sup-presses p53-mediated apoptosis in colon carcinogenesis[16] Bcl-2 a major anti-apoptotic regulatory proteinwas regulated by S-nitrosylation in various carcinomatissues [11 17 18] S-nitrosylation is shown to modu-late the activity stability and cellular localization of keyDNA repair proteins including O(6)-alkylguanine-DNA-alkyltransferase (AGT) 8-oxoguanine glycosylase (Ogg1)apurinic-apyrimidinic endonuclease 1 (APE1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [19]Inactivation of AGT by S-nitrosylation is found in hepatocar-cinogenesis [20] Moreover S-nitrosylated APE1 export fromthe nucleus to the cytoplasm is described in colon adenomasbreast cancer and hepatocellular carcinomas [21] AlsoS-nitrosylated DNA-PKcs shows increased transcriptionalexpression and activity in HEK-293 [22]

It is well known that arsenic a human carcinogen showedits chemical carcinogenesis activity by interaction with pro-tein -SH groups By its -SH binding activity thereforearsenic may compromise protein S-nitrosylations in cells Inaddition it is reported that arsenic has a significant effecton NO production in the endothelium [23] Arsenic remainsone of the most concerned environmental toxicants whichhas been classified as a group I human carcinogen by theInternational Agency for Research on Cancer (IARC) Epi-demiological studies demonstrated that long-term exposureto inorganic arsenic through either ingestion or inhalationis associated with an increased risk of malignant cancers inthe urinary bladder lung and especially skin since arsenictends to concentrate in keratinocytes [24] However the roleof S-nitrosylation induced by arsenic in skin carcinogenesisremains unclear In the present study we hypothesize thatarsenic can alter protein S-nitrosylation and NO productionin skin keratinocyte Our contribution is identifying inter-actions between arsenic and S-nitrosylation axis in ker-atinocytes that can provide the novel molecular and pharma-cological strategies for potential clinical applications

2 Materials and Methods

21 Human Keratinocyte Culture Freshly obtained prepucespecimenswere used to cultivate the primary human culturedkeratinocytes Briefly normal human prepuce specimenswere washed with PBS then cut into small pieces and incu-bated in medium containing 025 trypsin overnight at 4∘CThe epidermal sheet was lifted from the dermis using a fineforceps The epidermal cells were pelleted by centrifugation(500timesg 10min) and dispersed into individual cells byrepeated aspiration with a pipette Isolated keratinocytesmust be cultured in commercialized keratinocyte serum freemedium (Invitrogen Carlsbad USA) at 37∘C in a humidifiedincubator with 5 CO

2atmosphere with or without sodium

arsenite (Sigma St Louis USA) treatment (10120583M) Thisprimary keratinocyte culture protocol using human skinsamples has been approved by Institutional Review Boardof Kaohsiung Medical University Hospital (KMUH-IRB-960119)

22 Cell Lysis and Proteins Extraction Keratinocytes aftertreatment were washed with cord buffer [NaCl (014M) KCl(4mM) glucose (11mM) and HEPES (10mM pH 74)] andthen lysed with 100120583L of lysis buffer [Hepes (250mMpH 77) EDTA (1mM) neocuproine (01mM) and CHAPS(04 wv)] After centrifugation protein supernatant is col-lected and protein concentrations are determined with BCAassay reagent (Thermo Fisher Scientific Inc Rockford ILUSA)

23 Biotin SwitchMethod for Purifying S-Nitrosoproteins Thebiotin switch method was used according to previous study[22 25] Cells were washed with 1 times cord buffer (10mMHEPES pH 74 014M NaCl 4mM KCl and 11mM glu-cose) Protein lysates were obtained using ultrasound andlysis buffer (250mM HEPES pH 77 1mM EDTA 01mMneocuproine and 04 (wv) CHAPS) The free thiols weremethylated with blocking buffer (225mM HEPES pH 7709mM EDTA 009mM neocuproine 25 (wv) SDS and20mM MMTS) at the ratio of 08mg1mL and were incu-bated at 50∘C for 20min with agitation To remove residualMMTS the MMTS-treated lysate was precipitated with coldacetone and the resulting pellet was resuspended in HENSbuffer (250mM HEPES pH 77 1mM EDTA 01mMneocuproine and 1 (wv) SDS) This was followed by addi-tion of one-third of the HENS suspensionrsquos final volume of4mM N-[6-(biotinamido) hexyl]-31015840-(21015840-pyridyldithio) pro-pionamide (biotin-HPDPDMF)mixedwith 1mMascorbateThe protein lysatebiotin-HPDP mixture was incubated atroom temperature for 1 hour to allow biotinylation to occurThese mixtures were precipitated with cold acetone toremove excess biotin-HPDP and then resuspended in HENSbuffer The biotinylated proteins (ie the former S-nitro-soproteins) were recovered using neutravidin-agarose beads(15 120583Lpermg of initiated protein input) in neutralizationbuffer (20mM HEPES pH 77 100mM NaCl 1mM EDTAand 05 (vv) Triton X-100) The agarose beads were rinsedwith washing buffer (20mM HEPES pH 77 600mM NaCl1mMEDTA and 05 (vv) Triton X-100) The biotinylatedproteins were eluted by elution buffer (20mMHEPES pH 77100mM NaCl 1mM EDTA and 100mM 2-ME) We added100 femtomole of BSA in the eluent as a standard in thesubsequentMSMS analysisThe salt and detergent in eluentswere removed by SpinOUT and DetergentOUT resins (GenoTechnology Inc MO USA)

24 Mass Spectrometric Assay Around 100 ng eluents werereduced alkylated and trypsin digested according to theuserrsquos guideline (In-Gel Tryptic Digestion KitThermo FisherScientific IL USA) The peptide lysates were further desalt-ing with Proteomics C18 Column (Mass solution Ltd TaipeiTaiwan) and then subjected to mass analysis by nLCQ-TOF


(1) MMTS blocking



SH

SNO

SS

SNO

S SO

OCH

SS

SH

SS

SSBiotin CH3

CH3CH3

CH3

(a)

MW

(kD

a) 60

50

40

7090

100

30

0 10 0 10 0 10


20

As (1hr)

(120583M)

(b)











Ctrl 05 24 05 24

eNOS

Actin

(h)As (1120583M) As (10 120583M)

(a)

1 10

iNOS

Actin

CtrlAs (48h)

120583M)(

(b)

0

05

1

15

Ctrl 1 10

As

Rela

tive f

old

05h24h

120583M)(

lowastlowast

(c)

Rela

tive f

old

0

05

1

15

Ctrl 1 10As (48h)

120583M)(

lowastlowast

lowast

(d)




MW (kDa)pITheroc

MW (kDa)pIExpd

Sequencecoverage ()

MOWSEscore

PeptidesMatched

287Transformation


460789 51051 54549 12 268 3

63126S proteasome


157502193 42955 44352 19 380 8








4 7 4 7

MW

(kD

a)50

40

30

631

720 735 749

858

905899

287

631

720735

749

858

905899

287


(a)

287 631 720 735

749 858 899 905

As 0As 10

(b)







4 Conclusion




Acknowledgments


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(1) MMTS blocking



SH

SNO

SS

SNO

S SO

OCH

SS

SH

SS

SSBiotin CH3

CH3CH3

CH3

(a)

MW

(kD

a) 60

50

40

7090

100

30

0 10 0 10 0 10


20

As (1hr)

(120583M)

(b)











Ctrl 05 24 05 24

eNOS

Actin

(h)As (1120583M) As (10 120583M)

(a)

1 10

iNOS

Actin

CtrlAs (48h)

120583M)(

(b)

0

05

1

15

Ctrl 1 10

As

Rela

tive f

old

05h24h

120583M)(

lowastlowast

(c)

Rela

tive f

old

0

05

1

15

Ctrl 1 10As (48h)

120583M)(

lowastlowast

lowast

(d)




MW (kDa)pITheroc

MW (kDa)pIExpd

Sequencecoverage ()

MOWSEscore

PeptidesMatched

287Transformation


460789 51051 54549 12 268 3

63126S proteasome


157502193 42955 44352 19 380 8








4 7 4 7

MW

(kD

a)50

40

30

631

720 735 749

858

905899

287

631

720735

749

858

905899

287


(a)

287 631 720 735

749 858 899 905

As 0As 10

(b)







4 Conclusion




Acknowledgments


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Ctrl 05 24 05 24

eNOS

Actin

(h)As (1120583M) As (10 120583M)

(a)

1 10

iNOS

Actin

CtrlAs (48h)

120583M)(

(b)

0

05

1

15

Ctrl 1 10

As

Rela

tive f

old

05h24h

120583M)(

lowastlowast

(c)

Rela

tive f

old

0

05

1

15

Ctrl 1 10As (48h)

120583M)(

lowastlowast

lowast

(d)




MW (kDa)pITheroc

MW (kDa)pIExpd

Sequencecoverage ()

MOWSEscore

PeptidesMatched

287Transformation


460789 51051 54549 12 268 3

63126S proteasome


157502193 42955 44352 19 380 8








4 7 4 7

MW

(kD

a)50

40

30

631

720 735 749

858

905899

287

631

720735

749

858

905899

287


(a)

287 631 720 735

749 858 899 905

As 0As 10

(b)







4 Conclusion




Acknowledgments


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


4 7 4 7

MW

(kD

a)50

40

30

631

720 735 749

858

905899

287

631

720735

749

858

905899

287


(a)

287 631 720 735

749 858 899 905

As 0As 10

(b)







4 Conclusion




Acknowledgments


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



4 Conclusion




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

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