oxidation of lipids is widely regarded 
 as a contributing factor 
 to neurodegeneration , inflammation , and cancer . the polyunsaturated 
 fatty acid residues of membrane phospholipids are prime targets for 
 oxidative attack , as these molecules possess multiple bis - allylic hydrogens , which are sensitive to abstraction by oxidizing 
 agents . abstraction of a labile hydrogen in the presence of o2 leads to a radical chain oxidation mediated by lipid peroxyl 
 radicals , which consumes multiple fatty acid moieties and generates 
 a panoply of products . among these are reactive aldehydes [ e.g. , 4-hydroxynonenal 
 ( hne ) , figure 1a ] , which are capable of protein , 
 dna , and rna modification . lipid electrophile modification of nucleic 
 acids can induce base - pair substitutions , frameshift mutations , and 
 strand breaks , whereas modification of proteins distorts tertiary 
 structure and alters protein function . furthermore , free radical species 
 can recycle to oxidize neighboring phospholipids in a continuum until 
 the radical is intercepted . pin1 binds 
 the cis - phosphorylated serine - proline moiety via a ww binding domain . the ppiase domain of pin1 catalyzes the conversion of the prolyl bond 
 from cis to trans . hne is generated in vivo at low micromolar levels 
 endogenously and is elevated in tissues experiencing an oxidative 
 insult , such as the brains of alzheimer s disease ( ad ) patients 
 and the lungs of patients with chronic obstructive pulmonary disease . in these and other conditions , hne modification of cellular constituents 
 is associated with disease pathogenesis . however , the contribution 
 of hne to cellular fate is complex and incompletely understood . to 
 gain an understanding of the spectrum of biochemical consequences 
 of hne production , our laboratory used a multipronged approach to 
 compile an inventory of protein signaling networks activated by hne . using click chemistry and mass spectrometry , an inventory of protein 
 targets of hne was generated ; separately , 
 microarray analysis of hne - treated cells revealed up- or down - regulated 
 genes in response to treatment . using 
 bioinformatic analysis , protein targets of hne were linked to differentially 
 regulated genes via specific transcription factors . analysis of the protein 
 adduction inventory revealed that peptidyl - prolyl cis / trans - isomerases ( ppiases ) are a class 
 of proteins modified by hne . peptidylprolyl cis / trans - isomerase a1 ( pin1 ) is a unique ppiase that catalyzes only phosphoserine- 
 and phosphothreonine - proline conversions from cis to trans ( figure 1b ) . this enzyme is 
 particularly important in phosphorylation - dependent signaling pathways , 
 as proline - directed phosphatases are trans - specific . molecules that covalently modify pin1 s catalytic 
 or binding site residues have been previously demonstrated to induce 
 apoptosis and inhibit cell proliferation , possibly due to inhibition 
 of pin1 s actions on the cell cycle . epigallocatechin gallate 
 ( egcg ) , an anticancer polyphenol in green tea , inhibits cancer cell 
 growth by interacting with arg-17 of pin1 in the ww domain , preventing 
 pin1substrate interactions . additionally , pib , a derivative of juglone ( 1,4-naphthoquinone ) , 
 has been reported to induce cell death in a pin1-dependent fashion . the chemistry and biology of pin1 modification 
 by lipid electrophiles 
 have not been investigated . pin1 contains multiple exposed cys , his , 
 and lys residues capable of reacting with hne . using maldi - tof mass 
 spectrometry , we mapped the hne adduction sites on pin1 . data from 
 maldi - tof and maldi - tof / tof analysis with purified pin1 treated with 
 hne support the adduction of critical active site residues , with cys-113 
 being the most reactive . treatment of mda - mb-231 cells with hne , followed 
 by immunoprecipitation and high - resolution ms / ms analysis , also revealed 
 the presence of a cys-113hne michael adduct within the active 
 site of the protein . sirna - mediated knockdown of pin1 afforded protection 
 from hne - induced cell death . these studies demonstrate that pin1 is 
 an important target for modification by lipid electrophiles , and this 
 leads to alteration in cell signaling and viability . mda - mb-231 cells , a triple - negative 
 human breast carcinoma cell line , were obtained from the american 
 type culture collection ( atcc ) and were cultured in rpmi1640 medium 
 ( gibco ) supplemented with 10% fetal bovine serum ( fbs ) . mda - mb-231 
 cells were maintained at 37 c in a humidified cell culture incubator 
 under 5% co2 and 95% air . ahne , hne , or dmso ( vehicle control ) was added to cell culture medium 
 to achieve a final dmso concentration of less than 1% . purified 
 pin1 was obtained from genway biosciences ( gwb-523efe ) and was buffer - exchanged 
 once with dulbeccos - modified pbs ( gibco ) before use . protein 
 ( 2.5 g ) was diluted to 25 l with pbs and incubated for 
 the indicated times with hne at 37 c with agitation . reactions 
 were terminated with nabh4 and rotated end - over - end for 
 30 min at room temperature ( rt ) . samples were dried with a speedvac 
 and reconstituted in 10 l of 6 m guanidine hydrochloride for 
 45 min at rt . each sample was incubated with dtt ( 150 m ) for 
 30 min at 37 c , followed by 750 m iodoacetamide for 15 
 min at rt in the dark . samples were diluted 20-fold with 20 mm nh4hco3 and digested with 500 ng of chymotrypsin for 
 24 h at 37 c . the following day , chymotryptic digests were desalted 
 using c18 ziptips , followed by elution with 60% acetonitrile/0.1% 
 trifluoroacetic acid . samples were mixed 1:1 by volume with matrix 
 [ 20 mg / ml -cyano-4-hydroxycinnamic acid ( chca ) in 60% acetonitrile ] 
 and subjected to maldi mass spectrometry . mda - mb-231 cells were treated with 
 ahne for 1 h in serum - free medium . following treatment , cells were 
 washed with pbs and collected by scraping and centrifugation for 5 
 min at 1000 g . cell pellets were lysed in buffer containing 
 50 mm hepes , ph 7.5 , 150 mm nacl , 0.5% igepal , and mammalian protease 
 inhibitor cocktail ( sigma - aldrich ) . suspended cell pellets were sonicated 
 by 10 1 s pulses with a virsonic cell disruptor and then cleared by 
 centrifugation at 16000 g for 10 min . bicinchoninic 
 acid ( bca ) assay was used to determine the protein concentration and 
 was performed according to the manufacturer s instructions 
 ( thermo ) . each sample was diluted to 2 mg / ml in 3 ml of lysis buffer 
 and reduced with 2 mm nabh4 for 1 h at rt with agitation 
 to stabilize adducts . unreacted nabh4 was quenched with 
 3 l of acetone , and reduced lysates were subjected to huisgen 
 1,3-cycloaddition chemistry by the addition of 0.2 mm biotin benzoin 
 ( n3-biotin linker , porter laboratory ) , 1 mm tris(2-carboxyethyl)phosphine 
 ( tcep ) , 0.1 mm ligand tris((1-benzyl-1h-1,2,3-triazol-4-yl)methyl)amine 
 ( triazole ligand , porter laboratory ) , and 1 mm cuso4 . tubes 
 containing samples were covered with foil and rotated end - over - end 
 for 2 h at rt . proteins were precipitated by the addition of 6 ml 
 ( twice the volume of sample ) of ice - cold methanol for 30 min on ice 
 and pelleted by centrifugation . pellets were washed twice with cold 
 methanol , resuspended in 1 ml of 0.5% sds , and boiled for 5 min . after 
 they were boiled , 50 l of sample was aliquotted to serve as 
 the input fraction to demonstrate equal protein loading onto streptavidin 
 beads . the remaining 950 l was diluted to 10 ml with pbs containing 
 1 ml of streptavidin - agarose beads and 0.1% sds . the following day , beads were 
 washed with 1% sds , 4 m urea , pbs containing 1 m nacl , and pbs each . to elute ahne - modified proteins from the streptavidin beads , samples 
 were incubated under 365 nm uv light with rotation for 90 min at rt . 
 during this step eluted proteins 
 were separated from beads by centrifugation , concentrated , and subjected 
 to sds - page and western blot . samples for sds - page 
 were mixed 1:1 by volume with laemmli buffer containing 5% ( w / v ) -mercaptoethanol 
 and boiled for 5 min . proteins in the gel were transferred onto a 
 0.45 m nitrocellulose membrane and blocked with 5% nonfat dry 
 milk for 1 h. primary antibodies were incubated ( 1:1000 for pin1 , 
 flag , and gapdh ) with membranes overnight at 4 c . the following 
 day , blots were washed with tris - buffered saline containing 0.1% tween-20 
 ( tbst ) three times and then incubated with secondary antibody for 
 1 h at rt with shaking . blots were washed three times with tbst and 
 developed using luminol - based detection . flag and pin1 antibodies 
 were obtained from cell signaling , while gapdh antibody was obtained 
 from santa cruz biotechnology . all spectra were acquired 
 in positive ion mode on either an autoflex speed tof ms or an ultraflextreme 
 tof / tof ms ( bruker daltonics ) , both equipped with a nd : yag ( solid 
 state ) laser operating at 355 nm . peptide - chca solutions ( 1 l ) 
 were deposited on maldi target plates and air - dried prior to analysis . full mass spectra of digested peptides were obtained in reflectron 
 mode on the ultraflextreme , using a 5004000 mass range . peptide 
 masses and sequence ions were manually examined for mass shifts of 
 + 158 m / z or + 141 m / z , corresponding to reduced hne michael 
 adducts or reduced schiff base adducts , respectively . selected peptide 
 ions were dissociated using lift on the tof / tof . tof / tof fragmentation 
 data were interrogated using flexanalysis software and analyzed against 
 a theoretical peptide digest using protein prospector . a cdna clone of 
 pin1 ( mammalian gene collection accession number bc002899 ) was obtained 
 from the vanderbilt microarray shared resource cdna clone collection . pin1 was pcr - amplified from potb7 using the primers 5-cctgctagctccaccatggattacaaggatgacgacgataaggcggacgaggagaagctgccgc-3 
 ( includes an n - terminal flag tag ) and 5-cctggtacctcactcagtgcggaggatga-3. 
 the pcr product was digested with nhei and kpni and 
 ligated into pcdna3.1 hygro(+ ) ( invitrogen ) . mda - mb-231 cells at 80% 
 confluency were transfected using 10 g of expression construct 
 and 20 l of lipofectamine ( thermo - scientific , lafayette , co ) 
 for 24 h. after 24 h , cells were incubated with fresh medium for an 
 additional 48 h at 37 c before treatment and collection . the direct immunoprecipitation 
 kit ( pierce ) was used to immunoprecipitate flag - pin1 from mda - mb-231 
 cells . eighty microliters of anti - flag resin ( sigma - aldrich ) was loaded 
 onto columns and washed once with pbs . one milligram of protein lysate 
 was added per column in 500 l of total volume . columns were 
 plugged and rotated end - over - end overnight at 4 c . the following 
 day , columns were unplugged and centrifuged at 1000 rpm to elute nonspecific 
 proteins . beads containing flag - pin1 were washed three times with 
 ip / lysis wash buffer and once with conditioning buffer containing 
 0.5% protease inhibitors ( pierce ) . eluents were neutralized with the 
 addition of 1 m naoh and treated with nabh4 to stabilize 
 hne - adducted proteins . because multiple columns were necessary to 
 achieve sufficient protein quantities for proteomics , samples were 
 pooled and concentrated to 50 l using 3 kda molecular weight 
 cutoff filters immediately prior to sds - page . following sds - page of immunoprecipitated 
 flag - pin1 , the gel was stained with colloidal coomassie blue , and 
 the pin1 band was excised from the gel and cut into 1 mm pieces . the gel pieces were treated with 45 mm dtt for 45 min , and 
 available cys residues were carbamidomethylated with 100 mm iodoacetamide 
 for 45 min . after the gel pieces were destained with 50% acetonitrile 
 in 25 mm ammonium bicarbonate , pin1 was digested with endoproteinase 
 aspn ( 10 ng/l ) in 25 mm nh4hco3 overnight 
 at 37 c . peptides were extracted by gel dehydration ( 60% acetonitrile , 
 0.1% tfa ) , the extract was dried by vacuum centrifugation , and the 
 peptides were reconstituted in 0.1% formic acid . the peptide solution 
 was loaded onto a capillary reversed - phase analytical column ( 360 
 m o.d . the analytical column was packed with 20 cm 
 of c18 reversed - phase material ( jupiter , 3 m beads , 300  , 
 phenomenex ) , directly into a laser - pulled emitter tip . peptides were 
 gradient - eluted at a flow rate of 500 nl / min , and the mobile phase 
 solvents consisted of water containing 0.1% formic acid ( solvent a ) 
 and acetonitrile containing 0.1% formic acid ( solvent b ) . a 90 min 
 gradient was performed , consisting of the following : 010 min , 
 2% b ; 1050 min , 245% b ; 5060 min , 4590% 
 b ; 6065 min , 95% b ; 6570 min 952% b ; and 7090 
 min , 2% b. eluting peptides were mass analyzed on an ltq orbitrap 
 velos mass spectrometer ( thermo scientific ) , equipped with a nanoelectrospray 
 ionization source . the instrument was operated using a data - dependent 
 method with dynamic exclusion enabled . full - scan ( m / z 3002000 ) spectra were acquired with the 
 orbitrap ( resolution 60,000 ) , and the top 16 most abundant ions in 
 each ms scan were selected for fragmentation in the ltq . an isolation 
 width of 2 m / z , activation time 
 of 10 ms , and 35% normalized collision energy were used to generate 
 ms2 spectra . dynamic exclusion settings allowed for a repeat count 
 of 2 within a repeat duration of 10 s , and the exclusion duration 
 time was set to 15 s. for identification of pin1 peptides , tandem 
 mass spectra were searched with sequest ( thermo fisher scientific ) 
 against a human subset database created from the uniprotkb protein 
 database ( www.uniprot.org ) . variable modifications of + 57.0214 
 on cys ( carbamidomethylation ) , + 15.9949 on met ( oxidation ) , + 141.1279 
 on lys and arg ( corresponding to reduced schiff base ) , and + 158.1306 
 on cys , lys , and his residues ( corresponding to reduced hne modification ) 
 were included for database searching . spectra acquired of pin1 peptides 
 of interest were then inspected using xcalibur 2.1 qual browser software 
 ( thermo scientific ) . tandem mass spectra of hne - modified peptide precursors 
 and the spectra acquired of the corresponding unmodified peptide forms 
 were examined by manual interrogation . sirna was diluted 
 1:200 in optimem ( gibco ) for 5 min , and separately , dharmafect was 
 diluted 1:40 in optimem . after 5 min , solutions were combined and 
 incubated for 20 min at rt . the solution containing optimem , dharmafect , 
 and sirna was added to 4 ml of medium containing 2  10 mda - mb-231 cells and incubated overnight at 37 c . the medium 
 was changed the following day , and cells were incubated for an additional 
 48 h at 37 c . cells were lysed and processed for western blot . 
 for viability assays , 7500 cells / well were seeded in 96-well plates , 
 and wells were incubated with 3.75 l of sirna / dharmafect / optimem 
 solution for 24 h before the medium was changed . after an additional 
 48 h , the cell viability was assessed using 
 the calcein am assay , whereby a cell - permeable substrate ( calcein 
 am ) penetrates live cells and is converted to fluorescent calcein 
 by intracellular esterases . just prior to the assay , mda - mb-231 medium 
 was removed , and cells were washed twice with pbs . calcein am ( invitrogen ) 
 was dissolved in dmso for a stock concentration of 2 mm . calcein am 
 stock was diluted to 2 m in pbs and added to cells for 30 min 
 at rt . after incubation , the solution was removed from the wells , 
 and fluorescence was quantified using a spectramax plate reader ( excitation 
 = 494 nm , and emission = 517 nm ) . a one - way anova for treatment 
 followed by tukey posthoc test was used to assess statistical significance . to map the adduction sites 
 of pin1 by hne , we adapted a previously reported protocol for determining 
 the adduction sites of cytochrome c. purified pin1 was treated with a high concentration of 
 hne ( 2 mm ) for 3 h at 37 c , followed by nabh4-mediated 
 aldehyde reduction . although 2 mm greatly exceeds the pathophysiological 
 range of hne concentrations reported in cells , the aim of this experiment 
 was to compile all of the potential pin1 modification sites . samples 
 were carboxamidomethylated by treatment with iodoacetamide , then digested 
 with chymotrypsin , and analyzed by maldi - tof ms . several peaks with 
 mass shifts of + 158 m / z relative 
 to theoretical pin1 chymotryptic peptides were present in the digest , 
 corresponding to reduced michael adducts of hne ( figure 2a ) . in the case of hne modification at a cys residue , the 
 peak shift occurs + 101 m / z relative 
 to the alkylated peptide because of the replacement of the carboxamidomethyl 
 group ( figure 2a ) . michael adducts , as well as unmodified peptides , 
 were fragmented by maldi - tof / tof to confirm peptide identification 
 and identify the site of hne modification . tof / tof analysis of peak 
 1643 m / z revealed the presence of 
 an hne  cys reduced michael adduct occurring at cys-113 ( figure 2b , bottom panel ) , while his-157 adduction was observed 
 upon fragmentation of peak 2372 m / z ( figure s1 , bottom panel , in the supporting 
 information ) . interestingly , both cys-113 and his-157 are located 
 in the pin1 active site and are involved in its catalytic function . lys-132 adduct was also detected ( table 1 and figure s2 in the supporting 
 information ) . because peaks of modified and corresponding unmodified 
 pin1 peptides are detectable simultaneously in the maldi - tof spectra , 
 the relative reactivity of recovered peptides can be ascertained.as shown in figure 2a and table 1 , the intensities 
 of peaks corresponding to cys-113 and his-157 are increased approximately 
 3-fold relative to their respective unmodified peaks . by contrast , 
 the adducted to unadducted peak ratio corresponding to lys-132 adduction 
 was 0.23 , indicating a relatively lower sensitivity to hne modification . these data support the hypothesis that active site residues in pin1 
 are particularly susceptible to modification by hne . ( a ) maldi - tof 
 spectrum of chymotryptic digest from hne - treated pin1 . two peptides 
 are indicated , and arrows represent peaks with shifts of + 158 m / z from native pin1 chymotryptic digests . peak 1643 is shifted 101 m / z from 
 the cysteine - containing alkylated peptide indicated . ( b ) maldi - tof / tof 
 spectrum of peaks 1542 ( top ) and 1643 m / z ( bottom ) from hne - treated pin1 chymotryptic digest . spectral interrogation 
 reveals the presence of a mass shift of + 158 m / z between y12 and y13 ( or + 101 m / z as compared to top spectrum containing 
 alkylated cys-113 ) in the bottom spectrum corresponding to a reduced 
 michael adduct occurring at cys-113 . matched masses of b and y ions 
 in the spectrum to corresponding theoretical masses of pin1 peptide 
 sdcssakargdlgaf and sdc(+158 m / z)ssakargdlgaf are indicated by arrows . the asterisk within the peptide 
 sequence indicates the site of modification . we investigated the relative rate and extent of 
 adduction of the 
 two active site residues . purified pin1 was incubated with 25 , 100 , 
 or 200 m hne for 3 h , followed by nabh4 reduction , 
 digestion , and analysis by maldi - tof ms . spectral investigation revealed 
 that the formation of the cys-113 adduct increased with hne concentration , 
 whereas his-157 adduct formation was minimal ( figure 3a ) . incubation of pin1 with hne for increasing times revealed 
 complete modification of cys-113 by hne before any his-157 modification 
 was observed ( figure 3b ) . this indicates that 
 the catalytic cysteine of pin1 is the primary target for hne modification . relative 
 rate of formation of michael adducts with c113 vs h157 
 ( a ) as a function of hne concentration and ( b ) as a function of time 
 of hne incubation . for the graph in panel a , various concentrations 
 were incubated for 3 h before termination of the reaction with nabh4 . for function of time experiments , 200 m hne was incubated 
 with pin1 for indicated times . previous studies have 
 demonstrated that administration of hne to cells results in extensive 
 protein adduction . the use of alkynylated analogues 
 as taggable surrogates for electrophiles allows for the separation 
 of adducted proteins from unadducted proteins . cells treated 
 with ahne are lysed and exposed to click chemistry , in which the alkyne 
 at positions 89 of ahne reacts with an azido - biotin tag bearing 
 a uv - cleavable linker . only hne - adducted proteins are biotinylated , 
 so these targets can be purified by binding to streptavidin - coated 
 beads , followed by elution from the beads with uv light . western blotting of eluted fractions with antibodies 
 allows for detection of specific proteins that are adducted by ahne . mda - mb-231 cells were chosen as the model cell line for this study 
 because of a previously reported dependence on pin1 for cell proliferation . western blots of lysates from mda - mb-231 cells 
 dosed with 2.550 m ahne for 1 h revealed a dose - dependent 
 increase in pin1 adduction as a function of concentration ( figure 4b ) . a blot of the input fraction indicated equal 
 loading of cell lysate onto streptavidin beads , as well as an absence 
 of changes in pin1 protein levels with ahne treatment ( figure 4b ) . ( a ) schematic of detection of hne - adducted proteins in vitro , whereby 
 targets of ahne are separated from nontargets via click chemistry , 
 followed by streptavidin purification , uv light - induced elution , subject 
 to western blot , and probed with anti - pin1 antibody . treatment of mda - mb-231 cells with ahne and subjection 
 to click - mediated separation of electrophile - modified proteins followed 
 by western blot with anti - pin1 antibody reveal a concentration - dependent 
 adduction of pin1 in this cell line ( top blot ) ; the lower blot is 
 shown to demonstrate equal protein loading onto streptavidin beads . identification of electrophile - modified residues of pin1 
 in cells treated with hne would provide critical information into 
 the role the adduct plays in altering pin1 biochemical functions . our laboratory previously elucidated the hne adduction sites of hsp90 
 via geldanamycin - biotin affinity capture and lc - coupled tandem mass 
 spectrometry ( lc - ms / ms ) . in the current 
 study , we employed a similar strategy of pin1 affinity purification 
 and lc - ms / ms analysis to examine hne adduction of pin1 . to facilitate 
 isolation of pin1 from cells treated with hne , a flag - pin1 plasmid 
 was generated and transfected into mda - mb-231 cells ( figure 5a ) . immunoprecipitation using anti - flag resin enabled 
 purification of sufficient protein to observe with sds - page analysis 
 and subsequent colloidal coomassie staining ( figure 5b ) . the resolved pin1 band was excised and in - gel digested 
 with endoproteinase aspn , and then , peptides were extracted and analyzed 
 on an ltq - orbitrap velos mass spectrometer . the lc - ms base peak chromatogram 
 demonstrates thorough proteolytic digestion of the excised pin1 gel 
 band ( figure 5c , top panel ) . following lc - ms / ms 
 analysis , the resulting tandem mass spectra were searched via sequest 
 against a human database , and assigned spectra corresponding to aspn - derived 
 pin1 peptides of interest were validated via manual examination . among 
 those peptides detected , pin1 peptides containing cys-113 ( dcssakarg , 
 residues 112120 ) and his-157 ( dsgihiilrte- , residues 153163 ) 
 were identified ( figure 5c and figure s3 in 
 the supporting information ) . upon mass 
 spectral interrogation , no michael adducts of the peptide containing 
 his-157 were detected ( figure s3 in the supporting 
 information ) . however , in addition to detecting the unmodified 
 peptide containing cys-113 , an hne - adducted species of this peptide 
 was also observed ( figure 5c ) . relative to 
 the unmodified ( and reduced ) theoretical mass of the peptide , the 
 adducted peptide contained a mass shift of 158.13 da , corresponding 
 to reduced hne adduction having an elemental composition c9h18o2 . the theoretical m / z values of the observed peptide precursors were used to 
 generate extracted ion chromatograms ( xics figure 5c ) for each peptide form . the calculated mass errors of the 
 unmodified and hne - adducted peptide were each within 1 part per million 
 ( ppm ) of theoretical values , adding high confidence in the identification 
 of the hne - adducted peptide . in addition , the hne - adducted peptide 
 displayed the expected shift in retention time due to increased hydrophobicity , 
 relative to the unmodified peptide ( figure 5c ) . tandem mass spectra acquired from the modified peptides were 
 manually interrogated to determine the site of adduction , and for 
 comparison , the acquired ms / ms spectrum of the unmodified peptide 
 was also examined . michael adducts or schiff bases , 
 further demonstrating the sensitivity of the catalytic cysteine of 
 pin1 to electrophile adduction . pin1 is covalently modified at cys-113 
 in mda - mb-231 cells treated 
 with hne . ( a ) western blot of pin1 ( top ) and flag from mda - mb-231 
 cells transfected with flag - pin1 . ( b ) 1d gel of flag - pin1 immunoprecipitation 
 from mda - mb-231 cells treated with hne . ( c ) base peak chromatogram 
 of peptides derived from immunoprecipitated and in - gel - digested flag - pin1 
 and extracted ion chromatograms of observed forms of pin1 peptide , 
 dcssakarg . the base peak chromatogram ( top ) shows the lc - ms elution 
 profile of peptides generated with endoproteinase aspn digestion of 
 the excised pin1 band . the observed m / z values of a selection of peptides are provided above their corresponding 
 chromatographic peaks . extracted ion chromatograms ( xics ) of the in 
 vivo unmodified and hne - modified peptide dcssakarg , residues 112120 , 
 are shown in middle and lower panels , respectively . a tolerance of 
 10 ppm around the theoretical m / z values of the precursor ions observed for peptide forms of dcssakarg 
 was used to generate the xics . the theoretical 
 values of these precursor ions are adjacent to the observed peaks 
 and were used to calculate ppm mass errors ( shown in parentheses ) 
 for the monoisotopic ions of the identified peptide forms . note that 
 the pin1 gel band was treated with dtt and iodoacetamide , resulting 
 in carbamidomethylation of available cys residues . thus , the in vivo 
 unmodified form of the peptide dcssakarg contains a carbamidomethyl 
 group on cys-113 . ( d ) ms / ms spectra of in vivo unmodified ( top ) and 
 hne - modified peptide ( bottom ) , dcssakarg . the [ m + 2h ] precursor ions with m / z values 
 476.21 and 526.77 , respectively , were selected for fragmentation . the observed singly and doubly protonated b- and y - type product ions 
 are assigned to their corresponding m / z peaks in the tandem mass spectra . the amino acid sequence is provided 
 above the annotated spectra , and the interresidue - placed brackets 
 denote sites of fragmentation that occurred with collision - induced 
 dissociation ( cid ) to produce the observed product ions . an asterisk 
 is used to indicate the localization of the cysteine residue ( cys-113 ) 
 modified by hne . egcg and pib are pharmacological agents 
 capable of suppressing proliferation of some cell types in a pin1-dependent 
 manner . hne , which is produced endogenously and 
 appears to modify the catalytic cysteine of pin1 , is also cytotoxic . although hne has many targets besides pin1 , we reasoned that the toxicity 
 of hne in mda - mb-231 cells may be partly due to modification of this 
 protein . we used sirna to knock down pin1 in cells and verified the 
 knockdown by western blot ( figure 6a ) . cells 
 transfected with scrambled sirna and pin1 sirna were treated with 
 increasing doses of hne or the pin1 inhibitor egcg for 48 h , at which 
 point cell viability was measured . unsurprisingly , pin1 sirna - transfected 
 mda - mb-231 cells were less sensitive to egcg - induced growth inhibition 
 as compared to scrambled sirna - transfected cells ( ic50 values 
 for scrambled and pin1 sirna - tranfected cells were 21.53 and 34.80 , 
 respectively ) ( figure 6b ) . cell viability was 
 statistically higher in egcg - treated pin1 sirna cells as compared 
 to scrambled control cells at 25 , 30 , and 40 m ( figure 6b ) . interestingly , pin1 knockdown also afforded 
 protection from hne toxicity relative to scrambled sirna - transfected 
 ( control ) cells . the percent viability of pin1 sirna cells treated 
 with hne was statistically increased at 20 , 25 , and 30 m as 
 compared to scrambled control sirna cells , resulting in an increase 
 in the ic50 for hne ( 21.29 m for scrambled vs 29.24 
 m hne for pin1 sirna cells , respectively ) ( figure 6c ) . these data support the hypothesis that hne modification 
 of pin1 at cys-113 plays a role in the cellular response to lipid 
 electrophile production during conditions of oxidative stress . knockdown of 
 pin1 partially protects mda - mb-231 cells against hne - induced 
 growth inhibition . ( a ) western blot of pin1 sirna and scrambled control 
 ( gapdh loading control ) . ( b ) viability assay of scrambled and pin1 
 sirna - transfected mda - mb-231 cells treated with the pin1 inhibitor 
 egcg . knockdown of pin1 in mda - mb-231 cells results in protection 
 from egcg - induced growth inhibition ( 21.53 m for scrambled 
 vs 34.80 m for pin1-sirna transfected cells ) . ( c ) viability 
 assay of scrambled and pin1 sirna - transfected mda - mb-231 cells treated 
 with hne . knockdown of pin1 in mda - mb-231 cells results in a shift 
 in the ic50 of hne ( 21.29 m for scrambled vs 29.24 
 m for pin1-sirna transfected cells ) . n = 8/group ; 
 error bars represent standard deviation ; * p < 
 0.05 , * * p < 0.01 , and * * * p < 
 0.001 . the cis isomer of peptidyl - prolyl motifs 
 in protein sequences occurs 
 with a frequency of approximately 56% , a large majority of which appear at surface - exposed bend , coil , 
 or turn conformations . phosphorylation of serine or threonine preceding 
 proline renders the peptide bond resistant to conventional ppiases , 
 such as cyclophilin ( cyp18 ) and fkbp ( fkbp12 ) enzymes , while simultaneously 
 generating a pin1-bindable motif . the 
 tertiary structure and activity of proteins containing multiple ps - p 
 or pt - p motifs can be largely dictated by whether these bonds are 
 present in cis or trans . therefore , pin1 maintenance in cells is of 
 great importance , as protein substrate activity and/or stability can 
 be directly dependent on pin1 catalysis of pser - pro and pthr - pro bonds . here , we investigated the susceptibility of pin1 to modification 
 by hne . hne is a reactive aldehyde generated from the nonenzymatic 
 oxidation of arachidonic acid or linoleic acid . although hne is produced 
 endogenously at low micromolar concentrations , elevated levels of 
 hne are associated with a variety of diseases such as ad , carcinogenesis , and diabetes , among others . previous work from 
 our laboratory has shown that hne administration to cells activates 
 multiple signaling networks , such as the dna damage , antioxidant , 
 and heat shock response pathways . additionally , 
 hne modifies proteins at exposed nucleophilic sites , altering tertiary 
 structure and ultimately modifying activity . because of the importance 
 of pin1 to the stability of transcription factors ( e.g. , p53 , -catenin , 
 c - myc ) as well as its effects on cell cycle checkpoint kinetics , the 
 susceptibility of pin1 to modification during conditions of oxidative 
 stress warrants investigation . results of our experiments using 
 purified protein incubated with 
 hne , as well as from cells treated with hne , support that pin1 is 
 sensitive to electrophile adduction . we chose mda - mb-231 breast cancer 
 cells as our model to study the susceptibility of pin1 to hne modification , 
 as previous studies have reported suppressed growth rates of small 
 interfering rna ( sirna)-silenced pin1 mda - mb-231 cells in vitro and 
 decreased volume of tumors following orthotopic injection of short 
 hairpin rna ( shrna)-silenced pin1 mda - mb-231 cells into 
 the mammary fat pad of immunodeficient mice . mda - mb-231 cells incubated with ahne and subject to click chemistry 
 revealed a concentration - dependent increase in ahne adduction of pin1 , 
 with increasing modification observed as low as 2.5 m . in resting 
 cells , concentrations of hne are reported to range from 0.5 to 3 m ; however , in tissues and fluids experiencing 
 oxidative stress , levels of hne are elevated as much as 10-fold . levels of hne in ad ventricular fluid have been 
 reported at 15.2 m , and coincidentally , 
 pin1 has been identified as an excessively carbonylated ( 2,4-dinitrophyenylhydrazine - reactive , 
 dnph - reactive ) protein in brains of patients with ad and mild cognitive 
 impairment ( mci ) . although the site of pin1 modification 
 and adducting carbonyl - containing species was not determined in these 
 studies , hne adduction of pin1-active site residues is a possible 
 scenario . nevertheless , our data support that pin1 is covalently modified 
 by lipid electrophiles under conditions that mimic a pathogenic level 
 of oxidative stress . elucidation of amino acid sites of modification 
 by hne is critical 
 to determining the effect the modification has on cellular signaling 
 pathways . experiments incubating purified protein with electrophiles 
 have been valuable in categorizing proteins with hne - sensitive active 
 sites ( e.g. , thioredoxin and thioredoxin reductase ) and those whose modification occurs at residues outside 
 of the active site ( e.g. , hsp90 ) . active 
 site modifications of proteins by hne would theoretically have a greater 
 impact on protein function relative to modifications that occur outside 
 of the active site . our data from experiments incubating purified 
 pin1 with 2 mm hne revealed the presence of three michael adducts , 
 with two ( cys-113 and his-157 ) occurring at surface - exposed active 
 site residues . no schiff base modifications were observed , and although 
 a third michael adduct was detected ( lys-132 ) , the relative extent 
 of modification was significantly lower as compared to cys-113 and 
 his-157 adduction . data from time - dependent experiments support the 
 saturation of cys-113 by hne before quantifyable levels of his-157 
 adduction were observed . furthermore , cys-113 adduction was detectable 
 at lower concentrations of hne incubated with pin1 . we suspect that 
 the equivalent relative reactivity of cys-113 and his-157 observed 
 in mapping the amino acid modification sites ( figure 2a and table 1 ) is due to a stepwise 
 saturation of cys-113 , followed by modification of his-157 , resulting 
 from the large amount of hne used ( 2 mm ) in this experiment . this 
 suggestion is supported by results of the concentration - dependent 
 hne incubation experiments , in which the maximal concentration was 
 10-fold lower ( 200 m ) and revealed minimal his-157 adduction . cys-113 is the most important amino acid to pin1 function , and 
 cysteine residues are the most reactive amino acids with hne . cys-113 
 of pin1 is surface exposed , thereby accessible to electrophiles such 
 as hne ( figure 7a ) . a proposed mechanism of 
 isomerase action has suggested that his-59 abstracts a proton from 
 cys-113 , and the resulting thiolate interacts with the carbonyl carbon 
 of proline from the substrate , although 
 this mechanism has been challenged . mutagenesis 
 experiments have revealed that mutation of cys-113 to ala results 
 in 123-fold loss of protein activity . furthermore , this conserved cysteine is essential for inhibition 
 of parvulin ppiases by juglone . upon 
 observing that cys-113 is the primary site of adduction of purified 
 pin1 incubated with hne , we examined whether similar modifications 
 to pin1 would be observed in cells treated with hne . our proteomics 
 data of pin1 immunoprecipitated from flag - pin1 transfected mda - mb-231 
 cells treated with hne support that cys-113 is the primary site of 
 modification of pin1 upon cellular generation of hne . evidence of 
 adduction of this peptide is supported by low ppm ( 1.1 ) mass error 
 as well as a large shift in retention time of peptide elution ( resulting 
 from increased hydrophobicity of the reduced hne - bound peptide relative 
 to the unadducted peptide ) . furthermore , multiple chromatographic 
 peaks each having equivalent mass ( figure 5c ) were observed . it is likely that this is due to chiralty conferred 
 on the modified peptide by the reduced hne michael adduct , 
 causing slight but noticeable differences in retention time , as previously 
 reported . no other modified pin1 peptides were observed , 
 including his-157 , although the unmodified species of this peptide 
 was recovered ( figure s3 in the supporting information ) . our data support that pin1 is covalently modified in cells primarily 
 at the catalytic cysteine upon generation of hne . pymol image of pin1 surface 
 ( left ) and active site ( right ) including 
 hne adducted to cys-113 . ( a ) cys-113 , a surface exposed residue , is 
 indicated by the arrow and labeled in orange . also in ad , pin1 is oxidatively modified , down - regulated , 
 and has decreased activity as compared to brains from normal elderly 
 controls . in cancer , egcg , a green 
 tea polyphenol with potent anticancer activity , has recently been 
 identified as a pin1 inhibitor . egcg 
 interacts with both the ww and the ppiase domains of pin1 , interfering 
 with pin1-substrate binding and catalytic activity . furthermore , pin1 
 knockout mouse embryonic fibroblasts ( mefs ) were protected from egcg - induced 
 cell death as compared to pin1-expressing ( wt ) mefs . we observed that pin1 sirna partially protected mda - mb-231 
 cells against hne toxicity as compared to scrambled control sirna 
 cells . the magnitude of the increase in the ic50 of pin1 
 sirna cells treated with hne was less than the increase observed with 
 egcg ( figure 6b , c ) . this is likely due to the 
 fact that hne only modifies the catalytic cysteine , whereas egcg binds 
 to both the active site and the ww domain and to the fact that hne 
 does not modify all of the pin1 in the mda - mb-231 cells at the concentrations 
 used for the toxicity experiment . in addition , hne modifies many protein 
 targets so that pin1 is not the sole determinant of toxicity . unlike the pharmacological agents egcg , pib , and juglone , hne is 
 produced endogenously by cells and is elevated in conditions of oxidative 
 stress . the results presented demonstrate the modification of the 
 catalytic cysteine of pin1 by an endogenously produced species that 
 may have potential implications in disease where oxidative stress 
 and deregulation of pin1 coexist .