Kopelman Online Article Fall 2009

8/14/2019 Kopelman Online Article Fall 2009

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RESEARCHVolume 2 Issue 2 | Fall 2009

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Y-Box Binding Protein 1 is a Novel

Substrate of Granzyme A

Granzymes are the cell death eector serine proteases in the granules o natural killer (NK) cells and cytotoxic lymphocytes (CL) that target cells during an immune response. Among the ve human granzymes, Granzyme A(GzmA) is the most abundant. It initiates caspase-independent cell death that is morphologically indistinguishablerom apoptosis. Once delivered by killer cells into inected cells or tumor cells that have been targeted or elimina-tion, GzmA cleaves a number o proteins inside the cell, including multiple components o the SE complex, to in-duce apoptosis. Te Lieberman Laboratory perormed yeast two-hybrid screens that identied Y-box binding protein1 (YB-1) as a protein that specical ly interacts with two SE complex proteins, SE and pp32. YB-1 is a multiunc-tional nucleic acid-binding protein whose overexpression has been implicated in multiple cancers. We investigatedthe possibility that YB-1 might also be a substrate o GzmA. reatment o lysate rom cells over-expressing YB-1 withGzmA and treatment o whole cells with GzmA and perorin (PFN) demonstrated dose-dependent proteolysis o

YB-1. Puried recombinant YB-1 protein was cleaved by GzmA in the arginine-rich region between R234 and R253.

Introduction

Upon recognition o virally inected or tumor cells, naturalkil ler (NK) cells and cytotoxic lymphocytes (CL) release thecontents o their cytotoxic granules into the immunological syn-apse ormed with the cell singled out or elimination. Tese cy-totoxic granules contain a amily o serine proteases called gran-zymes (granule enzyme, Gzm) and a pore-orming protein calledperorin (PFN). PFN acilitates the entry o Gzms into the cytosolo the target cell, where they initiate a cascade o events ultimatelyinducing programmed cell death. Te dierent members o the Gzm

amily are remarkable in the diversity o substrates upon whichthey act and the distinct pathways o cell death that they initiate.

Te ve human Gzms are encoded on three gene clusters andeach Gzm has the ability to initiate distinct pathways o pro-grammed cell death; this redundancy likely evolved to allow theimmune system to combat a wide range o pathogens. While GzmAand GzmB are the most abundant members o the amily, the majority o research eorts historically have ocused on dissectingthe GzmB cell death pathway (Chowdhury and Lieberman, 2008).Granzymes are homologous to trypsin and other related serineproteases, named or the conserved serine residue in their activesite (Bell et al., 2003). High resolution crystal structures o some othe Gzms and the act that GzmA substrates do not share a com-

mon peptide sequence around their cleavage site suggest that subtledierences in active site conormation are responsible or the sub-strate specicity o each protease (Supplemental Figure 1) (Bell et al.,2003; Chowdhury and Lieberman, 2008; Hink-Schauer et al., 2003).

Expression o Gzm and PFN genes in nave CD8 cells is triggeredby cell receptor (CR) stimulation by antigen-presenting cells (Bossiand Griths, 2005). A signal sequence directs the nascent Gzms to theendoplasmic reticulum (ER) and begins the process o Gzm trackinginto cytotoxic granules, a specialized set o secretory lysosomes. A-ter cell recognition-mediated activation o cytotoxic cells, the lyticgranules o the killer cell migrate to the immunological synapse. Tegranule membrane uses with the immune cell plasma membrane,releasing PFN and Gzms into the immunological synapse. Entry o

Gzms into the target cell cytosol is mediated by PFN by a mecha-nism that is not ully-understood. Once inside the target cell, Gzmsare capable o initiating at least three distinct cell death pathways(Chowdhury and Lieberman, 2008). In the GzmA pathway, GzmAtranslocates to the interior o the mitochondria and nucleus to in-duce cell death. It has so ar been shown to cleave 8 protein substratesin the cell with a high degree o specicity (Supplemental able 1).

Inside the mitochondrial matrix, GzmA-mediated cleavage oa component o the electron transport chain complex I, NDUFS3,leads to generation o reactive oxygen species (ROS) (Martinvaletet al., 2008; Martinvalet et al., 2005), driving the ER-associated SE

complex into the nucleus. Tis complex is involved in the oxida-tive stress response and is currently known to be composed o sixproteins: three nucleases (Ape1, NM23-H1, and REX1), two chro-matin-modiying proteins (SE and pp32), and the DNA-bindingprotein HMGB2 (Beresord et al., 1997; Chowdhury et al., 2006; Fanet al., 2003a; Fan et al., 2002; Fan et al., 2003b). Inside the nucleus,GzmA cleaves SE, an inhibitor o the GzmA-Activated DNase(GAAD) NM23-H1. Tis activates the NM23-H1 endonuclease tocause single-stranded nicking o chromosomal DNA. DNA damageis then extended by the SE complex exonuclease REX-1 (Chow-dhury et al., 2006; Fan et al., 2003a). At the same time, GzmA alsocleaves two other SE complex proteins: HMGB2, a DNA bendingprotein, and Ape1, a crucial endonuclease in base excision repair

(BER) (Fan et al., 2003a; Fan et al., 2002). GzmA cleavage and in-activation o Ku70 and PARP-1 proteins in the nucleus also inter-eres with single- and double-strand DNA break repair pathways(Zhu et al., 2006). By wreaking havoc on the cells DNA repairmachinery, GzmA eectively commits the cell to cell death (Fig-ure 1). Cells subjected to GzmA and PFN loading die rapidly anddisplay morphological evidence o apoptosis, including membraneperturbation, nuclear condensation, and DNA damage. Further-more, caspase inhibition does not rescue targeted cells. GzmAthereore induces a novel apoptotic pathway that allows immunecells to wage war against inected cells that have developed meansto evade caspase pathways o cell death, as is the case or many tu-mors and some viruses (Beresord et al., 1999; Shresta et al., 1999).

David KopelmanHarvard College 09, [email protected]


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RESEARCH Volume 2 Issue 2 | Fall 2009

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In an ongoing eort to identiy new substrates o GzmA and un-derstand the unction o the SE complex, previous members o theLieberman Laboratory perormed yeast two-hybrid screens to iden-tiy proteins that interact with two components o the SE complex:SE and pp32. In these screens, the transcription actor Y-box bindingprotein 1 (YB-1) was shown to interact with both SE and pp32. Sub-sequent GS pull-down and co-immunoprecipitation assays in 293cells stably expressing HA-tagged YB-1 conrmed that SE and YB-1interact both in vitro and in cells, respectively (Zhang, unpublisheddata). YB-1 was original ly identied as a transcription actor (F) that

binds to the Y-box o major histocompatibility complex (MHC) class IIpromoters (Didier et al., 1988). Te 324 amino acid protein belongs tothe cold-shock domain (CSD) amily o proteins, which is conservedin nearly all living organisms and participates in environmentalstress responses in eukaryotes (Supplemental Figure 2) (Evdokimovaet al., 2006a). YB-1 is localized predominantly in the cytoplasm butrapidly translocates to the nucleus in response to anticancer drugs,hyperthermia, and UV radiation (Koike et al., 1997; Stein et al., 2001;Uchiumi et al., 1993). Tis illustrates a crucial potential link betweenYB-1 and the SE complex, which also translocates to the nucleus inresponse to oxidative stress. In the absence o stress, the C-terminuso YB-1 possesses a cytoplasmic retention signal (CRS) that prevailsover a nuclear localization signal (NLS) (Bader and Vogt, 2005; Jur-

chott et al., 2003). Since the CRS was rst characterized, nuclear trans-location has been shown to result rom cleavage o the C-terminus oYB-1 by both the 20S proteasome and thrombin (Sorokin et al., 2005;Stenina et al., 2001). Trombin and GzmA are both serine proteases.

YB-1 can regulate transcription in three ways: (1) direct bindingto the Y-box and related sequences, (2) interaction with other Fs(serving as a co-activator or co-repressor), or (3) binding to transcrip-tional promoters to either enhance or inhibit binding o other Fs.Te transcription o 21 genes has been shown to be regulated by YB-1.Interestingly, YB-1 down-regulates and up-regulates approximatelyequal numbers o genes (Kohno et al., 2003). One gene which YB-1was originally shown to upregulate is the multidrug resistance 1 gene(MDR1), which encodes an ABC transporter commonly associated

with increased drug resistance in cancer. Multiple studies demostrated that translocation o YB-1 into the nucleus results in upreglation o the MDR1 gene (Bargou et al., 1997; Oda et al., 1998; Ohgaal., 1996; Ohga et al., 1998). However, this theory has since been qutioned aer experiments utilizing RNA intererence (RNAi) indiced that YB-1 is not directly involved in MDR1 regulation (Kaszubet al., 2007). Although originally described as a transcription actYB-1 was later shown to also regulate translation in the cytoplas

where the majority o YB-1 is ound. YB-1 also infuences translatithrough multiple mechanisms, including (1) enhancing splicing, acilitating the ormation o messenger ribonucleoprotein partic(mRNPs), and (3) stabilizing nascent mRNAs in a cap-dependemanner (Evdokimova et al., 2001; Skabkin et al., 2004; Stickeleral., 2001). Microarray analysis has revealed that many YB-1-assoated mRNAs encode proteins regulating cell prolieration, oncogetransormation, and the stress response (Evdokimova et al., 2006

Recent studies concerning YB-1 have urther explored the cosequences o nuclear localization o YB-1, which is enhanced cancer cells. Increased nuclear localization in cancer correlawith tumor size, degree o invasion, lymph node metastasis, apoor clinical prognosis. Tis has been demonstrated in lung, prtate, and breast cancers; osteosarcoma; and multiple myelom(Chatterjee et al., 2008; Gimenez-Bonae et al., 2004; Oda et 1998; Saji et al., 2003; Shibahara et al., 2001). Similarly, high YBexpression in cells also correlates with drug resistance and potumor prognosis (Homer et al., 2005). Elucidation o the moleclar mechanisms behind these clinical observations will help termine whether YB-1 might be a potential therapeutic targ

Method

Cell Lines and Reagents

Cells were obtained rom the American ype Culture Colltion (Manassas, VA). HeLa cells were maintained in DMEM (Invrogen) supplemented with 10% FBS, 100 units/mL Penicillin G, 1

g/mL streptomycin sulate, 6 mM HEPES, 1.6 mM L-glutamiand 50 M -mercaptoethanol. K562 cells were maintained in RMI-1640 (Mediatech, Inc.) similarly supplemented. Te ollowiprimary antibodies were used: HA High Anity (Roche, 1:20working dilution), YB-1 (Epitomics, 1:5000), -tubulin (Sigm1:1000), -actin (rom J. Lin, University o Iowa; 1:1000), caspase(Stressgen Bioreagents, 1:1000), GS (GE Healthcare, 1:2000), aNM23-H1 (Santa Cruz Biotechnology, 1:1000). SE (1:1000) app32 (1:1000) antibodies were produced as described (Beresordal., 2001). Te ollowing secondary antibodies were used: -GoIg-HRP (Santa Cruz Biotechnology, 1:2000), -Rabbit-Ig-HRP (Healthcare, 1:2000), and -Mouse-Ig-HRP (GE Healthcare, 1:500

TransectionMammalian expression plasmid pCDNA6-YB1-HA was a kigi o Hans-Dieter Royer (Germany) (Supplemental Figure (Chatterjee et al., 2008). 5x105 HeLa cells were treated with 3 gplasmid and Lipoectamine 2000 Reagent (Invitrogen) at 37C 4 hours according to the manuacturers instructions in the prence o Opti-MEM (Invitrogen). Aer washing cells twice wPBS (Mediatech, Inc.), DMEM was added to wells and cells wincubated at 37C. Cells were plated 24 hours prior to transtion and transected cells were lysed 48 hours aer transectio

Cell Lysate Preparation

HeLa cells were washed twice with PBS and incubated with

Figure 1. Granzyme A cell death pathway. Upon entry into the tar-get cell, (1) GzmA targets the mitochondria and induces the produc-tion o reactive oxygen species (ROS). (2) ROS production leads tothe translocation o the SE complex into the nucleus. (3) GzmA

also enters the nucleus by an unknown mechanism and targets mem-bers o the SE complex and other proteins, resulting in irreversibleDNA damage and cell death. Adapted rom (Cullen and Martin, 2008).


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pLE Express (Invitrogen) at 37C or 5 minutes. Following washingwith DMEM, cells were centriuged at 500g or 5 minutes, the su-pernatant was removed, and the pellet was resuspended in 500 Lpolysome lysis buer (PLB) (100 mM KCl, 5 mM MgCl2, 10 mMHEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM D, 100 U ml-1 RNa-

sin RNase inhibitor, 2 mM vanadyl ribonucleoside complexes so-lution, and 25 L ml-1 protease inhibitor cocktail or mammaliantissues) (Peritz et al., 2006). Samples were incubated on ice or 10minutes and then centriuged at 14,000 rpm or 15 minutes. Su-pernatant (cell lysate) was collected and stored at -20C until use.

Western Blot

Samples were separated by SDS-PAGE and transerred to nitro-cellulose membrane (Millipore) using a rans-Blot SD Semi-DryElectrophoretic ranser Cell (Bio-Rad). Membranes were blockedin 5% non-at milk (Nestle) in 0.05% ween 20 with BS (BS) or30 minutes and then incubated in the indicated primary antibody in5% non-at milk BS either at 4C overnight or at room tempera-

ture or 1 hour. Membranes were washed three times with BS,incubated in secondary antibody in 5% non-at milk BS or 1 hourat room temperature, and washed again. Blots were visualized us-ing homemade ECL solution and Scientic Imaging Film (Kodak).

In vitro Cleavage Assays

Protein samples were suspended in 20-50 L PBS with indi-cated concentration o Gzms and incubated at 37C or indicatedlength o time. Cell lysate cleavage assays were perormed using0.2 g cell lysate protein/L and GS-YB1 cleavage assays were per-ormed using 0.1 g puried protein/L. All samples were equal-ized to nal working volume with PBS. Aer incubation, 5X re-ducing SDS sample buer was added and samples were boiled at

100C or 5 minutes. Samples were stored at -20C until analysis.

Cell Treatment with PFN and Gzms

K562 cells were washed three times with HBSS (Mediatech, Inc.)and 5x104 cells were resuspended in 60 L cell loading buer (HBSS

with 10 mM HEPES [pH7.2], 0.4% BSA, 3 mM CaCl2). A sublytic doseo PFN [dened as the concentration that lyses 5%-15% o cells (Mar-tinvalet et al., 2008)] and/or Gzms (concentration as indicated) wereadded and cells were incubated at 37C or 1 hour. In cells pretreatedwith a caspase-inhibitor, 50 M Caspase-3 Inhibitor II, Z-DEVD-FMK(Calbiochem) was added to the cells in suspension and then incubatedat 37C or 30 minutes prior to addition o granule enzymes. Te re-action was stopped by adding 15 L o 5X reducing SDS sample bu-er, boiling at 100C or 5 minutes, and storing at -20C until analysis.

Protein Expression and Purifcation

Recombinant GS-YB1 bacterial expression plasmid was a kindgi o Kimitoshi Kohno (Japan) (Ise et al., 1999; Okamoto et al., 2000).

One L plasmid was used to transorm BL21 DE3-competent E. coli.Cells were incubated on ice or 30 minutes, heat-shocked at 42C or30 seconds, and added to 250 L Super Optimal broth with Cataboliterepression (SOC) Medium (New England Biolabs). Cells were incubat-ed in a shaker at 37C or 1 hour and then 50 L o culture was spreadon an LB-ampicillin agar plate beore incubation overnight at 37C.A single colony was selected or inoculation in 500 mL LB Broth, Len-nox (Fisher Scientic) containing 50 g/mL ampicillin and incubatedat 37C overnight. Tis culture was used to seed 4 L o LB-ampicillin.Aer reaching an OD600 o 0.7, the large culture was induced by add-ing isopropyl -D-1-thiogalactopyranoside (IPG) (Sigma Aldrich)at a nal concentration o 250 g/mL. Four hours aer induction, thebacterial pellet was harvested, resuspended in lysis buer, sonicated,

Lysate + - + + + + +

GzmA (M) - 1 0.1 0.5 1 - -

S-AGzmA (M) - - - - - 1 -

GzmB (M) - - - - - - 1

HA

SET

-Tubulin50 kD

HA20 kDCleaved YB1-HA

50 kDYB1-HA

37 kDSET

25 kDCleaved SET SET

Figure 2. GzmA cleaves YB1-HA in HeLa cell lysate. Lysate rom HeLa cellsover-expressing YB1-HA was treated with increasing concentrations o ac-tive GzmA at 37C or 1 hour. Inactive S-AGzmA- and GzmB-treated celllysates served as negative controls. Immunoblots were probed with HA, SE,and -ubulin antibodies. GzmA cleaved YB1-HA in a dose-dependentmanner that was consistent with cleavage o SE, a known substrate o GzmA.

PFN - + - + + +

GzmA

(M)- - 1 0.05 0.2 1

SET

NM23-H1

YB-1

Figure 3. YB-1 is cleaved in K562 cells treated with PFN and GzmA.K562 cells were treated with PFN and varying concentrations o activeGzmA at 37C or 1 hour. Cells treated with PFN or GzmA alone served asnegative controls. Immunoblots were probed with YB-1, SE, and NM23-H1 antibodies. In the presence o PFN, GzmA cleaved YB-1 in intact K562cells in a dose-dependent manner that was consistent with cleavage o SE,a known substrate o GzmA. Tis experiment was perormed by DenisMartinvalet. Te immunoblot or SE was provided by Danielle Jensen.


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and centriuged at 20,000g at or 1 hour. Lysate was passed througha 0.45 m lter and then loaded onto a PBS-equilibrated columncontaining Gluathione Sepharose 4 Fast Flow (GE Healthcare) usingthe BioLogic DuoFlow FPLC (Bio-Rad). Column was washed with5 column volumes (CV) o PBS and then eluted using 1.5 CV o anincreasing linear gradient o elution buer (10 mM reduced glutathi-one) ollowed by 2 more CV o elution buer. In the second round opurication using the Glutathione Sepharose column, pooled sam-ples were reloaded onto the column, washed with 10 CV o PBS con-

taining 0.5 M NaCl, and eluted as previously described. In the nalround o purication, pooled samples were passed through a Sep-hadex 75 (S-75) gel ltration column (Amersham) equilibrated withPBS. Recombinant GzmA and inactive S-AGzmA were expressedand puried as reported (Beresord et al., 1997). PFN and GzmB wereexpressed and puried as reported (Shi et al., 1992; Shi et al., 2000).

Mass Spectrometry

Samples selected or analysis by mass spectrometry were excisedrom Coomassie-stained SDS-PAGE gel o samples rom the in vitroGS-YB1 cleavage assay and processed at the aplin Mass Spectrom-etry Facility (Harvard Medical School) using tryptic digestion. Pep-tides were aligned to the amino acid sequence or YB-1 as posted by the

National Center or Biotechnology Inormation (NCBI). Teoreticalmolecular weights o terminal peptide ragments were calculated usingthe ProtParam ool rom the ExPASy (Expert Protein Analysis Sys-tem) proteomics server o the Swiss Inst itute o Bioinormatics (SIB).

RESULTS

Measured by propidium iodine uptake and fow cytometry (datanot shown) (Martinvalet et al., 2008). Te level o ull-length YB-1decreased when cells were treated with increasing concentrationso GzmA in the presence o PFN, but did not change in cells thatwere treated with PFN, GzmA, or GzmB alone (Figure 3 and datanot shown). Unlike in HeLa cell lysates, the YB-1 cleavage productwas not detectable by YB-1 antibody, most likely due to the uniqueepitope recognized by the antibody or the act that cleavage rag-ments may be more labile in intact K562 cells. Cleavage o nativeYB-1 was not inhibited by pretreating the target cells with the cas-pase inhibitor Z-DEVD-FMK, which is consistent with the caspase-independence o GzmA-mediated cell death (data not shown).

Expression o Recombinant GST-YB1

o begin to characterize the GzmA cleavage site, we puried re-combinant YB-1 protein using an E. coli expression system. Te re-combinant protein could then be treated with GzmA to isolate cleav-age ragments or sequencing. A bacterial expression system wasused to express large quantities o YB-1 used at the N-terminus to

a glutathione S-transerase (GS) tag to allow purication o the re-combinant YB-1 protein (GS-YB1, Supplemental Figure 3A), as pre-viously reported (Hayakawa et al., 2002; Ise et al., 1999; Okamoto etal., 2000). Te usion protein has an expected MW o ~75 kDa, whichis consistent with the usion o the ~26 kDa GS tag to the ~50 kDaYB-1 protein. Purication by ast protein liquid chromatography(FPLC) using glutathione resin yielded large quantities o recombi-nant protein, as determined by raction analysis using SDS-PAGEand Coomassie staining. Te indicated ractions o eluted proteinwere pooled and subjected to two urther rounds o purication in-volving a high salt wash (to remove non-specically bound proteins)and gel ltration (to remove low WV contaminants) (Supplemen-tal Figure 4). Upon elution, the chromatogram showed a dominant

peak in ractions #5 and 6 with an approximate MW consistent wthe expected MW o GS-YB1 o ~75 kDa (Supplemental FigureScreening o the peaks rom the nal purication step demonstrathat a doublet o GS-YB1 was obtained in early ractions (Suppmental Figure 4D). We do not understand why the GS-YB-1 usprotein elutes as a doublet, but other investigators have reported silar results (Ise et al., 1999). Western Blot conrmed that both o dominant bands rom Supplemental Figure 4D reacted with GS YB-1 antibodies (data not shown). Fraction #5 contained 0.1 g pued protein/L and was selected or use in subsequent experimen

GzmA Cleaves GST-YB1 in vitro

Puried protein was subjected to in vitro GzmA cleavage asssimilar to those perormed using HeLa cell lysates. Samples o purirecombinant GS-YB1 were treated with increasing concentrationGzmA and appropriate controls, separated by SDS-PAGE, and stainwith Coomassie Blue. GzmA, GzmB, and GS alone were incubaalone to allow dierentiation between ull-length protein, enzytag, and potential cleavage ragment bands (Figure 4). When puriprotein was incubated with increasing concentrations o GzmA, length GS-YB1 (~75 kDa) decreased at the same time that two nbands appeared with apparent molecular weights o ~50 kDa and ~

kDa. Te intensity o the GzmA band at ~25 kDa also increased as concentration o GzmA increased. GzmB did not have a signiceect on the level o ull-length GS-YB1. o characterize the nbands, the GS-YB1 cleavage assay samples were reanalyzed by Wern Blot, probing with YB-1 and GS antibodies (Supplemental Fure 6). In the GS-YB1 cleavage assay, the YB-1 antibody recognithe new ~50 kDa band in GzmA-treated samples, but not the ~40 kband. wo new bands reacted with GS antibody in the GzmA-treasamples. Te only new band that appeared in GzmA-treated sampand reacted with GS antibody migrated at ~26 kDa, the MW o G

Based on these results, we suspect that in addition to cleavYB-1, GzmA also cleaved the GS-YB1 usion protein between Yand the GS tag. Tis resulted in the accumulation o W-len

GST-YB1 + - - + + + + -

GzmA

(M)- 1 - 0.25 0.5 1 - -

GzmB

(M)- - 1 - - - 1 -

GST - - - - - - - +

75 kD

37 kD

50 kD

25 kD

Full-length

GST-YB1

Cleavage

fragment

GzmB

GzmAGST

Full-length Y

Figure 4. GzmA cleaves recombinant GST-YB1 in vitro. Puried GYB1 was treated with varying concentrations o active GzmA GzmB at 37C or 2 hours. Samples were separated by SDS-PAand the gel was stained with Coomassie Blue. GzmA cleaved recobinant GS-YB1 and cleavage product accumulated at ~40 kBands outlined in red were submitted or mass spectrometry analy


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YB-1 at ~50 kDa and an accumulation o GS monomer at ~26 kDa.Furthermore, based on the act that the YB-1 antibody recognizes theC-terminus o the protein (amino acids 280-301 o 324) and did notrecognize the ~40 kDa band in GzmA-treated samples (SupplementalFigure 6A), we hypothesized that this Coomassie-stained band rep-resents the N-terminal YB-1 cleavage ragment lacking the GS tag.Since GzmB did not cleave GS-YB1, this suggests that GzmA cleav-age o YB-1 is specic. Te size o the N-terminal cleavage ragmentappears consistent with our previous ndings in HeLa cell lysate. InFigure 2, GzmA cleavage o YB1-HA produced a ~20 kDa HA-taggedC-terminal ragment and here we report that the N-terminal cleavageragment o GS-YB1 is ~40 kDa. Although we would predict thatthe weights o N- and C-terminal cleavage ragments would add upto ~50 kDa, the accepted MW o YB-1, the slight variation in electro-phoretic mobility may not be an accurate refection o actual MW.

GzmA Cleaves YB-1 at the C-Terminus

Cleaved recombinant GS-YB1 bands were subjected to trypsindigest and mass spectrometry analysis to identiy the location o theGzmA cleavage site. Specically, the top band o the GS-YB1-alonecontrol sample observed at ~75 kDa and the cleavage ragments inthe three GzmA-treated samples observed at ~40 kDa (submitted asa single sample) were excised and submitted or analysis (submitted

samples outlined in red) (Figure 4). Peptide ragments were alignedaccording to the amino acid sequence o YB-1 obtained rom the Na-tional Center or Biotechnology Inormation (NCBI) (Figure 5). ryp-tic peptides covering the YB-1 sequence rom V54 to the C-terminalend o the protein (E324) were recovered in the u ll-length sample, butonly rom V54 to R234 in the cleavage ragment sample. Te regionbetween Q235 and R253, in which no peptides were identied in eithersample, is arginine-rich and might have resulted in small try ptic prod-ucts that were too small to be resolved by mass spectrometry. Surpris-ingly, no peptides corresponding to the rst 53 amino acids o YB-1were recovered rom either sample. Peptides corresponding to GSwere only recovered in the ull-length sample (data not shown), con-sistent with our hypothesis that the ~40 kDa band lacked the GS tag.

We interpret these data to mean that GzmA cleaves YB-1 withinthe arginine-rich region between Q235 and R253. Cleavage in thisregion would produce a C-terminal ragment o between 70 and 89amino acids in length. Te theoretical molecular weights predictedby the amino acid sequences o these two potential cleavage rag-ments (measured rom Q235 to E324 and rom R253 to E324) are~8.1 kDa and ~10.6 kDa, respectively. Hence, cleavage o peptideragments o these weights is consistent with the ~10 kDa dierencein electrophoretic mobility between W YB-1 (~50 kDa) and thecleavage ragment (~40 kDa) (Supplemental Figure 7). However, theregion between R234 and R253 contains seven arginine residues aerwhich GzmA, a tryptase, could potentially cleave YB-1. Tereore, we

were unable to identiy the precise preerred cleavage site. While ourresults eectively narrow the location o the GzmA cleavage site to aregion within the C-terminus o YB-1, urther experiments are re-quired to identiy the specic amino acid aer which YB-1 is cleaved.

Discussion

Based on preliminary data which suggest that YB-1 interacts withSE, a known substrate o the protease GzmA, in the present studywe investigated whether the nucleic acid-binding protein YB-1 is alsoa GzmA substrate. We ound that GzmA cleaves YB-1 in mamma-lian cell lysate and in whole cells treated with PFN and GzmA. Pu-rication o recombinant YB-1 using a bacterial expression systemand subsequent treatment with GzmA demonstrated that recombi-nant YB-1 is specically cleaved in a dose-dependent manner. Massspectrometry analysis o isolated YB-1 cleavage ragments suggestedthat GzmA cleaves YB-1 at the C-terminus, most likely between ami-no acid residues R234 and R253. aken together, our data strong-ly suggest that YB-1 is a direct physiological substrate o GzmA.

In the immediate uture, eorts should ocus on identiying theprecise GzmA cleavage site in YB-1 within the range o possible resi-dues suggested by our mass spectrometry analysis. Tis could be ac-complished by analyzing ragment samples without enzymatic digestor sequentially mutating each o the arginine residues in the range opotential cleavage sites and assaying or the mutant recombinant pro-teins ability to withstand GzmA-induced cleavage. Future experi-ments should next aim to determine whether cleavage o YB-1 is nec-

essary or the induction o apoptosis by GzmA. o test this, researchersshould explore the eect o over-expressing a GzmA-uncleavable mu-tant orm o YB-1 on GzmAs ability to induce cell death. Cells sta-bly over-expressing W and GzmA-uncleavable YB-1 would rst betreated with PFN and GzmA and apoptosis would then be assayed byfow cytometry ollowing annexin V and propidium iodide (PI) stain-ing. I cleavage o YB-1 is necessary or the induction o apoptosis, wewould expect that over-expression o the GzmA-uncleavable orm oYB-1 would render cells more resistant to GzmA-induced cell death.

Initial investigations into the hallmarks o apoptosis centeredon the eects o caspases, which were thought to be as the prima-ry mediators o this innate cellular process; however, it was latershown that caspase inhibition was not sucient to block the in-

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Full-length YB-1

MSSEAETQQP PAAPPAAPAL SAADTKPGTT GSGAGSGGPG

GLTSAAPAGG DKKVIATKVL GTVKWFNVRN GYGFINRNDT

KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKGA

EAANVTGPGG VPVQGSKYAA DRNHYRRYPR RRGPPRNYQQ

NYQNSESGEK NEGSESAPEG QAQQRRPYRR RRFPPYYMRR

PYGRRPQYSN PPVQGEVMEG ADNQGAGEQG RPVRQNMYRG

YRPRFRRGPP RQRQPREDGN EEDKENQGDE TQGQQPPQRR

YRRNFNYRRR RPENPKPQDG KETKAADPPA ENSSAPEAEQ

GGAE

324 amino acid sequence of YB-1

Peptide coveragePeptide present in full-length sample but missing from

cleavage fragment sample

Predicted region containing cleavage site

Cleavage Product

MSSEAETQQP PAAPPAAPAL SAADTKPGTT GSGAGSGGPG

GLTSAAPAGG DKKVIATKVL GTVKWFNVRN GYGFINRNDT

KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKGA

EAANVTGPGG VPVQGSKYAA DRNHYRRYPRRRGPPRNYQQ

NYQNSESGEK NEGSESAPEG QAQQRRPYRR RRFPPYYMRR

PYGRRPQYSN PPVQGEVMEG ADNQGAGEQG RPVRQNMYRG

YRPRFRRGPP RQRQPREDGN EEDKENQGDE TQGQQPPQRR

YRRNFNYRRR RPENPKPQDG KETKAADPPA ENSSAPEAEQ

GGAE

1

41

81

121

161

201

241

281

321

Figure 5. GzmA cleaves YB-1 at the C-terminus. Full-length. GS-YB1 andYB-1 N-terminal cleavage ragment bands were excised rom the gel de-picted in Figure 10 and submitted or tryptic digestion and mass spectrom-etry analysis. Peptide coverage was aligned according to the sequence orYB-1 obtained rom the National Center or Biotechnology Inormation(NCBI). Recovered peptides are indicated in bold. Highlighted in yelloware peptides recovered in the ull-length protein samples but missing romthe cleavage ragment sample. Highlighted in turquoise is the predictedrange within which GzmA cleaves YB-1. GzmA appears to cleave YB-1

between amino acids R234 and R253.


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duction o apoptosis and that caspase-independent cell death waspossible (ait and Green, 2008). Researchers also discovered thatinhibition o ROS generation aorded cells some protection romapoptosis, particularly in the context o cell deletion during de- velopment o the immune system (Hildeman et al., 1999; Sand-strom et al., 1994). reating cells targeted or destruction by CLswith superoxide scavengers and antioxidants completely blockedGzmA-induced cell death. ROS generation is now considered es-sential to GzmA-mediated cell death (Martinvalet et al., 2005).

ROS are the endogenous byproduct o respiration and oxidativemetabolism and are induced by a variety o environmental agents,such as ultraviolet (UV) radiation. ROS generation causes DNAdamage by creating abasic sites and plays an important role in theinduction o various diseases, including cancer. YB-1 was recentlyshown to stimulate the base excision activity o NEIL2, a recentlycharacterized oxidative base-specic DNA glycosylase (Das et al.,2007). YB-1s ability to inhibit translation o damaged RNA has alsobeen demonstrated. Researchers ound that ull-length YB-1 hadenhanced binding anity or 8-oxoguanine-containing RNA. Tisoxidized orm o guanine is generated in the nucleotide pool by theaction o oxygen radicals produced in cells and is capable o pair-ing with both cytosine and adenine. Studies in E. coli demonstrated

that when W YB-1, capable o binding 8-oxoguanine-containingRNAs, was expressed, cells acquired resistance against paraquat, adrug that induces oxidative stress in cells. Cells expressing a trun-cated orm o YB-1 that lacked binding anity remained sensitiveto the drug (Hayakawa et al., 2002). Tus, unctional YB-1 may pro-tect cells against oxidative stress by multiple mechanisms. Cleavageo YB-1 by GzmA raises the possibility that the human immunesystem may have evolved to cleave YB-1 to prevent the targetedcell rom recovering rom the damage induced by killer lympho-cytes and NK cells. Tis idea is supported by the act that GzmAhas already been shown to target a number o components o theSE complex, which is thought to be involved in oxidative DNA re-pair, as well as other proteins involved in repairing DNA damage.

Acknowledgments

I would like to thank Judy or providing me with the oppor-tunity to engage in what has easily proven to be one o my great-est learning experiences. Tis work would not have been possiblewithout your kindness and I would not be as proud o it as I amwithout your dedication to a higher standard. Tank you Denis,Ashish, Danielle, Perry, and all members o the Lieberman Labo-ratory or your assistance, advice, insight, and riendship. Fi-nally, I thank my parents, without whom nothing I accomplishwould be possible; thank you or providing me with the meansto explore and the ability to put the journey into perspective.

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Appendix

Supplemental Figures

Supplemental Figure 1. Homodimer structure o Granzyme A. Reproducedrom (Bell et al., 2003).

Supplemental Table 1. Granzyme A substrates

Target Functon in Cell

SET Inhibion of NM23-H1 endonuclease

HMGB2 Recognion of distorted DNA

Ape1 Endonuclease

Ku70 dsDNA break repair

PARP-1 DNA break recognion

Histones: H1, H2B, and H3 DNA condensaon

Lamins: A/C and B Structural stabil ity of nuclear envelope

NDUFS3 Subunit of ETC complex I


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Supplemental Figure 2. Y-box binding protein 1. (A) Ribbon structure and

(B) domain structure o YB-1. Variable N-terminal domain (V), Cold ShockDomain (CSD), and alternating basic/acidic residue repeats (B, A). Re-produced rom http://www.pdb.org and (Kloks et al., 2002), respectively.

Supplemental Figure 3. Transient transection with YB1-HA plasmid

leads to YB-1 expression. (A) Schematic representation o the two plas-mids used in experiments. (B) Cell lysates rom HeLa cells transientlytransected with either YB1-HA or empty vector plasmid were analyzedby Western Blot. Immunoblots were probed with HA and -ubulinantibodies. YB1-HA was only expressed in YB1-HA-transected cellsand migrated at the expected apparent molecular weight o ~52 kDa.

Supplemental Figure 4. Expression o recombinant GST-YB1. (A) Schemrepresentation o GS-YB1 purication scheme. Coomassie-stained SPAGE gels show ractions o GS-YB1 puried by (B) glutathione an(C) glutathione anity with 0.5 M NaCl wash, and (D) gel ltration. In ull-length GS-YB1 is indicated by asterisk. Elution raction #5 rom purication was used in subsequent experiments, indicated by yellow

Supplemental Figure 5. Elution profle o recombinant GST-Y

rom gel fltration column. FPLC chromatogram o gel ltration rication o GS-YB1 shows elution o GS-YB1 between tions #3 and 8, which is consistent with raction screeningSDS-PAGE and Coomassie staining in Supplemental Figure

A

B

1 324

Absorbance(260nm)

Time (min)


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Supplemental Figure 6. N-terminal cleavage ragment o YB-1 protein was

submitted or mass spectrometry analysis. Samples rom GS-YB1 cleav-age assay depicted in Figure 4 were reanalyzed by Western Blot. Immuno-blots were probed or (A) YB-1 and (B) GS. GzmA cleaved recombinantGS-YB1 and cleavage product accumulated at ~40 kDa.

Supplemental Figure 7. Schematic representation o predicted YB-1

cleavage ragments. We interpret the results o the mass spectrometry

analysis to mean that ull-length GS-YB1 was cleaved by GzmA be-tween YB-1 and its GS tag and at some point within the arginine-rich region at the C-terminus o YB-1. Tis schematic representsthe predicted YB-1 cleavage ragments that would result i GzmAwere to cleave YB-1 rom the GS tag or C-terminal to the earli-est and latest potential cleavage sites within the region between R234and R253. Cleavage o such ragments rom W YB-1 is consistentwith the observed change in electrophoretic mobility o ~10 kDa.

Current contact information: FDA/CBER/DH/LH,National Institutes o Health, 8800 Rockville PikeBethesda, MD 20892

GST-YB1 + - - + + + + -

GzmA (M) - 1 - 0.25 0.5 1 - -

GzmB (M) - - 1 - - - 1 -

GST - - - - - - - +

GST

Full-length

GST-YB175 kD

37 kD

50 kD

25 kD

75 kD

37 kD

50 kDYB-1

A

B

Full-length

GST-YB1

Full-length

YB-1

GST

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