Volume 2 Issue 2 | Fall 2009 RESEARCH

Y-Box Binding Protein 1 is a Novel

Substrate of Granzyme A
David Kopelman
Harvard College ‘09, dbkopelman@gmail.com
Granzymes are the cell death effector serine proteases in the granules of natural killer (NK) cells and cytotoxic T
lymphocytes (CTL) that target cells during an immune response. Among the five human granzymes, Granzyme A
(GzmA) is the most abundant. It initiates caspase-independent cell death that is morphologically indistinguishable
from apoptosis. Once delivered by killer cells into infected cells or tumor cells that have been targeted for elimina-
tion, GzmA cleaves a number of proteins inside the cell, including multiple components of the SET complex, to in-
duce apoptosis. The Lieberman Laboratory performed yeast two-hybrid screens that identified Y-box binding protein
1 (YB-1) as a protein that specifically interacts with two SET complex proteins, SET and pp32. YB-1 is a multifunc-
tional nucleic acid-binding protein whose overexpression has been implicated in multiple cancers. We investigated
the possibility that YB-1 might also be a substrate of GzmA. Treatment of lysate from cells over-expressing YB-1 with
GzmA and treatment of whole cells with GzmA and perforin (PFN) demonstrated dose-dependent proteolysis of
YB-1. Purified recombinant YB-1 protein was cleaved by GzmA in the arginine-rich region between R234 and R253.

Introduction
Upon recognition of virally infected or tumor cells, natural
killer (NK) cells and cytotoxic T lymphocytes (CTL) release the
contents of their cytotoxic granules into the immunological syn-
apse formed with the cell singled out for elimination. These cy-
totoxic granules contain a family of serine proteases called gran-
zymes (granule enzyme, Gzm) and a pore-forming protein called
perforin (PFN). PFN facilitates the entry of Gzms into the cytosol
of the target cell, where they initiate a cascade of events ultimately
inducing programmed cell death. The different members of the Gzm
family are remarkable in the diversity of substrates upon which
they act and the distinct pathways of cell death that they initiate.
The five human Gzms are encoded on three gene clusters and
each Gzm has the ability to initiate distinct pathways of pro-
grammed cell death; this redundancy likely evolved to allow the
immune system to combat a wide range of pathogens. While GzmA
and GzmB are the most abundant members of the family, the ma-
jority of research efforts historically have focused on dissecting
the GzmB cell death pathway (Chowdhury and Lieberman, 2008).
Granzymes are homologous to trypsin and other related serine
proteases, named for the conserved serine residue in their active
site (Bell et al., 2003). High resolution crystal structures of some of
the Gzms and the fact that GzmA substrates do not share a com-
mon peptide sequence around their cleavage site suggest that subtle
differences in active site conformation are responsible for the sub-
strate specificity of each protease (Supplemental Figure 1) (Bell et al.,
2003; Chowdhury and Lieberman, 2008; Hink-Schauer et al., 2003).
Expression of Gzm and PFN genes in naïve CD8 T cells is triggered
by T cell receptor (TCR) stimulation by antigen-presenting cells (Bossi
and Griffiths, 2005). A signal sequence directs the nascent Gzms to the
endoplasmic reticulum (ER) and begins the process of Gzm trafficking
into cytotoxic granules, a specialized set of secretory lysosomes. Af-
ter T cell recognition-mediated activation of cytotoxic cells, the lytic
granules of the killer cell migrate to the immunological synapse. The
granule membrane fuses with the immune cell plasma membrane,
releasing PFN and Gzms into the immunological synapse. Entry of
Immunology
Gzms into the target cell cytosol is mediated by PFN by a mecha-
nism that is not fully-understood. Once inside the target cell, Gzms
are capable of initiating at least three distinct cell death pathways
(Chowdhury and Lieberman, 2008). In the GzmA pathway, GzmA
translocates to the interior of the mitochondria and nucleus to in-
duce cell death. It has so far been shown to cleave 8 protein substrates
in the cell with a high degree of specificity (Supplemental Table 1).
Inside the mitochondrial matrix, GzmA-mediated cleavage of
a component of the electron transport chain complex I, NDUFS3,
leads to generation of reactive oxygen species (ROS) (Martinvalet
et al., 2008; Martinvalet et al., 2005), driving the ER-associated SET
complex into the nucleus. This complex is involved in the oxida-
tive stress response and is currently known to be composed of six
proteins: three nucleases (Ape1, NM23-H1, and TREX1), two chro-
matin-modifying proteins (SET and pp32), and the DNA-binding
protein HMGB2 (Beresford et al., 1997; Chowdhury et al., 2006; Fan
et al., 2003a; Fan et al., 2002; Fan et al., 2003b). Inside the nucleus,
GzmA cleaves SET, an inhibitor of the GzmA-Activated DNase
(GAAD) NM23-H1. This activates the NM23-H1 endonuclease to
cause single-stranded nicking of chromosomal DNA. DNA damage
is then extended by the SET complex exonuclease TREX-1 (Chow-
dhury et al., 2006; Fan et al., 2003a). At the same time, GzmA also
cleaves two other SET complex proteins: HMGB2, a DNA bending
protein, and Ape1, a crucial endonuclease in base excision repair
(BER) (Fan et al., 2003a; Fan et al., 2002). GzmA cleavage and in-
activation of Ku70 and PARP-1 proteins in the nucleus also inter-
feres with single- and double-strand DNA break repair pathways
(Zhu et al., 2006). By wreaking havoc on the cell’s DNA repair
machinery, GzmA effectively commits the cell to cell death (Fig-
ure 1). Cells subjected to GzmA and PFN loading die rapidly and
display morphological evidence of apoptosis, including membrane
perturbation, nuclear condensation, and DNA damage. Further-
more, caspase inhibition does not rescue targeted cells. GzmA
therefore induces a novel apoptotic pathway that allows immune
cells to wage war against infected cells that have developed means
to evade caspase pathways of cell death, as is the case for many tu-
mors and some viruses (Beresford et al., 1999; Shresta et al., 1999).

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 RESEARCH
Figure 1. Granzyme A cell death pathway. Upon entry into the tar-
get cell, (1) GzmA targets the mitochondria and induces the produc-
tion of reactive oxygen species (ROS). (2) ROS production leads to
the translocation of the SET complex into the nucleus. (3) GzmA
also enters the nucleus by an unknown mechanism and targets mem-
Immunology
bers of the SET complex and other proteins, resulting in irreversible
DNA damage and cell death. Adapted from (Cullen and Martin, 2008).
In an ongoing effort to identify new substrates of GzmA and un-
derstand the function of the SET complex, previous members of the
Lieberman Laboratory performed yeast two-hybrid screens to iden-
tify proteins that interact with two components of the SET complex:
SET and pp32. In these screens, the transcription factor Y-box binding
protein 1 (YB-1) was shown to interact with both SET and pp32. Sub-
sequent GST pull-down and co-immunoprecipitation assays in 293
cells stably expressing HA-tagged YB-1 confirmed that SET and YB-1
interact both in vitro and in cells, respectively (Zhang, unpublished
data). YB-1 was originally identified as a transcription factor (TF) that
binds to the Y-box of major histocompatibility complex (MHC) class II
promoters (Didier et al., 1988). The 324 amino acid protein belongs to
the cold-shock domain (CSD) family of proteins, which is conserved
in nearly all living organisms and participates in environmental
stress responses in eukaryotes (Supplemental Figure 2) (Evdokimova
et al., 2006a). YB-1 is localized predominantly in the cytoplasm but
rapidly 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). This illustrates a crucial potential link between
YB-1 and the SET complex, which also translocates to the nucleus in
response to oxidative stress. In the absence of stress, the C-terminus
of YB-1 possesses a cytoplasmic retention signal (CRS) that prevails
over a nuclear localization signal (NLS) (Bader and Vogt, 2005; Jur-
chott et al., 2003). Since the CRS was first characterized, nuclear trans-
location has been shown to result from cleavage of the C-terminus of
YB-1 by both the 20S proteasome and thrombin (Sorokin et al., 2005;
Stenina et al., 2001). Thrombin and GzmA are both serine proteases.
YB-1 can regulate transcription in three ways: (1) direct binding
to the Y-box and related sequences, (2) interaction with other TFs
(serving as a co-activator or co-repressor), or (3) binding to transcrip-
tional promoters to either enhance or inhibit binding of other TFs.
The transcription of 21 genes has been shown to be regulated by YB-1.
Interestingly, YB-1 down-regulates and up-regulates approximately
equal numbers of genes (Kohno et al., 2003). One gene which YB-1
was originally shown to upregulate is the multidrug resistance 1 gene
(MDR1), which encodes an ABC transporter commonly associated
2 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
with increased drug resistance in cancer. Multiple studies demon-
strated that translocation of YB-1 into the nucleus results in upregu-
lation of the MDR1 gene (Bargou et al., 1997; Oda et al., 1998; Ohga et
al., 1996; Ohga et al., 1998). However, this theory has since been ques-
tioned after experiments utilizing RNA interference (RNAi) indicat-
ed that YB-1 is not directly involved in MDR1 regulation (Kaszubiak
et al., 2007). Although originally described as a transcription factor,
YB-1 was later shown to also regulate translation in the cytoplasm,
where the majority of YB-1 is found. YB-1 also influences translation
through multiple mechanisms, including (1) enhancing splicing, (2)
facilitating the formation of messenger ribonucleoprotein particles
(mRNPs), and (3) stabilizing nascent mRNAs in a cap-dependent
manner (Evdokimova et al., 2001; Skabkin et al., 2004; Stickeler et
al., 2001). Microarray analysis has revealed that many YB-1-associ-
ated mRNAs encode proteins regulating cell proliferation, oncogenic
transformation, and the stress response (Evdokimova et al., 2006b).
Recent studies concerning YB-1 have further explored the con-
sequences of nuclear localization of YB-1, which is enhanced in
cancer cells. Increased nuclear localization in cancer correlates
with tumor size, degree of invasion, lymph node metastasis, and
poor clinical prognosis. This has been demonstrated in lung, pros-
tate, and breast cancers; osteosarcoma; and multiple myeloma
(Chatterjee et al., 2008; Gimenez-Bonafe et al., 2004; Oda et al.,
1998; Saji et al., 2003; Shibahara et al., 2001). Similarly, high YB-1
expression in cells also correlates with drug resistance and poor
tumor prognosis (Homer et al., 2005). Elucidation of the molecu-
lar mechanisms behind these clinical observations will help de-
termine whether YB-1 might be a potential therapeutic target.
Method
Cell Lines and Reagents
Cells were obtained from the American Type Culture Collec-
tion (Manassas, VA). HeLa cells were maintained in DMEM (Invit-
rogen) supplemented with 10% FBS, 100 units/mL Penicillin G, 100
µg/mL streptomycin sulfate, 6 mM HEPES, 1.6 mM L-glutamine,
and 50 µM β-mercaptoethanol. K562 cells were maintained in RP-
MI-1640 (Mediatech, Inc.) similarly supplemented. The following
primary antibodies were used: HA High Affinity (Roche, 1:2000
working dilution), YB-1 (Epitomics, 1:5000), β-tubulin (Sigma,
1:1000), β-actin (from J. Lin, University of Iowa; 1:1000), caspase 3
(Stressgen Bioreagents, 1:1000), GST (GE Healthcare, 1:2000), and
NM23-H1 (Santa Cruz Biotechnology, 1:1000). SET (1:1000) and
pp32 (1:1000) antibodies were produced as described (Beresford et
al., 2001). The following secondary antibodies were used: α-Goat-
Ig-HRP (Santa Cruz Biotechnology, 1:2000), α-Rabbit-Ig-HRP (GE
Healthcare, 1:2000), and α-Mouse-Ig-HRP (GE Healthcare, 1:5000).
Transfection
Mammalian expression plasmid pCDNA6-YB1-HA was a kind
gift of Hans-Dieter Royer (Germany) (Supplemental Figure 3)
(Chatterjee et al., 2008). 5x105 HeLa cells were treated with 3 µg of
plasmid and Lipofectamine 2000 Reagent (Invitrogen) at 37°C for
4 hours according to the manufacturer’s instructions in the pres-
ence of Opti-MEM (Invitrogen). After washing cells twice with
PBS (Mediatech, Inc.), DMEM was added to wells and cells were
incubated at 37°C. Cells were plated 24 hours prior to transfec-
tion and transfected cells were lysed 48 hours after transfection.
Cell Lysate Preparation
HeLa cells were washed twice with PBS and incubated with Try-
 Volume 2 Issue 2 | Fall 2009 RESEARCH

Lysate + - + + + + + PFN - + - + + +

GzmA
GzmA (µM) - 1 0.1 0.5 1 - - - - 1 0.05 0.2 1
(µM)
S-AGzmA (µM) - - - - - 1 -

GzmB (µM) - - - - - - 1
YB-1

YB1-HA 50 kD HA

SET
Cleaved YB1-HA 20 kD HA

SET 37 kD SET

Cleaved SET 25 kD SET

NM23-H1
50 kD β-Tubulin

Immunology
Figure 3. YB-1 is cleaved in K562 cells treated with PFN and GzmA.
K562 cells were treated with PFN and varying concentrations of active
Figure 2. GzmA cleaves YB1-HA in HeLa cell lysate. Lysate from HeLa cells
GzmA at 37°C for 1 hour. Cells treated with PFN or GzmA alone served as
over-expressing YB1-HA was treated with increasing concentrations of ac-
negative controls. Immunoblots were probed with YB-1, SET, and NM23-
tive GzmA at 37°C for 1 hour. Inactive S-AGzmA- and GzmB-treated cell
H1 antibodies. In the presence of PFN, GzmA cleaved YB-1 in intact K562
lysates served as negative controls. Immunoblots were probed with HA, SET,
cells in a dose-dependent manner that was consistent with cleavage of SET,
and β-Tubulin antibodies. GzmA cleaved YB1-HA in a dose-dependent
a known substrate of GzmA. This experiment was performed by Denis
manner that was consistent with cleavage of SET, a known substrate of GzmA.
Martinvalet. The immunoblot for SET was provided by Danielle Jensen.

pLE Express (Invitrogen) at 37°C for 5 minutes. Following washing
with DMEM, cells were centrifuged at 500g for 5 minutes, the su-
pernatant was removed, and the pellet was resuspended in 500 µL
polysome lysis buffer (PLB) (100 mM KCl, 5 mM MgCl2, 10 mM
HEPES, pH 7.0, 0.5% Nonidet P-40, 1 mM DTT, 100 U ml-1 RNa-
sin RNase inhibitor, 2 mM vanadyl ribonucleoside complexes so-
lution, and 25 µL ml-1 protease inhibitor cocktail for mammalian
tissues) (Peritz et al., 2006). Samples were incubated on ice for 10
minutes and then centrifuged at 14,000 rpm for 15 minutes. Su-
pernatant (cell lysate) was collected and stored at -20°C until use.
Western Blot
Samples were separated by SDS-PAGE and transferred to nitro-
cellulose membrane (Millipore) using a Trans-Blot SD Semi-Dry
Electrophoretic Transfer Cell (Bio-Rad). Membranes were blocked
in 5% non-fat milk (Nestle) in 0.05% Tween 20 with TBS (TBST) for
30 minutes and then incubated in the indicated primary antibody in
5% non-fat milk TBST either at 4°C overnight or at room tempera-
ture for 1 hour. Membranes were washed three times with TBST,
incubated in secondary antibody in 5% non-fat milk TBST for 1 hour
at room temperature, and washed again. Blots were visualized us-
ing homemade ECL solution and Scientific Imaging Film (Kodak).
In vitro Cleavage Assays
Protein samples were suspended in 20-50 µL PBS with indi-
cated concentration of Gzms and incubated at 37°C for indicated
length of time. Cell lysate cleavage assays were performed using
0.2 µg cell lysate protein/µL and GST-YB1 cleavage assays were per-
formed using 0.1 µg purified protein/µL. All samples were equal-
ized to final working volume with PBS. After incubation, 5X re-
ducing SDS sample buffer was added and samples were boiled at
100°C for 5 minutes. Samples were stored at -20°C 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 µL cell loading buffer (HBSS
with 10 mM HEPES [pH7.2], 0.4% BSA, 3 mM CaCl2). A sublytic dose
of PFN [defined as the concentration that lyses 5%-15% of cells (Mar-
tinvalet et al., 2008)] and/or Gzms (concentration as indicated) were
added and cells were incubated at 37°C for 1 hour. In cells pretreated
with a caspase-inhibitor, 50 µM Caspase-3 Inhibitor II, Z-DEVD-FMK
(Calbiochem) was added to the cells in suspension and then incubated
at 37°C for 30 minutes prior to addition of granule enzymes. The re-
action was stopped by adding 15 µL of 5X reducing SDS sample buf-
fer, boiling at 100°C for 5 minutes, and storing at -20°C until analysis.
Protein Expression and Purification
Recombinant GST-YB1 bacterial expression plasmid was a kind
gift of Kimitoshi Kohno (Japan) (Ise et al., 1999; Okamoto et al., 2000).
One µL plasmid was used to transform BL21 DE3-competent E. coli.
Cells were incubated on ice for 30 minutes, heat-shocked at 42°C for
30 seconds, and added to 250 µL Super Optimal broth with Catabolite
repression (SOC) Medium (New England Biolabs). Cells were incubat-
ed in a shaker at 37°C for 1 hour and then 50 µL of culture was spread
on an LB-ampicillin agar plate before incubation overnight at 37°C.
A single colony was selected for inoculation in 500 mL LB Broth, Len-
nox (Fisher Scientific) containing 50 µg/mL ampicillin and incubated
at 37°C overnight. This culture was used to seed 4 L of LB-ampicillin.
After reaching an OD600 of 0.7, the large culture was induced by add-
ing isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich)
at a final concentration of 250 µg/mL. Four hours after induction, the
bacterial pellet was harvested, resuspended in lysis buffer, sonicated,

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 RESEARCH
and centrifuged at 20,000g at for 1 hour. Lysate was passed through
a 0.45 µm filter and then loaded onto a PBS-equilibrated column
containing Gluathione Sepharose 4 Fast Flow (GE Healthcare) using
the BioLogic DuoFlow FPLC (Bio-Rad). Column was washed with
5 column volumes (CV) of PBS and then eluted using 1.5 CV of an
increasing linear gradient of elution buffer (10 mM reduced glutathi-
one) followed by 2 more CV of elution buffer. In the second round of
purification using the Glutathione Sepharose column, pooled sam-
ples were reloaded onto the column, washed with 10 CV of PBS con-
taining 0.5 M NaCl, and eluted as previously described. In the final
round of purification, pooled samples were passed through a Sep-
hadex 75 (S-75) gel filtration column (Amersham) equilibrated with
PBS. Recombinant GzmA and inactive S-AGzmA were expressed
and purified as reported (Beresford et al., 1997). PFN and GzmB were
expressed and purified as reported (Shi et al., 1992; Shi et al., 2000).
Mass Spectrometry
Samples selected for analysis by mass spectrometry were excised
from Coomassie-stained SDS-PAGE gel of samples from the in vitro
GST-YB1 cleavage assay and processed at the Taplin Mass Spectrom-
etry Facility (Harvard Medical School) using tryptic digestion. Pep-
tides were aligned to the amino acid sequence for YB-1 as posted by the
National Center for Biotechnology Information (NCBI). Theoretical
Immunology
molecular weights of terminal peptide fragments were calculated using
the ProtParam Tool from the ExPASy (Expert Protein Analysis Sys-
tem) proteomics server of the Swiss Institute of Bioinformatics (SIB).
RESULTS
Measured by propidium iodine uptake and flow cytometry (data
not shown) (Martinvalet et al., 2008). The level of full-length YB-1
decreased when cells were treated with increasing concentrations
of GzmA in the presence of PFN, but did not change in cells that
were treated with PFN, GzmA, or GzmB alone (Figure 3 and data
not shown). Unlike in HeLa cell lysates, the YB-1 cleavage product
was not detectable by YB-1 antibody, most likely due to the unique
epitope recognized by the antibody or the fact that cleavage frag-
ments may be more labile in intact K562 cells. Cleavage of native
YB-1 was not inhibited by pretreating the target cells with the cas-
pase inhibitor Z-DEVD-FMK, which is consistent with the caspase-
independence of GzmA-mediated cell death (data not shown).
Expression of Recombinant GST-YB1
To begin to characterize the GzmA cleavage site, we purified re-
combinant YB-1 protein using an E. coli expression system. The re-
combinant protein could then be treated with GzmA to isolate cleav-
age fragments for sequencing. A bacterial expression system was
used to express large quantities of YB-1 fused at the N-terminus to
a glutathione S-transferase (GST) tag to allow purification of the re-
combinant YB-1 protein (GST-YB1, Supplemental Figure 3A), as pre-
viously reported (Hayakawa et al., 2002; Ise et al., 1999; Okamoto et
al., 2000). The fusion protein has an expected MW of ~75 kDa, which
is consistent with the fusion of the ~26 kDa GST tag to the ~50 kDa
YB-1 protein. Purification by fast protein liquid chromatography
(FPLC) using glutathione resin yielded large quantities of recombi-
nant protein, as determined by fraction analysis using SDS-PAGE
and Coomassie staining. The indicated fractions of eluted protein
were pooled and subjected to two further rounds of purification in-
volving a high salt wash (to remove non-specifically bound proteins)
and gel filtration (to remove low WV contaminants) (Supplemen-
tal Figure 4). Upon elution, the chromatogram showed a dominant
4 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
GST-YB1 + - - + + + + -
GzmA
- 1 - 0.25 0.5 1 - -
(µM)
GzmB
- - 1 - - - 1 -
(µM)
GST - - - - - - - +
Full-length
75 kD GST-YB1
Full-length YB-1
50 kD
Cleavage
37 kD fragment
GzmB
GST
25 kD GzmA
Figure 4. GzmA cleaves recombinant GST-YB1 in vitro. Purified GST-
YB1 was treated with varying concentrations of active GzmA and
GzmB at 37°C for 2 hours. Samples were separated by SDS-PAGE
and the gel was stained with Coomassie Blue. GzmA cleaved recom-
binant GST-YB1 and cleavage product accumulated at ~40 kDa.
Bands outlined in red were submitted for mass spectrometry analysis.
peak in fractions #5 and 6 with an approximate MW consistent with
the expected MW of GST-YB1 of ~75 kDa (Supplemental Figure 5).
Screening of the peaks from the final purification step demonstrated
that a doublet of GST-YB1 was obtained in early fractions (Supple-
mental Figure 4D). We do not understand why the GST-YB-1 fusion
protein elutes as a doublet, but other investigators have reported sim-
ilar results (Ise et al., 1999). Western Blot confirmed that both of the
dominant bands from Supplemental Figure 4D reacted with GST and
YB-1 antibodies (data not shown). Fraction #5 contained 0.1 µg puri-
fied protein/µL and was selected for use in subsequent experiments.
GzmA Cleaves GST-YB1 in vitro
Purified protein was subjected to in vitro GzmA cleavage assays
similar to those performed using HeLa cell lysates. Samples of purified
recombinant GST-YB1 were treated with increasing concentrations of
GzmA and appropriate controls, separated by SDS-PAGE, and stained
with Coomassie Blue. GzmA, GzmB, and GST alone were incubated
alone to allow differentiation between full-length protein, enzyme,
tag, and potential cleavage fragment bands (Figure 4). When purified
protein was incubated with increasing concentrations of GzmA, full-
length GST-YB1 (~75 kDa) decreased at the same time that two new
bands appeared with apparent molecular weights of ~50 kDa and ~40
kDa. The intensity of the GzmA band at ~25 kDa also increased as the
concentration of GzmA increased. GzmB did not have a significant
effect on the level of full-length GST-YB1. To characterize the new
bands, the GST-YB1 cleavage assay samples were reanalyzed by West-
ern Blot, probing with YB-1 and GST antibodies (Supplemental Fig-
ure 6). In the GST-YB1 cleavage assay, the YB-1 antibody recognized
the new ~50 kDa band in GzmA-treated samples, but not the ~40 kDa
band. Two new bands reacted with GST antibody in the GzmA-treated
samples. The only new band that appeared in GzmA-treated samples
and reacted with GST antibody migrated at ~26 kDa, the MW of GST.
Based on these results, we suspect that in addition to cleaving
YB-1, GzmA also cleaved the GST-YB1 fusion protein between YB-1
and the GST tag. This resulted in the accumulation of WT-length
 Volume 2 Issue 2 | Fall 2009 RESEARCH

Full-length YB-1 Figure 5. GzmA cleaves YB-1 at the C-terminus. Full-length. GST-YB1 and
1 MSSEAETQQP PAAPPAAPAL SAADTKPGTT GSGAGSGGPG YB-1 N-terminal cleavage fragment bands were excised from the gel de-
41 GLTSAAPAGG DKKVIATKVL GTVKWFNVRN GYGFINRNDT picted in Figure 10 and submitted for tryptic digestion and mass spectrom-
81 KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKGA etry analysis. Peptide coverage was aligned according to the sequence for
121 EAANVTGPGG VPVQGSKYAA DRNHYRRYPR RRGPPRNYQQ YB-1 obtained from the National Center for Biotechnology Information
(NCBI). Recovered peptides are indicated in bold. Highlighted in yellow
161 NYQNSESGEK NEGSESAPEG QAQQRRPYRR RRFPPYYMRR
are peptides recovered in the full-length protein samples but missing from
201 PYGRRPQYSN PPVQGEVMEG ADNQGAGEQG RPVRQNMYRG
the cleavage fragment sample. Highlighted in turquoise is the predicted
241 YRPRFRRGPP RQRQPREDGN EEDKENQGDE TQGQQPPQRR
range within which GzmA cleaves YB-1. GzmA appears to cleave YB-1
281 YRRNFNYRRR RPENPKPQDG KETKAADPPA ENSSAPEAEQ
between amino acids R234 and R253.
321 GGAE
We interpret these data to mean that GzmA cleaves YB-1 within
Cleavage Product the arginine-rich region between Q235 and R253. Cleavage in this
1 MSSEAETQQP PAAPPAAPAL SAADTKPGTT GSGAGSGGPG region would produce a C-terminal fragment of between 70 and 89
41 GLTSAAPAGG DKKVIATKVL GTVKWFNVRN GYGFINRNDT amino acids in length. The theoretical molecular weights predicted
81 KEDVFVHQTA IKKNNPRKYL RSVGDGETVE FDVVEGEKGA by the amino acid sequences of these two potential cleavage frag-
121 EAANVTGPGG VPVQGSKYAA DRNHYRRYPR RRGPPRNYQQ ments (measured from Q235 to E324 and from R253 to E324) are
161 NYQNSESGEK NEGSESAPEG QAQQRRPYRR RRFPPYYMRR ~8.1 kDa and ~10.6 kDa, respectively. Hence, cleavage of peptide
201 PYGRRPQYSN PPVQGEVMEG ADNQGAGEQG RPVRQNMYRG fragments of these weights is consistent with the ~10 kDa difference
241 YRPRFRRGPP RQRQPREDGN EEDKENQGDE TQGQQPPQRR in electrophoretic mobility between WT YB-1 (~50 kDa) and the
281 YRRNFNYRRR RPENPKPQDG KETKAADPPA ENSSAPEAEQ cleavage fragment (~40 kDa) (Supplemental Figure 7). However, the
GGAE region between R234 and R253 contains seven arginine residues after
321
which GzmA, a tryptase, could potentially cleave YB-1. Therefore, we
•324 amino acid sequence of YB-1
were unable to identify the precise preferred cleavage site. While our

•Peptide coverage
•Peptide present in full-length sample but missing from
cleavage fragment sample
•Predicted region containing cleavage site
YB-1 at ~50 kDa and an accumulation of GST monomer at ~26 kDa.
Furthermore, based on the fact that the YB-1 antibody recognizes the
C-terminus of the protein (amino acids 280-301 of 324) and did not
recognize the ~40 kDa band in GzmA-treated samples (Supplemental
Figure 6A), we hypothesized that this Coomassie-stained band rep-
resents the N-terminal YB-1 cleavage fragment lacking the GST tag.
Since GzmB did not cleave GST-YB1, this suggests that GzmA cleav-
age of YB-1 is specific. The size of the N-terminal cleavage fragment
appears consistent with our previous findings in HeLa cell lysate. In
Figure 2, GzmA cleavage of YB1-HA produced a ~20 kDa HA-tagged
C-terminal fragment and here we report that the N-terminal cleavage
fragment of GST-YB1 is ~40 kDa. Although we would predict that
the weights of N- and C-terminal cleavage fragments would add up
to ~50 kDa, the accepted MW of YB-1, the slight variation in electro-
phoretic mobility may not be an accurate reflection of actual MW.
GzmA Cleaves YB-1 at the C-Terminus
Cleaved recombinant GST-YB1 bands were subjected to trypsin
digest and mass spectrometry analysis to identify the location of the
GzmA cleavage site. Specifically, the top band of the GST-YB1-alone
control sample observed at ~75 kDa and the cleavage fragments in
the three GzmA-treated samples observed at ~40 kDa (submitted as
a single sample) were excised and submitted for analysis (submitted
samples outlined in red) (Figure 4). Peptide fragments were aligned
according to the amino acid sequence of YB-1 obtained from the Na-
tional Center for Biotechnology Information (NCBI) (Figure 5). Tryp-
tic peptides covering the YB-1 sequence from V54 to the C-terminal
end of the protein (E324) were recovered in the full-length sample, but
only from V54 to R234 in the cleavage fragment sample. The region
between Q235 and R253, in which no peptides were identified in either
sample, is arginine-rich and might have resulted in small tryptic prod-
ucts that were too small to be resolved by mass spectrometry. Surpris-
ingly, no peptides corresponding to the first 53 amino acids of YB-1
were recovered from either sample. Peptides corresponding to GST
were only recovered in the full-length sample (data not shown), con-
sistent with our hypothesis that the ~40 kDa band lacked the GST tag.
Immunology
results effectively narrow the location of the GzmA cleavage site to a
region within the C-terminus of YB-1, further experiments are re-
quired to identify the specific amino acid after which YB-1 is cleaved.
Discussion
Based on preliminary data which suggest that YB-1 interacts with
SET, a known substrate of the protease GzmA, in the present study
we investigated whether the nucleic acid-binding protein YB-1 is also
a GzmA substrate. We found that GzmA cleaves YB-1 in mamma-
lian cell lysate and in whole cells treated with PFN and GzmA. Pu-
rification of recombinant YB-1 using a bacterial expression system
and subsequent treatment with GzmA demonstrated that recombi-
nant YB-1 is specifically cleaved in a dose-dependent manner. Mass
spectrometry analysis of isolated YB-1 cleavage fragments suggested
that GzmA cleaves YB-1 at the C-terminus, most likely between ami-
no acid residues R234 and R253. Taken together, our data strong-
ly suggest that YB-1 is a direct physiological substrate of GzmA.
In the immediate future, efforts should focus on identifying the
precise GzmA cleavage site in YB-1 within the range of possible resi-
dues suggested by our mass spectrometry analysis. This could be ac-
complished by analyzing fragment samples without enzymatic digest
or sequentially mutating each of the arginine residues in the range of
potential cleavage sites and assaying for the mutant recombinant pro-
teins’ ability to withstand GzmA-induced cleavage. Future experi-
ments should next aim to determine whether cleavage of YB-1 is nec-
essary for the induction of apoptosis by GzmA. To test this, researchers
should explore the effect of over-expressing a GzmA-uncleavable mu-
tant form of YB-1 on GzmA’s ability to induce cell death. Cells sta-
bly over-expressing WT and GzmA-uncleavable YB-1 would first be
treated with PFN and GzmA and apoptosis would then be assayed by
flow cytometry following annexin V and propidium iodide (PI) stain-
ing. If cleavage of YB-1 is necessary for the induction of apoptosis, we
would expect that over-expression of the GzmA-uncleavable form of
YB-1 would render cells more resistant to GzmA-induced cell death.
Initial investigations into the hallmarks of apoptosis centered
on the effects of caspases, which were thought to be as the prima-
ry mediators of this innate cellular process; however, it was later
shown that caspase inhibition was not sufficient to block the in-

www.thurj.org 5
 RESEARCH
duction of apoptosis and that caspase-independent cell death was
possible (Tait and Green, 2008). Researchers also discovered that
inhibition of ROS generation afforded cells some protection from
apoptosis, particularly in the context of T cell deletion during de-
velopment of the immune system (Hildeman et al., 1999; Sand-
strom et al., 1994). Treating cells targeted for destruction by CTLs
with superoxide scavengers and antioxidants completely blocked
GzmA-induced cell death. ROS generation is now considered es-
sential to GzmA-mediated cell death (Martinvalet et al., 2005).
ROS are the endogenous byproduct of respiration and oxidative
metabolism and are induced by a variety of environmental agents,
such as ultraviolet (UV) radiation. ROS generation causes DNA
damage by creating abasic sites and plays an important role in the
induction of various diseases, including cancer. YB-1 was recently
shown to stimulate the base excision activity of NEIL2, a recently
characterized oxidative base-specific DNA glycosylase (Das et al.,
2007). YB-1’s ability to inhibit translation of damaged RNA has also
been demonstrated. Researchers found that full-length YB-1 had
enhanced binding affinity for 8-oxoguanine-containing RNA. This
oxidized form of guanine is generated in the nucleotide pool by the
action of oxygen radicals produced in cells and is capable of pair-
ing with both cytosine and adenine. Studies in E. coli demonstrated
that when WT YB-1, capable of binding 8-oxoguanine-containing
Immunology
RNAs, was expressed, cells acquired resistance against paraquat, a
drug that induces oxidative stress in cells. Cells expressing a trun-
cated form of YB-1 that lacked binding affinity remained sensitive
to the drug (Hayakawa et al., 2002). Thus, functional YB-1 may pro-
tect cells against oxidative stress by multiple mechanisms. Cleavage
of YB-1 by GzmA raises the possibility that the human immune
system may have evolved to cleave YB-1 to prevent the targeted
cell from recovering from the damage induced by killer lympho-
cytes and NK cells. This idea is supported by the fact that GzmA
has already been shown to target a number of components of the
SET 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 for providing me with the oppor-
tunity to engage in what has easily proven to be one of my great-
est learning experiences. This work would not have been possible
without your kindness and I would not be as proud of it as I am
without your dedication to a higher standard. Thank you Denis,
Ashish, Danielle, Perry, and all members of the Lieberman Labo-
ratory for your assistance, advice, insight, and friendship. Fi-
nally, I thank my parents, without whom nothing I accomplish
would be possible; thank you for providing me with the means
to explore and the ability to put the journey into perspective.
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3821-3830.

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1 324
Supplemental Figure 2. Y-box binding protein 1. (A) Ribbon structure and
Immunology
(B) domain structure of YB-1. Variable N-terminal domain (V), Cold Shock
Domain (CSD), and alternating basic/acidic residue repeats (B, A). Re-
produced from http://www.pdb.org and (Kloks et al., 2002), respectively.
Supplemental Figure 3. Transient transfection with YB1-HA plasmid
leads to YB-1 expression. (A) Schematic representation of the two plas-
mids used in experiments. (B) Cell lysates from HeLa cells transiently
transfected with either YB1-HA or empty vector plasmid were analyzed
by Western Blot. Immunoblots were probed with HA and β-Tubulin
antibodies. YB1-HA was only expressed in YB1-HA-transfected cells
and migrated at the expected apparent molecular weight of ~52 kDa.
8 The Harvard Undergraduate Research Journal
Volume 2 Issue 2 | Fall 2009
Supplemental Figure 4. Expression of recombinant GST-YB1. (A) Schematic
representation of GST-YB1 purification scheme. Coomassie-stained SDS-
PAGE gels show fractions of GST-YB1 purified by (B) glutathione affinity,
(C) glutathione affinity with 0.5 M NaCl wash, and (D) gel filtration. In (D),
full-length GST-YB1 is indicated by asterisk. Elution fraction #5 from final
purification was used in subsequent experiments, indicated by yellow star.
Absorbance (260 nm)
Time (min)
Supplemental Figure 5. Elution profile of recombinant GST-YB1
from gel filtration column. FPLC chromatogram of gel filtration pu-
rification of GST-YB1 shows elution of GST-YB1 between frac-
tions #3 and 8, which is consistent with fraction screening by
SDS-PAGE and Coomassie staining in Supplemental Figure 4.
Volume 2 Issue 2 | Fall 2009 RESEARCH

GST-YB1 + - - + + + + -

GzmA (µM) - 1 - 0.25 0.5 1 - -

GzmB (µM) - - 1 - - - 1 -

GST - - - - - - - +

A
Full-length
75 kD GST-YB1

YB-1 Full-length
50 kD
YB-1

37 kD

B Full-length
75 kD GST-YB1

50 kD
Supplemental Figure 7. Schematic representation of predicted YB-1
GST cleavage fragments. We interpret the results of the mass spectrometry

37 kD
GST
25 kD
Supplemental Figure 6. N-terminal cleavage fragment of YB-1 protein was
submitted for mass spectrometry analysis. Samples from GST-YB1 cleav-
age assay depicted in Figure 4 were reanalyzed by Western Blot. Immuno-
blots were probed for (A) YB-1 and (B) GST. GzmA cleaved recombinant
GST-YB1 and cleavage product accumulated at ~40 kDa.
Immunology
analysis to mean that full-length GST-YB1 was cleaved by GzmA be-
tween YB-1 and its GST tag and at some point within the arginine-
rich region at the C-terminus of YB-1. This schematic represents
the predicted YB-1 cleavage fragments that would result if GzmA
were to cleave YB-1 from the GST tag or C-terminal to the earli-
est and latest potential cleavage sites within the region between R234
and R253. Cleavage of such fragments from WT YB-1 is consistent
with the observed change in electrophoretic mobility of ~10 kDa.
Current contact information: FDA/CBER/DH/LH,
National Institutes of Health, 8800 Rockville Pike
Bethesda, MD 20892

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