See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/279631549

Thioredoxin-dependent regulation of AIF-

mediated DNA damage

Article in Free Radical Biology and Medicine June 2015

DOI: 10.1016/j.freeradbiomed.2015.06.029 Source: PubMed

CITATIONS READS

6 50

10 authors, including:

Kamila Kaminska Victor C Yu

Lund University National University of Singapore
6 PUBLICATIONS 28 CITATIONS 79 PUBLICATIONS 4,428 CITATIONS

SEE PROFILE SEE PROFILE

Jun Lu Arne Holmgren

Karolinska Institutet Karolinska Institutet
56 PUBLICATIONS 3,726 CITATIONS 485 PUBLICATIONS 37,031 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Redox Control of Skin and Mucosal Inflammatory Diseases including Psoriasis, Atopy and SJS/TEN
View project

cell death mechanism; mitochondria; liver diseases View project

All content following this page was uploaded by Eng-Hui Chew on 07 February 2016.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Free Radical Biology and Medicine 87 (2015) 125136

Contents lists available at ScienceDirect

Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Thioredoxin-dependent regulation of AIF-mediated DNA damage

Sandeep B. Shelar a,1, Kamila K. Kaminska a, Shridhivya A. Reddy a, Dilip Kumar b,
Chong-Teik Tan a, Victor C. Yu a, Jun Lu c, Arne Holmgren c, Thilo Hagen d, Eng-Hui Chew a,n
a
Department of Pharmacy, Faculty of Science, National University of Singapore, S117543, Republic ofSingapore
b
Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (AnSTAR), S138648, Republic of Singapore
c
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden
d
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, S117597, Republic of Singapore

art ic l e i nf o a b s t r a c t

Article history: The thioredoxin (Trx) system is one major redox system in mammalian cells. One of its component, Trx,
Received 25 January 2015 is involved in redox homeostasis and many cellular biological processes through participating in disulde
Received in revised form reduction, S-nitrosylation/S-denitrosylation reactions and protein-protein interactions. In this study, we
21 May 2015
report the identication of a novel interaction between cytosolic/nuclear Trx1 and apoptosis inducing
Accepted 5 June 2015
Available online 25 June 2015
factor (AIF), and the redox sensitivity and biological signicance of the Trx-AIF interaction was char-
acterized. Cytosolic Trx1 but not mitochondrial Trx2 was observed to interact with AIF under physio-
Keywords: logical conditions and Trx1's active site cysteines were crucial for the interaction. Under oxidative stress
Apoptosis inducing factor conditions, Trx-AIF interaction was disrupted. When the treated cells were allowed to recover from
Thioredoxin oxidative stress by means of removal of the oxidants, interaction between Trx1 and AIF was re-estab-
Oxidative stress
lished time-dependently, which underpins the biological relevance of a Trx-dependent redox regulation
Protein-protein interaction
of AIF-mediated cell death. Indeed, in times of oxidative stress, nuclear translocation of AIF was found to
occur concurrently with perturbations to the Trx-AIF interaction. Once localized in the nucleus, reduced
Trx1 hindered the interaction between AIF and DNA, thereby bringing about an attenuation of AIF-
mediated DNA damage. In conclusion, characterization of the Trx-AIF interaction has led to an under-
standing of the effect of reduced Trx1 on possibly regulating AIF-dependent cell death through impeding
AIF-mediated DNA damage. Importantly, identication of the novel interaction between Trx1 and AIF has
provided opportunities to design and develop therapeutically relevant strategies that either promote or
prevent this protein-protein interaction for the treatment of different disease states.
& 2015 Published by Elsevier Inc.

1. Introduction normal functioning of cells, their aberrant production during ex-

Redox signaling is known to play fundamental roles in phy-
siological processes such as embryogenesis, cellular differentiation
and tissue regeneration [13]. The messengers orchestrating re-
dox-dependent processes include reactive oxygen species (ROS),
reactive nitrogen species (RNS) and other reactive electrophilic
species. Although these redox active species are important for the
Abbreviations: AIF, apoptosis inducing factor; ASK1, apoptosis signal-regulating
kinase 1; CypA, cyclophilin A; GR, glutathione reductase; Trx, thioredoxin; TrxR,
thioredoxin reductase
n
Correspondence to: Department of Pharmacy, Faculty of Science, National
University of Singapore, 18 Science Drive 4, Singapore 117543, Republic of Singa-
pore. fax: 6567791554.
E-mail address: phaceh@nus.edu.sg (E.-H. Chew).
1 to cope with the daily load of ROS and RNS produced from es-
S.B. Shelar's present address: Cardiac Aging & Redox Signaling Laboratory,
Division of Molecular & Cellular Pathology, University of Alabama at Birmingham,
Birmingham, Alabama 35294, United States
aggerated or defective metabolic activities can cause oxidative
stress to the cell, which leads to development of pathophysiolo-
gical conditions. Indeed, increasing evidence has suggested that
apoptosis caused by oxidative stress is involved in the pathogen-
esis of several diseases such as Alzheimer's disease [4], Parkinson's
disease [5], and diabetes mellitus [6,7]. In the context of cancer,
the effect of redox active species, particularly ROS, has been
known to contribute towards carcinogenesis, tumor progression
and cancer cell survival through redox control of signaling path-
ways dictating proliferation and apoptosis [8,9]. It is therefore
important to achieve a better understanding of the possible roles
of endogenous redox molecules in regulating oxidative stress-
triggered apoptosis induction.
Various redox defense systems are in place in mammalian cells
sential cellular activities. Among them is the thioredoxin (Trx)
system comprising of thioredoxin reductase (TrxR), Trx and

http://dx.doi.org/10.1016/j.freeradbiomed.2015.06.029
0891-5849/& 2015 Published by Elsevier Inc.
126 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136

NADPH. Oxidative stress causes oxidation of protein thiols to form
disuldes. Such thiol-to-disulde changes in cellular proteins re-
sult in alterations in their biological activities. Trx, a ubiquitous
12 kDa dithiol protein, maintains dithiol/disulde homeostasis
through reducing the disuldes based on a dithiol/disulde ex-
change reaction between the dithiol at the active site of Trx (Cys32
and Cys35 in hTrx1) and the disulde in the protein substrate. The
two isoforms of mammalian Trx, namely cytosolic Trx1 and mi-
tochondrial Trx2, contain a common active site motif Trp-Cys-Gly-
Pro-Cys- in a Trx-fold structure. Upon reducing the disulde in a
protein substrate, the active site disulde in Trx1 and Trx2 is re-
cycled back to the reduced form by cytosolic TrxR1 and mi-
tochondrial TrxR2 respectively [10]. Besides being a disulde re-
ductase, Trx is also reported to mediate S-denitrosylation and
trans-S-nitrosylation reactions [11], as well as participate in pro-
tein-protein interactions [reviewed in 10,12]. Through these reac-
tions, Trx participates in the regulation of many cellular processes.
As part of a preliminary effort in our laboratory to identify
novel cellular proteins that could be associated with and under
redox regulation by the Trx system, a breast carcinoma MCF-7 cell
line stably expressing FLAG-Trx1 was generated and a FLAG im-
munoprecipitation reaction performed had led to the identica-
tion of a novel interaction between Trx1 and apoptosis inducing
factor (AIF) (presented in Fig. 1A). AIF is a phylogenetically con-
served mitochondrial avoprotein that mediates cell death in a
caspase-independent process. Following the application of an
apoptotic stimulus, AIF translocates from the mitochondria,
through the cytosol and into the nucleus to trigger chromatin
condensation and DNA degradation [13,14]. Although several
studies have reported the occurrence of AIF-mediated cell death in
cells exposed to oxidative stress [1518], a potential redox reg-
ulation of this apoptotic process remains largely elusive. In this
study, the redox sensitivity of the Trx-AIF interaction was
characterized, and the possible role of Trx1 in the regulation of
AIF-mediated apoptosis in response to oxidative stress was in-
vestigated. It was found that cytosolic Trx1, but not mitochondrial
Trx2 interacted with AIF under physiological conditions. Disrup-
tion of the Trx-AIF interaction under oxidative stress conditions
had demonstrated that the redox state of Trx1 was crucial for the
protein-protein interaction. In isolated nuclei, AIF-mediated DNA
damaging effects was attenuated by reduced Trx1 likely through
impeding the interaction between AIF and DNA. In conclusion,
characterization of the Trx-AIF interaction has led to an under-
standing of the effect of reduced Trx1 on impeding AIF-mediated
DNA damage, which underpins the biological relevance of a Trx-
dependent redox regulation of AIF-mediated cell death.
2. Materials and methods
2.1. Chemicals, antibodies and cell culture
FLAG M2 agarose beads, as well as anti-FLAG M2 and -tubulin
antibodies were purchased from Sigma-Aldrich (St Louis, MO).
Recombinant human Trx1 (rhTrx1) and anti-human Trx1 anti-
bodies (polyclonal ATRX-03; monoclonal ATRX-04) were obtained
from IMCO Corporation (Stockholm, Sweden), while anti-V5 anti-
body was from Serotec Inc. (Kidlington, Oxford, UK). Antibodies
against AIF (monoclonal sc-13116), HSP60 (sc-13115), Tom20 (sc-
11415) and -actin (monoclonal sc-47778) were obtained from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-CypA anti-
body (AB58144) was from Abcam (Cambridge, UK). Lamin A/C
antibody (2032S) was from Cell Signaling Technology (Beverly,
MA). Horseradish peroxidase (HRP)-conjugated anti-mouse and
anti-rabbit secondary antibodies were acquired from Pierce
s
(Rockford, IL), while Alexa Fluor 488-conjugated anti-mouse IgG

Fig. 1. Interaction between Trx1 and AIF in cells. (A) FLAG immunoprecipitation using lysates of untransfected and FLAG-Trx1 expressing MCF-7 cells, followed by coomassie
blue staining of the resolved immunoprecipitates on a 15% SDS-polyacrylamide gel revealed a protein band identied as programmed cell death 8 isoform 2 (accession
number NP_665811), which corresponded to the amino acid sequence of AIF. (B) HEK293, HCT116 and Hela cell lysates were subjected to Trx immunoprecipitation using
Trx1 antibody, and the immunoprecipitates were resolved by SDS-PAGE followed by western blot analysis using the indicated antibodies. Lysates of HEK293 cells transiently
transfected with (C) the indicated FLAG-tagged proteins, or (D) V5-tagged Trx1 or Trx2 were subjected to Trx and V5 immunoprecipitation using Trx1 and V5 antibody
respectively, and the immunoprecipitates were resolved by SDS-PAGE followed by western blot analysis using the indicated antibodies. All western blots images shown are
representative of 3 independent experiments.
 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136
s
and Alexa Fluor 568-conjugated anti-rabbit IgG were obtained
from Molecular Probes.
Human embryonic kidney HEK293, human-derived cervical
carcinoma Hela cells (ATCC, Rockville, MD) and stably transfected
T-Rex293 cells expressing mAIF101-612-FLAG upon tet-induction
were cultured in high glucose DMEM supplemented with 10% fetal
bovine serum (FBS), 100 units/ml penicillin, 100 mg/ml strepto-
mycin and 1 mM of sodium pyruvate (pyruvate added only to
HEK293 and T-Rex293 cells), while human-derived breast carci-
noma MCF-7 and colon carcinoma HCT116 cells (ATCC, Rockville,
MD) were maintained in RPMI 1640 medium supplemented with
10% FBS, 100 units/ml penicillin and 100 mg/ml streptomycin. The
cells were incubated at 37 C in a humidied atmosphere of 95%
air and 5% CO2. The respective cell lines were transfected or co-
transfected with one or more of the mammalian expression plas-
mids described below for 2448 h.
2.2. Plasmids constructs and mutagenesis
Trx1 (NM_003329) was cloned from HCT116 cDNA and Trx2
(NM_012473) and HSP60 (NM_199440) were cloned from HEK293
cDNA. Murine AIF (BC003292) and human AIF (BC111065) were
amplied from cDNA I.M.A.G.E. clones purchased from Gene-
service Ltd (I.M.A.G.E. ID 3500037, 5767978, respectively) and in-
serted into modied pcDNA3.1/Zeo including N-terminal V5- or
2xFLAG epitope tags (Trx1 constructs), pcDNA3 including
C-terminal V5- or 2xFLAG tags (HSP60, Trx1, Trx2 and AIF con-
structs), or pcDNA4/TO including a C-terminal 2xFLAG tags (AIF
construct). DNA inserts encoding cytosolic forms of murine AIF
(mAIF101-612; amino acids 1-100) and human AIF (hAIF121-
613; amino acids 1-120) were cloned into the pcDNA3FLAG or
pcDNA4TOFLAG vector. The T-Rex system (Invitrogen, Carlsbad,
CA) was used to generate cell lines with tetracycline-inducible
expression of mAIF101-612-FLAG according to the manufacturer's
instructions. The cysteine mutant constructs of Trx1 and mAIF101-
612 were obtained by performing mutagenesis using the site-di-
rected mutagenesis kit following manufacturer's instructions
(Stratagene, La Jolla, CA). For recombinant protein expression, an
N-terminally hexahistidine-tagged expression clone of hAIF1-
120 (His-hAIF121-613) was generated by PCR-amplication of
hAIF1-120 using forward primer 5'- ag cat atg cat cac cat cac cat
cac gag gaa gtt cct c (incorporates NdeI site as underlined (includes
start codon) and 6xHis tag sequence) and reverse primer 5'- ag act
agt tca gtc ttc atg aat g (incorporates SpeI site as underlined). The
resultant fragment was inserted into NdeI- and SpeI-digested
pET41a (Novagen, Nottingham, UK).
2.3. Purication of His-tagged hAIF1-120
His-tagged hAIF1-120 was expressed in BL21(DE3) cells in
2-liter LB medium containing 50 mg/ml kanamycin. Briey, cul-
tures were grown at 37 C and induced by 0.5 mM isopropyl -D-
thiogalactopyranoside (IPTG) for 4 h when A600 reached 0.6. Cell
pellets, resuspended in Buffer A (50 mM sodium phosphate,
500 mM NaCl, pH 8) containing 10 mM imidazole, 1 mM phe-
nylmethylsulfonyl uoride (PMSF), 0.1 mg/ml lysozyme and
0.1 mM FAD were lysed by sonication. Claried lysates were in-
cubated with Ni-nitrilotriacetic acid (NTA) resin at 4 C for 1 h,
followed by resin washing (2 volumes of Buffer A containing
20 mM imidazole, and nally 1 volume of Buffer A containing
50 mM imidazole). The protein was eluted in Buffer A containing
250 mM imidazole, dialyzed overnight against 50 mM sodium
phosphate, 150 mM NaCl, pH 8 (Buffer B), concentrated by ultra-
ltration using a 10 kDa molecular mass cutoff lter (Microsep,
Pall Corp.) and nally loaded on a Sephadex G75 gel ltration
column (60  1.5 cm) preequilibrated with Buffer B containing
127
5 mM DTT. Protein purity was determined by SDS-PAGE and His-
hAIF1-120 containing fractions were pooled and concentrated
by ultraltration.
2.4. Lysate preparation and Immunoblotting
For collection of cell lysates for immunoprecipitation reactions
or immunoblotting, cells were lysed in triton-X lysis buffer
[25 mM TrisHCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM
EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM sodium -glyceropho-
sphate, 10 mM sodium pyrophosphate, 0.5% triton X-100 con-
taining freshly added protease inhibitor cocktail (Roche Diag-
nostics)]. Lysates were precleared by centrifugation and protein
concentrations were determined using a modied Bradford assay
(Bio-Rad Laboratories) as described in the manufacturer's manual.
Equal amounts of protein in lysate samples were separated by
SDS-PAGE and resolved proteins were electroblotted onto ni-
trocellulose membranes. Nuclear extracts obtained from lysis of
isolated nuclei were also subjected to Bradford assay prior to re-
solution of nuclear proteins by SDS-PAGE and western blot ana-
lysis. The blots were probed with a primary antibody followed by a
secondary antibody conjugated to horseradish peroxidase, and
then developed using enhanced chemiluminescence system
[Western Lighning, Perkin-Elmer (Boston, MA) or SuperSignal
West Femto, Pierce]. All Western blots shown are representative of
three independent experiments.
2.5. Determination of redox states of Trx
The freshly collected whole cell lysates or nuclear extracts in ice
cold triton-X lysis buffer were immediately subjected to non-re-
ducing native PAGE. Briey, equal amounts of proteins in lysate
samples were mixed with native Laemmli's sample buffer and
resolved on an 8% native polyacrylamide gel. The resolved proteins
were transferred from the gel onto a nitrocellulose membrane and
the reduced and oxidized forms of Trx were detected by western
blot analysis using Trx1 antibody.
2.6. Immunoprecipitation and pull-down assay
For immunoprecipitation reactions, mouse monoclonal or goat
polyclonal anti-Trx1 antibody (1 mg), or V5 antibody (1 mg), cou-
pled to 20 ml of protein G sepharose slurry (Amersham Bios-
ciences), or 20 ml of anti-FLAG M2 agarose (Sigma) was used.
Briey, volumes of pre-cleared cell lysates containing equal
amounts of proteins were tumbled with the agarose or sepharose
beads at 4 C for 2 h. The beads were washed three times in 1 ml of
cold washing buffer containing 20 mM Tris (pH 7.5), 0.5 M NaCl
and 1 mM EGTA and once in buffer containing 50 mM Tris (pH 7.5).
The immunoprecipitated proteins were then eluted in Laemmli's
sample buffer containing 5% -mercaptoethanol and subjected to
SDS-PAGE and Western blotting. All immunoprecipitation experi-
ments shown are representative of three independent
experiments.
For pull-down assays, recombinant His-hAIF1-120 protein
(5 mM) was mixed with 5 mM oxidized or reduced rhTrx1 in PBS
buffer at 37 C for 1 h. To obtain reduced rhTrx1, oxidized rhTrx1
was reduced by treatment with 10 mM DTT for 15 min and the
reduced rhTrx1 was desalted by passing through NAP-5 column.
The protein mixture was next tumbled with 1 mg of anti-Trx1 an-
tibody and protein G sepharose beads for 1-2 h at 4 C. The beads
were washed with PBS and pull-down proteins were eluted in
Laemmli's sample buffer containing 5% of -mercaptoethanol.
128 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136
2.7. Nuclei isolation and preparation of nuclear extracts
HEK293 or Hela cells were harvested in ice cold PBS and pel-
leted by centrifugation at 1000 rpm, for 5 min a 4 C. The cell
pellets were suspended and incubated in nuclei isolation buffer A
(10 mM HEPES pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM EGTA) for
20-30 min on the ice bath. The suspended cells were treated with
the NP-40 (nal concentration 0.5% v/v) and further incubated
on ice for 5-10 min. The mixtures were centrifuged at 1000 rpm
for 1 min at 4 C to sediment unlysed cells and the supernatant
was centrifuged at 0.7 g for 10 min at 4 C to obtain the nuclear
pellet. The supernatant was collected as cytosolic extract, while
the nuclear pellet was washed with nuclei isolation buffer A and
nally suspended in CSF buffer (220 mM mannitol, 68 mM su-
crose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2,
5 mM pyruvate and 10 mM HEPES pH 7.2) containing freshly ad-
ded protease inhibitor cocktail. To obtain nuclear extracts, the
collected nuclear pellets were lysed in triton-X lysis buffer.
2.8. Extraction of genomic DNA and agarose gel electrophoresis
HEK293 cells cultured in 6-well plates to 80-90% conuence
were treated with diamide at specied concentrations for 3 h. The
collected cell pellets were incubated overnight at 55 C with
constant shaking in 200 ml of SDS lysis buffer (50 mM Tris pH 8,
100 mM NaCl, 100 mM EDTA, 1% w/v SDS) and 6 ml of proteinase K
(20 mg/ml). An aliquot of 70 ml of saturated NaCl solution was
added, followed by removal of precipitated proteins by cen-
trifugation at 13,000 rpm at room temperature for 20 min. The
supernatant was mixed with 500 ml of absolute ethanol. The pre-
cipitated DNA was pelleted by centrifugation at 13,000 rpm at
room temperature for 12 min, washed once with 70% ethanol and
re-dissolved in an appropriate volume of sterile dH2O. A volume of
each sample equivalent to 10 mg of DNA was mixed with 6x DNA
loading dye and subjected to gel electrophoresis on a 1% agarose
gel. DNA bands on the gel were visualized by UV trans-illumina-
tion and gel images were captured on the Gel Doc instrument
(Bio-Rad Laboratories, Hercules, CA).
2.9. Immunocytochemistry
Hela and HEK293 cells were cultured on coverslips (pre-coated
with 0.01% w/v poly-L-lysine). Upon reaching 80-90% conuence,
the cells were xed with 4% para-formaldehyde. Fixed cells were
permeabilized using 0.2% triton-X in PBS, followed by blocking
with 5% horse serum, then probed with primary antibodies against
AIF and Tom20 (mitochondrial marker) followed by Alexa
s s
Fluor 488- and Alexa Fluor 568-conjugated IgGs. The coverslips
were mounted on glass slides using Vectashield mounting med-
ium containing 4',6-diamidino-2-phenylindole (DAPI; Vectorlabs,
Burlingame, CA). Microscopic images were obtained using Olym-
pus FluoView FV10i-DOC confocal laser scanning microscope, and
co-localization analysis was performed using the Olympus Fluo-
View software. To the experiment set up to quantify nuclei with
damaged nuclear morphology, an aliquot of a mixture of nuclei
isolated from the cells was mounted onto a glass slide using DAPI-
containing Vectashield. Microscopic images were obtained at 100X
magnication using a Nikon Eclipse Ti-S epiuorescence micro-
scope connected to a Nikon DS-Ri1 camera. Results were ex-
pressed as a percentage of nuclei with damaged nuclear mor-
phology over a total of 50 nuclei stained with DAPI in a number of
spots taken randomly from the slide. Results were representative
of three independent experiments.
2.10. Flow cytometric analysis of DNA content
Nuclei from Hela cells were isolated and suspended in CFS
buffer [19], equal aliquots of isolated nuclei were incubated in the
presence or absence of reduced or oxidized rhTrx1 (5 mM) for
30 min at 37 C, followed by the addition of rHis-hAIF121 (nal
concentration 1 mM) for further incubation of 2 h at 37 C. Quan-
tication of nuclear apoptosis was carried out by cytouorometric
determination of propidium iodide-stained DNA content [19,20].
Briey, the nuclei were incubated with propidium iodide (50 mg/
ml) for 30 min in the dark at 4 C and analyzed for DNA content
using Beckman coulter ow cytometer.
3. Results
3.1. Trx physically interacts with AIF under physiological conditions
We initially generated a MCF-7 cell line stably expressing FLAG-
Trx1. Whole cell lysates of untransfected and FLAG-Trx1 expres-
sing MCF-7 cells were subjected to FLAG immunoprecipitation
reaction. On a coomassie stained SDS-PAGE gel containing sepa-
rated immunoprecipitates, a protein band in the region of the
65 kDa marker was observed exclusively in the FLAG-Trx1 lane
(Fig.1A). MALDI-TOF analysis identied the protein as programmed
cell death 8 isoform 2 (accession number NP_665811) (data not
shown), which corresponded to the amino acid sequence of AIF.
The interaction of endogenous Trx1 and AIF was further conrmed
by immunoprecipitation with Trx1 antibody in human-derived
HEK293, HCT116 and Hela cells (Fig. 1B). The full length of AIF
resides in the mitochondria and it is the truncated form that is
released into the cytosol [13]. As shown in Fig. 1C, both transfected
full length [mAIF(FL)-FLAG] and mature [mAIF101-612-FLAG]
forms of mAIF interacted with endogenous Trx1 while HSP60, a
protein reported to have mitochondrial and extramitochondrial
localization [21] displayed no existence of such interaction. These
results further afrmed the specicity of the Trx-AIF interaction.
Furthermore, V5 immunoprecipitation performed using lysates of
HEK293 cells transfected with C-terminally V5-tagged Trx1 or
Trx2 showed that endogenous AIF interacted only with cytosolic
Trx1, but not with mitochondrial Trx2 (Fig. 1D).
3.2. Active site cysteine residues in Trx1 are required for interaction
with AIF
To characterize the interaction between Trx1 and AIF, we gen-
erated a HEK293 cell line with tetracycline-inducible expression of
C-terminally FLAG-tagged murine truncated AIF1-100 (mAIF101-
612-FLAG), reported to exhibit cytosolic distribution [22]. Con-
sistent with as previously reported [23], we observed no induction
of cell death in these tet-on AIF-overexpressing cells. To examine
whether Trx1 binding to AIF was mediated via active site cysteine
residues, lysates of HEK293 cells transiently transfected with
hAIF121-613-FLAG and V5-tagged Trx1 single (C32S or C35S) or
double (C32SC35S) cysteine mutants were immunoprecipitated
and immunoprecipitates were analyzed for co-im-
munoprecipitated FLAG-tagged truncated AIF. As shown in Fig. 2,
interaction was abolished by single mutation of Cys32 in Trx1, as
well as in redox-inactive Trx1C32SC35S. These results indicated
that the catalytic Cys32 in Trx1 was involved in AIF binding, where
its loss had prevented binding.
To identify the cysteine residue(s) engaged in Trx binding, a
panel of single, double and triple mouse AIF cysteine mutants was
constructed. AIF has 3 cysteines in its sequence, namely Cys255
and Cys440 in the NAD(H) binding domain and Cys316 in the FAD
binding domain (numbering positions with reference to murine
 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136
Fig. 2. Involvement of cysteine residues at the active site of Trx1 in the Trx-AIF
interaction. Lysates of HEK293 cells transiently co-transfected with hAIF121-613-
FLAG and V5-tagged wild-type Trx1 or Trx1 carrying one or both point-mutation
(s) at the active site cysteines (Cys32, Cys35) were subjected to V5 im-
munoprecipitation, and the immunoprecipitates were resolved by SDS-PAGE fol-
lowed by western blot analysis using the indicated antibodies. All western blots
images shown are representative of 3 independent experiments.
AIF; the respective cysteine residues in human AIF are numbered
Cys256, Cys441 and Cys317). Immunoprecipitation assays of en-
dogenous Trx showed that as compared to wild-type mAIF101-
612, Trx binding to all single AIF cysteine mutants [mAIF(101-612)
C255S, C316S and C440S] was negligibly affected, while all double
AIF cysteine mutants (C255SC316S, C255SC440S and C316SC440S)
displayed a drastic reduction in Trx binding (Fig. 3). Interestingly,
triple cysteine mutants bound to the same extent as double cy-
steine mutants and complete abolishment of binding could not be
achieved. Collectively, these ndings suggested that two cysteine
residues were involved in the interaction, which explained that
mutation of the third cysteine to serine could not further reduce
binding. These results were consistent with the earlier observation
that active site cysteines in Trx1 were critical for binding (Fig. 2). It
could be argued that as was observed for single Trx Cys32 mutant
and double cysteine mutant C32SC35S (Fig. 2), double AIF cysteine
mutants would likely fail to bind Trx1 completely. We reasoned
that besides interaction between the two protein molecules at the
cysteine residues, other non-covalent interactions might exist at
the binding interface that contributed to the residual binding as
observed between Trx1 and double/ triple AIF cysteine mutants.
Fig. 3. Cysteine residues in AIF are not crucial for the Trx-AIF interaction. Lysates of HEK293 cells transiently transfected with FLAG-tagged wild-type truncated mAIF
(mAIF101-612) or truncated AIF carrying single, double or triple cysteine-to-serine mutations at Cys255, Cys316 and Cys440 were subjected to Trx immunoprecipitation
using Trx1 antibody, and the immunoprecipitates were resolved by SDS-PAGE followed by western blot analysis using the indicated antibodies. All western blots images
shown are representative of 3 independent experiments.
129
3.3. Oxidative stress reversibly abrogates Trx-AIF interaction and the
redox state of Trx1 is crucial for the interaction
Having to demonstrate that the cysteines in the active site of
Trx1 were involved in interacting with AIF, it was postulated that
the interaction was redox-sensitive. The effect of oxidative stress
caused by H2O2 and HOCl on the Trx-AIF interaction in tet-on
HEK293 cells overexpressing mAIF101-612-FLAG was therefore
examined. As shown in Fig. 4A, treatment with H2O2 and HOCl at a
lethal dose that could cause cell death had caused apparent dis-
turbances in the AIF-Trx interaction. Interestingly, in a time-chase
study that involved treatment of transfected HEK293 cells ex-
pressing hAIF121-613-FLAG with 1 mM H2O2 for 2 h followed by
replacement of H2O2-containing medium with oxidant-free cul-
ture medium, the disrupted Trx-AIF interaction was observed to
recover in a time-dependent manner (Fig. 4B). The recovery of
disturbed Trx-AIF interaction suggested that redox defense sys-
tems might be in place to facilitate the reversal of oxidative stress-
mediated modications in AIF and Trx1 so as to favor their re-
interaction.
Trx1 is known to be susceptible to oxidative stress where Cys32
and Cys35 at the active site undergo oxidation to form an in-
tramolecular disulde bond. We further examined the association
between thiol-dependent oxidation of Trx1 and concurrent dis-
turbances in the Trx-AIF interaction. Immunoprecipitation reac-
tions revealed that T-Rex293 cells expressing tetracycline-in-
ducible mAIF101-612-FLAG and transiently transfected with Trx1-
V5 produced a disrupted interaction between mAIF101-612-FLAG
and Trx1-V5 when treated with a lethal dose of 1 mM diamide for
5 min, whereas a non-toxic dose of 0.5 mM diamide was not
strong enough to disrupt interaction between the two proteins
(Fig. 4C). The reversibility of the AIF-Trx interaction upon removal
of 1 mM diamide treatment for 5 min was further investigated. To
this end, medium containing diamide was removed and replaced
by fresh culture medium. At time points of 0, 5, 10 and 15 min
following replacement of fresh medium, which would be in-
dicative of the removal of an oxidant to allow cells to recover from
an oxidative stress insult, immunoprecipitation reactions had
found that the disrupted AIF-Trx interaction was regained as soon
as within 5 min of oxidative stress recovery (Fig. 4D). In agree-
ment, lysates run alongside in a native PAGE gel indeed showed a
time-dependent reversal of oxidized Trx1 to the reduced state
(lower panel in Fig. 4D). Taken together, the redox state of Trx1
was found to be critical in the Trx-AIF interaction, where reduced
Trx1 interacts with AIF and oxidized Trx1 dissociates from AIF.
130 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136

Fig. 4. Redox sensitivity of the Trx-AIF interaction. T-Rex293 cells (HEK293 cells stably expressing tetracycline (tet) repressor) stably transfected with pcDNA4/TO-mAIF101-
612-FLAG were used in (A) and (C). These cells were transiently transfected with pcDNA3-Trx1-V5 and were incubated for 24-48 h, after which mAIF121-612-FLAG ex-
pression was induced by addition of tetracycline 2 mg/ml for 24 h. The cells were then treated with (A) 2 mM of H2O2 or HOCl for 2 h, or (C) 0.5 or 1 mM diamide for 5 min.
Freshly collected lysates were subjected to V5 immunoprecipitation. Immunoprecipitates resolved by SDS-PAGE were analyzed by western blotting using the indicated
antibodies. (B) HEK293 cells co-transfected with pcDNA3-hAIF121-613-FLAG and pcDNA3-Trx1-V5 plasmids were treated with 1 mM H2O2 for 2 h, after which they were
subjected to oxidative stress recovery by changing the medium with fresh medium and cultured further for indicated timepoints. Freshly collected lysates were subjected to
V5 immunoprecipitation. Immunoprecipitates resolved by SDS-PAGE were analyzed by western blotting using the indicated antibodies. (D) HEK293 cells were treated with
1 mM diamide for 5 min, after which they were subjected to oxidative stress recovery by changing the medium with fresh medium and cultured further for indicated
timepoints. Upper panel: Freshly collected lysates were subjected to Trx immunoprecipitation using Trx1 antibody, and the immunoprecipitates were resolved by SDS-PAGE
followed by western blot analysis using the indicated antibodies. Lower panel: Freshly collected lysates were resolved on a native PAGE gel followed by western blot analysis
using Trx1 antibody. All western blots images shown are representative of 3 independent experiments.

3.4. Trx1 protects AIF-mediated DNA damage through modulating
interaction between AIF and DNA
It was observed that endogenous AIF and Trx1 translocated into
the nuclei of cells exposed to varying oxidative stress upon
treatment with 0.5 and 1 mM diamide for 1 h (Fig. 5A). As shown
in Fig. 5B, on a native PAGE gel, while nuclear Trx1 remained re-
latively reduced when the cells were challenged with mild to
moderate level of oxidative stress inicted by a sublethal dose of
0.5 mM diamide, it was fully oxidized when cells were exposed to
highly oxidizing conditions caused by treatment with a cytotoxic
dose of 1 mM diamide. When genomic DNA collected from these
cells was subjected to electrophoresis on an agarose gel, a nucleic
acid smear was observed in the lane containing genomic DNA of
cells in the 1 mM diamide treatment group (Fig. 5C). Taken to-
gether, the results presented in Figs. 4C and 5A-C suggested the
following cellular events: mild to moderate level of oxidative
stress neither caused signicant Trx oxidation nor sufcient dis-
ruption of the Trx-AIF interaction, whereas severe oxidative stress
resulted in oxidation of cytosolic and nuclear Trx1 and disruption
of the Trx-AIF interaction to bring about DNA damage in the cells.
We therefore deduced that nuclear Trx1 might play a role in
modulating AIF-mediated DNA damaging effects, and led us to
investigate the possible effect Trx1 would have on the interaction
between AIF and DNA. Initially, the interaction between re-
combinant His-hAIF1-120 and reduced or oxidized hTrx1 re-
combinant proteins was investigated in a pull-down assay. As
shown in Fig. 6A, rHis-hAIF1-120 was found to interact with only
the reduced form of rhTrx1. Furthermore, in an in vitro experi-
ment, His-hAIF1-120 recombinant protein and plasmid DNA
were allowed to interact in the presence or absence of reduced or
oxidized rhTrx1 and the mobility of the DNA was examined using
agarose gel electrophoresis. As depicted in Fig. 6B, while incuba-
tion of plasmid DNA with rHis-hAIF1-120, recombinant rat TrxR
and NADPH brought about retarded DNA mobility, pre-incubation
of oxidized rhTrx1 with rHis-hAIF1-120 in the presence of re-
combinant rat TrxR and NADPH and subsequent treatment with
plasmid DNA brought about a partial alleviation of this retarding
effect on DNA mobility. However, oxidized rhTrx1 alone with
NADPH was found not to have any reversal effect on retardation of
DNA mobility caused by AIF (Fig.6B). As reported previously that
AIF would execute DNA fragmentation when in association with
Cyclophilin A (CypA) [24] and H2AX [25], the experiment was
repeated with the addition of CypA co-immunoprecipitated from
freshly collected Hela cell lysates. As shown in Fig. 6C, fragmen-
tation of plasmid DNA treated with rHis-hAIF1-120 in the pre-
sence of reduced rhTrx1 and CypA was blocked. On the other hand,
treatment of DNA with rHis-hAIF1-120 alone with CypA or rHis-
hAIF1-120 along with oxidized rhTrx1 and CypA produced a
nucleic acid smear on the lanes of the agarose gel when sub-
jected to electrophoresis, indicating that DNA fragmentation had
set in.
To further investigate the postulation that nuclear Trx1 could
modulate AIF-mediated DNA damaging effects, we examined
whether Trx1 could attenuate nuclear DNA damage induced by
translocated AIF. In the experiment, isolated nuclei from Hela cells
were preincubated with reduced or oxidized recombinant hTrx1
for 30 min, followed by addition of recombinant His-hAIF1-120.
As shown in Fig. 7, nuclei incubated with rHis-hAIF1-120 dis-
played distorted morphology; the nuclei looked shrunken, lacked
a clearly dened nuclear membrane and DAPI-stained nucleic acid
content appeared condensed and fragmented. Pre-treatment of
 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136 131

Fig. 5. Effect of oxidative stress on oxidation of nuclear Trx1 and cellular DNA damage. (A) HEK293 cells were treated with 0.5 or 1 mM diamide for 1 h. Cytosolic and nuclear
extracts were obtained and subjected to SDS-PAGE, followed by western blot analysis using the indicated antibodies. (B) HEK293 cells were treated with 0.5 or 1 mM diamide
for 3 h, after which freshly collected nuclear extracts and whole cell lysates were resolved on a native PAGE gel and analyzed by western blotting using Trx1 antibody.
(C) Following treatment of HEK293 cells with 0.5 or 1 mM diamide for 3 h, genomic DNA was extracted and 10 mg of DNA in each sample was electrophoresed on a 1% SYBRs
Green-stained agarose gel. All western blots images shown are representative of 3 independent experiments.

nuclei with reduced rhTrx1, but not oxidized rhTrx1, was able to
reverse the distorted nuclear morphology brought about by rHis-
hAIF1-120. When the DNA content in these nuclei was analyzed
by ow cytometry, it was found that nuclei incubated with rHis-
hAIF1-120 contained around 47% of DNA content in the sub-G1
phase (Fig. 8). Apparently, the percentage of nuclei in sub-G1
phase was reduced signicantly upon pre-incubating the nuclei
with reduced rhTrx1. Taken together, results obtained from uor-
escence microscopic observations and ow cytometry had in-
dicated that Trx1 in its reduced form was capable of attenuating
DNA damage induced by AIF.
4. Discussion
Oxidative stress caused by increased ROS levels during meta-
bolic disorders, disease states or xenobiotic insults are known to
activate apoptotic signaling cascades. H2O2, an endogenous ROS is
a key activator of numerous signaling cascades including apoptotic
cell death pathways. For example, H2O2 is reported to activate
apoptosis signal regulating kinase 1 (ASK-1)-mediated caspase-
dependent [26,27], as well as AIF-mediated caspase-independent
[1517] apoptotic cell death. Under physiological conditions,
apoptosis is tightly regulated by various proteins involved in vital
metabolic functions [28] and redox control [12,29,30]. Pertaining
to the Trx system, Trx has been reported to exert redox control of
cell death processes through protein-protein interaction with
several cellular proteins [reviewed in 12,29]. Of note, reduced Trx1
and Trx2 are known to inhibit ASK-mediated apoptosis activation
through a direct association via their active site cysteines with
ASK1 and ASK2 respectively [3133]. Furthermore, Trx interacting
protein [TXNIP, thioredoxin binding protein 2 (TBP2) or vitamin D3
up-regulated protein 1 (VDUP1)], an endogenous inhibitor of Trx1
[34], is known to bind to reduced Trx1 but not oxidized Trx1 [35].
This raises the notion that Trx-TXNIP interaction promotes apop-
tosis through competing with ASK1 for reduced Trx1 and con-
tributes to the dissociation of Trx1/Trx2 from the Trx1-ASK1/Trx2-
ASK2 complexes [12].
Given the current understanding of redox regulation of apop-
totic signaling remains incomplete, we had proposed that there
were novel proteins that could be associated with and under redox
regulation by Trx. In this study, we report the identication of AIF
interacting with Trx1, but not with Trx2. The results were in
agreement with a study by Joza et al. that reported a novel in-
teraction between Drosophila melanogaster AIF without the mi-
tochondrial localization sequence (N-DmAIF; protein observed
to display a non-mitochondrial distribution) and D. melanogaster
Trx2 (DmTrx2) [36]. It was also noted in the study that a yeast
two-hybrid screen of a mouse brain cDNA library using mouse AIF
(mAIF) as bait had identied Trx1 as a binding partner. Between
DmTrx1 and DmTrx2, despite the numbering, DmTrx2 shares
greater sequence identity and is the preferred substrate of thior-
edoxin peroxidase [37]. Its physiological functions are thus per-
ceived to be analogous to that of Trx1 in human. Recently, Nakao
et al. had also reported full-length AIF as an interacting protein
with Trx1 in HEK293 and Hela cells [38], which was in agreement
132 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136

Fig. 6. Modulating effect of reduced Trx1 on retardation of rHis-hAIF1-120-mediated electrophoretic mobility of DNA. (A) His-hAIF1-120 recombinant protein (1 mM) and
reduced or oxidized form of hTrx1 recombinant protein (5 mM) were co-incubated in PBS buffer at 37 C for 1 h, followed by Trx1 pull-down using anti-Trx1 antibody. The
pulled-down proteins were resolved by SDS-PAGE and detected by western blot analysis. (B) Recombinant His-hAIF1-120 (1 mM) was incubated with recombinant rat TrxR
(100 nM) and NADPH, oxidized rhTrx1 (5 mM) and NADPH, or recombinant rat TrxR (100 nM), oxidized rhTrx1 (5 mM) and NADPH in PBS at 4 C for 1 h. The mixtures and
rHis-hAIF121 (1 mM) only sample were further incubated with supercoiled plasmid DNA (2.5 mg) at 37 C for 1 h. The samples were electrophoresed on a 1% SYBRs Green-
stained agarose gel. (C) Recombinant His-hAIF1-120 (1 mM) was incubated with oxidized rhTrx1 (5 mM) in PBS in the presence or absence of 10 mM DTT at 4 C for 1 h. The
mixtures and rHis-hAIF1-120 (1 M) only sample were then incubated with CypA freshly immunoprecipitated from lysates of Hela cells cultured in a 100- mm culture dish
for 15 min at 4 C, followed by incubation with supercoiled DNA plasmid (2.5 mg) at 37 C for 1 h. The samples were electrophoresed on a 1% SYBRs Green-stained agarose
gel.

with our experimental ndings presented in Fig. 1B.
The full-length AIF protein (613 and 612 amino acids in human
and mouse AIF respectively), a 67 kDa precursor protein that
contains a N-terminal mitochondrial localization sequence (MLS),
is encoded by the AIF gene known as AIFM1 or PCDC8 [39]. Besides
the MLS (aa 154), the precursor protein possesses two nuclear
localization sequences (NLSs), two FAD-binding domains (aa. 128
262 and 401480), a NAD(H)-binding domain (aa. 263400) and a
C-terminal region (aa. 481-608) [40,41]. Once imported into the
mitochondria, the AIF precursor protein is cleaved to form mature
AIF (AIF1-54) of  62 kDa [42]. Due to presence of the FAD- and
NAD(H)-binding motifs and that AIF has a glutathione reductase
(GR) fold [43] with a close structural homology to bacterial oxy-
genase-coupled NADH-dependent ferredoxin reductases [44], AIF
has been suggested to possess oxidoreductase activity and studies
have reported that this activity is independent of its apoptogenic
activity [22,40,45]. Lines of evidence have also shed light on the
dual physiological role of AIF. Although it remains to be fully
elucidated, it is currently understood that under physiological
conditions, AIF residing in the IMS is vital for cell survival [46,47].
Conversely, when an appropriate caspase-independent cell death
stimulus is applied, mature AIF gets cleaved at amino acid 102
(murine numbering) by calpain-mediated proteolysis to form
truncated AIF of  57 kDa [42,48]. Upon permeabilization of the
outer mitochondrial membrane, truncated AIF then translocates
from the IMS through the cytosol into the nucleus to induce
chromatin condensation and large scale DNA fragmentation
[13,14]. In this study, we had conducted investigations on human
and murine AIF, which are most studied in published studies thus
far. For murine AIF, the full-length AIF precursor that resides in the
mitochondria [denoted as mAIF(FL)] and the apoptogenic trun-
cated AIF consisting of residues 101612 [denoted as mAIF101
612] were examined. For human AIF, the truncated AIF fragment
consisting of residues 121613 [denoted as hAIF121-613/hAIF1-
120] that has been demonstrated to trigger nuclear apoptosis in
cell-free systems [13,45] was investigated. Our ndings had re-
vealed that it was cytosolic Trx1 but not mitochondrial Trx2 that
interacted with endogenous AIF (Fig. 1D), which attested that the
Trx-AIF interaction likely occurred in the cytoplasm and nucleus
instead of the mitochondria. One would raise the argument how
mitochondrial AIF protein would interact with cytosolic Trx1 un-
der basal conditions. We performed immunocytochemical analysis
of the location of AIF in two cell lines used in our experiments,
namely Hela and HEK293 cells. Consistent with what has been
reported in the literature, AIF was localized in the mitochondria
(Fig. S1 in supplementary data). Similar analysis was attempted to
track the cellular localization of Trx1. However, due to the abun-
dance of Trx1, it was difcult to distinguish the sole localization of
Trx1 in the cytosol (results not shown). Therefore, as discussed
previously that Trx1 could be found in the IMS [38], we deduced
that interaction between Trx1 and AIF could occur in the mi-
tochondria under basal conditions. Nevertheless, it remains to be
investigated the biological signicance of a potential Trx1-AIF in-
teraction in the mitochondria. There also remained a possibility
that the association between Trx1 and AIF under basal conditions
could partly have been exemplied by a post-lysis event when
whole cell lysates were collected for immunoprecipitation reac-
tions. Indeed, the two recombinant proteins had demonstrated
ready association in a Trx1 pull-down assay (Fig. 6A). Regardless,
as this study was focused on investigating the association with and
redox regulation of apoptosis inducing cytoplasm-released forms
of AIF by cytosolic and nuclear Trx1, subsequent investigations
were carried out in transfected cells expressing cytosolic mAIF101-
612-FLAG and hAIF121-613-FLAG.
Several cellular proteins have been identied as pro-survival or
pro-death binding partners of AIF in the cytosol and/or nucleus
[reviewed in 41]. For instance, heat shock protein 70 (HSP70)
binds to AIF in the cytosol and restricts its nuclear translocation,
thereby inhibiting AIF-mediated cell death [4952]. On the other
hand, AIF is reported to interact with CypA in the cytosol [24] and
 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136 133

Fig. 7. Reduced Trx1 in nucleus impedes AIF's interaction with DNA. Nuclei isolated from Hela cells were pre-incubated with 5 mM of rhTrx1 (reduced or oxidized) in CFS
buffer for 30 min, followed by addition of rHis-hAIF1-120 to a nal concentration of 1 mM. The mixture was incubated at 37 C for 2 h and an aliquot of this mixture was
mounted onto a glass slide using Vectashield mounting medium containing DAPI for uorescence microscopic imaging. Upper panel: Representative microscopic images of
DAPI-stained nuclei in each sample as indicated. Lower panel: Bar graph depicts the percentage of number of damaged nuclei over a total of 50 nuclei observed under
microscope at various randomly selected spots on each coverslip in each treatment group. Data points are means 7SD (n 3). * indicates p o 0.01 versus rHis-hAIF1-120
treatment group; ns indicates p 40.05 versus rHis-hAIF1-120 treatment group.

cotranslocate into the nucleus [53]. As AIF is devoid of en-
donuclease activity and is unable to induce signicant DNA da-
mage by itself, studies have suggested that the co-binding of CypA
and histone protein H2AX to nuclear AIF forms a AIF/H2AX/CypA
DNA-degrading complex that directs nuclear DNA degradation
[24,25,54]. In this study, the redox sensitivity and biological sig-
nicance of the Trx-AIF interaction was investigated. Mutation of
the catalytic cysteine in Trx1 abolished AIF binding, whereas the
complete mutation of the cysteines in AIF could only reduce but
not abrogate the Trx-AIF interaction. In view that reduced binding
was observed between Trx1 and double/triple AIF cysteine mu-
tants, while we deduce that cysteines in Trx1 are involved in the
interface contact on the AIF protein, we also acknowledge that
other amino acids may be involved in non-covalent interactions at
the binding surface between the proteins. The Trx-AIF interaction
was redox-sensitive; under oxidative stress conditions achieved by
administration of cytotoxic concentrations of endogenous oxidants
H2O2 and HOCl or exogenous oxidant diamide, Trx-AIF interaction
was abrogated. Interestingly, the disrupted Trx-AIF interaction was
found to recover upon oxidative stress recovery (Fig. 4B and D),
which indicated the possible engagement of cellular redox defense
mechanism(s) in preserving the Trx-AIF interaction. Hence, we
postulate the following events: when truncated AIF is released
into the cytosol, it interacts with Trx1 in a redox-dependent
manner. Under severe oxidizing conditions caused by lethal con-
centrations of oxidants, Trx1 becomes oxidized and the Trx-AIF
complex dissociates (illustrated in Fig. 9). It has been reported that
under oxidative stress, Trx1 translocates from cytosol to nucleus
where it may impart cytoprotective effects through its disulde
reductase activity or by interactions with other proteins [55,56]. In
agreement with the published studies, we observed that en-
dogenous AIF and Trx1 translocated into the nuclei of cells sub-
jected to mild/moderate and severe oxidative stress upon treat-
ment with sublethal (0.5 mM) and lethal (1 mM) concentrations of
diamide respectively (Fig. 5A). Severe oxidative stress imposed by
treatment with 1 mM diamide caused oxidation of cytosolic and
nuclear Trx1 (Fig. 5B), as well as abrogation of the Trx-AIF inter-
action (Fig. 4C), which coincided with increased signs of cellular
DNA damage (Fig. 5C). We next deduced that nuclear Trx1 could
modulate AIF-mediated DNA damaging effects. This led us to ex-
amine the effect of Trx1 on the interaction between AIF and DNA
in vitro. The reduced form of Trx1, but not oxidized Trx1 was able
to disturb AIF-DNA interaction (Fig.6B). The retarding effect of
reduced Trx1 against AIF-mediated DNA damage was further de-
monstrated in the failure of recombinant hAIF1-120 protein to
(1) degrade plasmid DNA in the presence of CypA im-
munoprecipitated from freshly collected Hela cell lysates (Figs. 6C),
and (2) produce DNA fragmentation in isolated nuclei from Hela
cells (Figs. 7 and 8).
In general, reduced Trx is viewed upon as an anti-apoptotic
protein. Recently, inhibition of TrxR1 is reported to induce nuclear
translocation of AIF, suggesting the cytoprotective role of the Trx
system in the regulation of AIF-mediated apoptosis [57]. Therefore,
in this study, we had proposed that the Trx system, through a
physical association between Trx1 and AIF, would regulate AIF-
dependent cell death. Indeed, results presented had shown that
134 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136

Fig. 8. Reduced Trx1 in nucleus prevents AIF-mediated DNA damage. Nuclei isolated from Hela cells were pre-incubated with 5 mM of rhTrx1 (reduced or oxidized) in CFS
buffer for 30 min; followed by addition of rHis-hAIF1-120 to a nal concentration of 1 mM. The mixture was incubated at 37 C for 2 h and an aliquot of this mixture was
incubated with propidium iodide (50 mg/ml) for 30 min, followed by analysis of DNA content using ow cytometry. Upper panel: Representative DNA histograms of isolated
nuclei in each sample as indicated. Lower panel: Bar graph depicts percentage hypoploidy (% DNA content in Sub-G1 phase) of nuclei in each treatment group. Columns show
means of three experiments 7 SD. * indicates p o0.01 versus rHis-hAIF1-120 treatment group; # indicates p o 0.05 versus rHis-hAIF1-120 treatment group; ns indicates
p 40.05 versus rHis-hAIF1-120 treatment group.

when localized in the nucleus, nuclear Trx1 in its reduced form
was capable of attenuating AIF-mediated DNA damage possibly
through hindering the interaction between AIF and DNA (Fig. 9).
Importantly, illumination of the Trx1-dependent redox regulation
of AIF-mediated cell death has offered insights into designs of
therapeutic strategies against different disease states. For neuro-
degenerative diseases where cell death caused by oxidative stress
is undesirable, therapeutic strategies targeted to promote Trx-AIF
in the cytoplasm, or to enhance the reducing capacity of nuclear
Trx1 can be derived. On the other hand, in cancer, especially for
tumors where components of the Trx system Trx and TrxR are
overexpressed and related to cancer drug resistance and poor
prognosis [5862], targeting the cytosolic and nuclear Trx-AIF in-
teraction may serve as an effective anti-cancer strategy. All in all, a
better understanding of the pathways involved in redox signaling
and identication of a complete array of redox-sensitive proteins
will greatly facilitate opportunities for novel drug design.
Conict of Interest
The authors state that they have no conict of interest.
 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136
Fig. 9. Proposed mechanism of Trx1-dependent redox regulation of AIF-mediated
cell death. Under physiological conditions, reduced Trx1 interacts with AIF. When
subjected to pro-apoptotic oxidative stress conditions, Trx1 is oxidized, resulting in
the dissociation of the Trx-AIF complex, which likely leads to increased nuclear
translocation of AIF. In the nucleus, reduced Trx1 is proposed to attenuate inter-
action between AIF and DNA and prevents AIF-mediated DNA damage.
Acknowledgements
We thank Dr Elias Arner and Dr Qing Cheng (Division of Bio-
chemistry, Karolinksa Institutet) for providing recombinant rat
TrxR and Ms Mei-Yoke Lim (Department of Pharmacy, National
University of Singapore) for her help in gures construction. This
work was supported by research grants National University of
Singapore Start-Up Grant R-148-000-107-133 and National Uni-
versity of Singapore Academic Research Fund Tier 1 R-148-000-
110-112 to E-H. Chew, National Medical Research Council (NMRC)
of Singapore NMRC/1317/2011 to Y. C. Yu and Swedish Cancer
Society Grant 961 to A. Holmgren.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.freeradbiomed.
2015.06.029.
References
[1] W.C. Burhans, N.H. Heintz., The cell cycle is a redox cycle: linking phase-
specic targets to cell fate, Free Radic. Biol. Med. 47 (2009) 12821293.
[2] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress,
Curr. Biol. 24 (2014) R453R462.
[3] C. Ufer, C.C. Wang, A. Borchert, D. Heydeck, H. Kuhn, Redox control in mam-
malian embryo development, Antioxid. Redox Signal. 13 (2010) 833875.
[4] M.A. Lovell, W.R. Markesbery, Amyloid beta peptide, 4-hydroxynonenal and
apoptosis, Curr. Alzheimer Res. 3 (2006) 359364.
[5] G.S. Gaki, A.G. Papavassiliou, Oxidative stress-induced signaling pathways
implicated in the pathogenesis of Parkinson's disease, Neuromolecular Med.
16 (2014) 217230.
[6] R. Anuradha, M. Saraswati, K.G. Kumar, S.H. Rani, Apoptosis of beta cells in
diabetes mellitus, DNA Cell. Biol. 33 (2014) 743748.
[7] L. Rochette, M. Zeller, Y. Cottin, C. Vergely, Diabetes, oxidative stress and
therapeutic strategies, Biochim. Biophys. Acta 2709-2729 (1840) 2014.
135
[8] G.I. Giles, The redox regulation of thiol dependent signaling pathways in
cancer, Curr. Pharm. Des 12 (2006) 44274443.
[9] M. Valko, C.J. Rhodes, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals
and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160
(2006) 140.
[10] C.H. Lillig, A. Holmgren, Thioredoxin and related moleculesfrom biology to
health and disease, Antioxid. Redox Signal. 9 (2007) 2547.
[11] R. Sengupta, A. Holmgren, The role of thioredoxin in the regulation of cellular
processes by S-nitrosylation, Biochim. Biophys. Acta 689-700 (2012) 1820.
[12] J. Lu, A. Holmgren, Thioredoxin system in cell death progression, Antioxid.
Redox Signal. 17 (2012) 17381747.
[13] S.A. Susin, H.K. Lorenzo, N. Zamzami, I. Marzo, B.E. Snow, G.M. Brothers,
J. Mangion, E. Jacotot, P. Costantini, M. Loefer, N. Larochette, D.R. Goodlett,
R. Aebersold, D.P. Siderovski, J.M. Penninger, G. Kroemer, Molecular char-
acterization of mitochondrial apoptosis-inducing factor, Nature 397 (1999)
441446.
[14] S.A. Susin, N. Zamzami, M. Castedo, T. Hirsch, P. Marchetti, A. Macho,
E. Daugas, M. Geuskens, G. Kroemer, Bcl-2 inhibits the mitochondrial release
of an apoptogenic protease, J. Exp. Med. 184 (1996) 13311341.
[15] D. Arnoult, P. Parone, J.C. Martinou, B. Antonsson, J. Estaquier, J.C. Ameisen,
Mitochondrial release of apoptosis-inducing factor occurs downstream of cy-
tochrome c release in response to several proapoptotic stimuli, J. Cell. Biol. 159
(2002) 923929.
[16] G.P. Colo, M.F. Rubio, I.M. Nojek, S.E. Werbajh, P.C. Echeverra, C.V. Alvarado, V.
E. Nahmod, M.D. Galigniana, M.A. Costas, The p160 nuclear receptor co-acti-
vator RAC3 exerts an anti-apoptotic role through a cytoplasmatic action, On-
cogene 27 (2008) 24302444.
[17] Y.O. Son, Y.S. Jang, J.S. Heo, W.T. Chung, K.C. Choi, J.C. Lee, Apoptosis-inducing
factor plays a critical role in caspase-independent, pyknotic cell death in hy-
drogen peroxide-exposed cells, Apoptosis 14 (2009) 796808.
[18] N. Doti, C. Reuther, P.L. Scognamiglio, A.M. Dolga, N. Plesnila, M. Ruvo,
C. Culmsee, Inhibition of the AIF/CypA complex protects against intrinsic death
pathways induced by oxidative stress, Cell Death Dis 5 (2014) e993.
[19] H.K. Lorenzo, S.A. Susin, G. Kroemer, Cytouorometric quantitation of nuclear
apoptosis induced in a cell-free system, Methods Enzymol 322 (2000)
198201.
[20] S.A. Susin, N. Zamzami, M. Castedo, E. Daugas, H.G. Wang, S. Geley, F. Fassy, J.
C. Reed, G. Kroemer, The central executioner of apoptosis: multiple connec-
tions between protease activation and mitochondria in Fas/APO-1/CD95- and
ceramide-induced apoptosis, J Exp Med 186 (1997) 2537.
[21] C.C. Deocaris, S.C. Kaul, R. Wadhwa, On the brotherhood of the mitochondrial
chaperones mortalin and heat shock protein 60, Cell Stress Chaperones 11
(2006) 116128.
[22] M. Loefer, E. Daugas, S.A. Susin, N. Zamzami, D. Metivier, A.L. Nieminen,
G. Brothers, J.M. Penninger, G. Kroemer, Dominant cell death induction by
extramitochondrially targeted apoptosis-inducing factor, FASEB J 15 (2001)
758767.
[23] M. Wu, L.G. Xu, X. Li, Z. Zhai, H.B. Shu, AMID, an apoptosis-inducing factor-
homologous mitochondrion-associated protein, induces caspase-independent
apoptosis, J. Biol. Chem. 277 (2002) 2561725623.
[24] C. Cand, N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.
T. Kroemer, F. Giordanetto, C. Garrido, J.M. Penninger, G. Kroemer, AIF and
cyclophilin A cooperate in apoptosis-associated chromatinolysis, Oncogene 23
(2004) 15141521.
[25] C. Artus, H. Boujrad, A. Bouharrour, M.N. Brunelle, S. Hoos, V.J. Yuste,
P. Lenormand, J.C. Rousselle, A. Namane, P. England, H.K. Lorenzo, S.A. Susin,
AIF promotes chromatinolysis and caspase-independent programmed ne-
crosis by interacting with histone H2AX, EMBO J 29 (2010) 15851599.
[26] K. Tobiume, M. Saitoh, H. Ichijo, Activation of apoptosis signal-regulating ki-
nase 1 by the stress-induced activating phosphorylation of pre-formed oli-
gomer, J. Cell. Physiol. 191 (2002) 95104.
[27] P.J. Nadeau, S.J. Charette, M.B. Toledano, J. Landry, Disulde Bond-mediated
multimerization of Ask1 and its reduction by thioredoxin-1 regulate H(2)O(2)-
induced c-Jun NH(2)-terminal kinase activation and apoptosis, Mol. Biol. Cell
18 (2007) 39033913.
[28] D.R. Green, L. Galluzzi, G. Kroemer, Cell biology. Metabolic control of cell
death, Science 345 (2014) 1250256.
[29] M.L. Circu, T.Y. Aw, Reactive oxygen species, cellular redox systems, and
apoptosis, Free Radic. Biol. Med. 48 (2010) 749762.
[30] S. Ueda, H. Masutani, H. Nakamura, T. Tanaka, M. Ueno, J. Yodoi, Redox control
of cell death, Antioxid. Redox Signal. 4 (2002) 405414.
[31] M. Saitoh, H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M. Kawabata,
K. Miyazono, H. Ichijo, Mammalian thioredoxin is a direct inhibitor of apop-
tosis signal-regulating kinase (ASK) 1, EMBO J 17 (1998) 25962606.
[32] Y. Liu, W. Min, Thioredoxin promotes ASK1 ubiquitination and degradation to
inhibit ASK1-mediated apoptosis in a redox activity-independent manner,
Circ. Res. 90 (2002) 12591266.
[33] R. Zhang, R. Al-Lamki, L. Bai, J.W. Streb, J.M. Miano, J. Bradley, W. Min,
Thioredoxin-2 inhibits mitochondria-located ASK1-mediated apoptosis in a
JNK-independent manner, Circ. Res. 94 (2004) 14831491.
[34] A. Nishiyama, M. Matsui, S. Iwata, K. Hirota, H. Masutani, H. Nakamura,
Y. Takagi, H. Sono, Y. Gon, J. Yodoi, Identication of thioredoxin-binding pro-
tein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thior-
edoxin function and expression, J. Biol. Chem. 274 (1999) 2164521650.
[35] P. Patwari, L.J. Higgins, W.A. Chutkow, J. Yoshioka, R.T. Lee, The interaction of
thioredoxin with Txnip. Evidence for formation of a mixed disulde by
136 S.B. Shelar et al. / Free Radical Biology and Medicine 87 (2015) 125136
disulde exchange, J. Biol. Chem. 281 (2006) 2188421891.
[36] N. Joza, K. Galindo, J.A. Pospisilik, P. Benit, M. Rangachari, E.E. Kanitz,
Y. Nakashima, G.G. Neely, P. Rustin, J.M. Abrams, G. Kroemer, J.M. Penninger,
The molecular archaeology of a mitochondrial death effector: AIF in Droso-
phila, Cell Death Differ. 15 (2008) 10091018.
[37] H. Bauer, S.M. Kanzok, R.H. Schirmer, Thioredoxin-2 but not thioredoxin-1 is a
substrate of thioredoxin peroxidase-1 from Drosophila melanogaster: isola-
tion and characterization of a second thioredoxin in D. Melanogaster and
evidence for distinct biological functions of Trx-1 and Trx-2, J. Biol. Chem. 277
(2002) 1745717463.
[38] L.S. Nakao, R.A. Everley, S.M. Marino, S.M. Lo, L.E. de Souza, S.P. Gygi, V.
N. Gladyshev, Mechanism-based proteomic screening identies targets of
thioredoxin-like proteins, J. Biol. Chem. 290 (2015) 56855695.
[39] E. Daugas, D. Nochy, L. Ravagnan, M. Loefer, S.A. Susin, N. Zamzami,
G. Kroemer, Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxi-
doreductase involved in apoptosis, FEBS Lett. 476 (2000) 118123.
[40] H. Ye, C. Cande, N.C. Stephanou, S. Jiang, S. Gurbuxani, N. Larochette, E. Daugas,
C. Garrido, G. Kroemer, H. Wu, DNA binding is required for the apoptogenic
action of apoptosis inducing factor, Nat. Struct. Biol 9 (2002) 680684.
[41] I.F. Sevrioukova, Apoptosis-inducing factor: structure, function, and redox
regulation, Antioxid. Redox Signal. 14 (2011) 25452579.
[42] H. Otera, S. Ohsakaya, Z. Nagaura, N. Ishihara, K. Mihara, Export of mi-
tochondrial AIF in response to proapoptotic stimuli depends on processing at
the intermembrane space, EMBO J 24 (2005) 13751386.
[43] M.J. Mat, M. Ortiz-Lombarda, B. Boitel, A. Haouz, D. Tello, S.A. Susin,
J. Penninger, G. Kroemer, P.M. Alzari, The crystal structure of the mouse
apoptosis-inducing factor AIF, Nat. Struct. Biol 9 (2002) 442446.
[44] T. Senda, T. Yamada, N. Sakurai, M. Kubota, T. Nishizaki, E. Masai, M. Fukuda,
Y. Mitsuidagger, Crystal structure of NADH-dependent ferredoxin reductase
component in biphenyl dioxygenase, J. Mol. Biol. 304 (2000) 397410.
[45] M.D. Miramar, P. Costantini, L. Ravagnan, L.M. Saraiva, D. Haouzi, G. Brothers, J.
M. Penninger, M.L. Peleato, G. Kroemer, S.A. Susin, NADH oxidase activity of
mitochondrial apoptosis-inducing factor, J. Biol. Chem. 276 (2001)
1639116398.
[46] J.A. Klein, C.M. Longo-Guess, M.P. Rossmann, K.L. Seburn, R.E. Hurd, W.
N. Frankel, R.T. Bronson, S.L. Ackerman, The harlequin mouse mutation
downregulates apoptosis-inducing factor, Nature 419 (2002) 367374.
[47] N. Vahsen, C. Cand, J.J. Brire, P. Bnit, N. Joza, N. Larochette, P.
G. Mastroberardino, M.O. Pequignot, N. Casares, V. Lazar, O. Feraud, N. Debili,
S. Wissing, S. Engelhardt, F. Madeo, M. Piacentini, J.M. Penninger, H. Schgger,
P. Rustin, G. Kroemer, AIF deciency compromises oxidative phosphorylation,
EMBO J 23 (2004) 46794689.
[48] B.M. Polster, G. Basaez, A. Etxebarria, J.M. Hardwick, D.G. Nicholls, Calpain I
induces cleavage and release of apoptosis-inducing factor from isolated mi-
tochondria, J. Biol. Chem. 280 (2005) 64476454.
[49] L. Ravagnan, S. Gurbuxani, S.A. Susin, C. Maisse, E. Daugas, N. Zamzami, T. Mak,
M. Jttel, J.M. Penninger, C. Garrido, G. Kroemer, Heat-shock protein 70
View publication stats
antagonizes apoptosis-inducing factor, Nat. Cell Biol. 3 (2001) 839843.
[50] S. Gurbuxani, E. Schmitt, C. Cande, A. Parcellier, A. Hammann, E. Daugas,
I. Kouranti, C. Spahr, A. Pance, G. Kroemer, C. Garrido, Heat shock protein 70
binding inhibits the nuclear import of apoptosis-inducing factor, Oncogene 22
(2003) 66696678.
[51] Y. Matsumori, S.M. Hong, K. Aoyama, Y. Fan, T. Kayama, R.A. Sheldon, Z.
S. Vexler, D.M. Ferriero, P.R. Weinstein, J. Liu, Hsp70 overexpression sequesters
AIF and reduces neonatal hypoxic/ischemic brain injury, J. Cereb. Blood Flow
Metab. 25 (2005) 899910.
[52] J.C. Lui, S.K. Kong, Heat shock protein 70 inhibits the nuclear import of
apoptosis-inducing factor to avoid DNA fragmentation in TF-1 cells during
erythropoiesis, FEBS Lett. 581 (2007) 109117.
[53] C. Zhu, X. Wang, J. Deinum, Z. Huang, J. Gao, N. Modjtahedi, M.R. Neagu,
M. Nilsson, P.S. Eriksson, H. Hagberg, J. Luban, G. Kroemer, K. Blomgren, Cy-
clophilin A participates in the nuclear translocation of apoptosis-inducing
factor in neurons after cerebral hypoxia-ischemia, J Exp Med 204 (2007)
17411748.
[54] M. Baritaud, H. Boujrad, H.K. Lorenzo, S. Krantic, S.A. Susin, Histone H2AX: The
missing link in AIF-mediated caspase-independent programmed necrosis, Cell
Cycle 9 (2010) 31663173.
[55] K. Hirota, M. Matsui, S. Iwata, A. Nishiyama, K. Mori, J. Yodoi, AP-1 tran-
scriptional activity is regulated by a direct association between thioredoxin
and Ref-1, Proc. Natl. Acad. Sci. USA 94 (1997) 36333638.
[56] K. Hirota, M. Murata, Y. Sachi, H. Nakamura, J. Takeuchi, K. Mori, J. Yodoi,
Distinct roles of thioredoxin in the cytoplasm and in the nucleus. A two-step
mechanism of redox regulation of transcription factor NF-kappaB, J. Biol.
Chem. 274 (1999) 2789127897.
[57] R.L. Poerschke, P.J. Moos, Thioredoxin reductase 1 knockdown enhances se-
lenazolidine cytotoxicity in human lung cancer cells via mitochondrial dys-
function, Biochem. Pharmacol. 81 (2011) 211221.
[58] M. Berggren, A. Gallegos, J.R. Gasdaska, P.Y. Gasdaska, J. Warneke, G. Powis,
Thioredoxin and thioredoxin reductase gene expression in human tumors and
cell lines, and the effects of serum stimulation and hypoxia, Anticancer Res. 16
(1996) 34593466.
[59] K. Kahlos, Y. Soini, M. Saily, P. Koistinen, S. Kakko, P. Paakko, A. Holmgren, V.
L. Kinnula, Up-regulation of thioredoxin and thioredoxin reductase in human
malignant pleural mesothelioma, Int. J. Cancer 95 (2001) 198204.
[60] Y. Soini, K. Kahlos, U. Napankangas, R. Kaarteenaho-Wiik, M. Saily, P. Koistinen,
P. Paaakko, A. Holmgren, V.L. Kinnula, Widespread expression of thioredoxin
and thioredoxin reductase in non-small cell lung carcinoma, Clin. Cancer Res.
7 (2001) 17501757.
[61] J. Raffel, A.K. Bhattacharyya, A. Gallegos, H. Cui, J.G. Einspahr, D.S. Alberts,
G. Powis, Increased expression of thioredoxin-1 in human colorectal cancer is
associated with decreased patient survival, J. Lab. Clin. Med. 142 (2003) 4651.
[62] S.J. Kim, Y. Miyoshi, T. Taguchi, Y. Tamaki, H. Nakamura, J. Yodoi, K. Kato,
S. Noguchi, High thioredoxin expression is associated with resistance to
docetaxel in primary breast cancer, Clin. Cancer Res. 11 (2005) 84258430.