Differences in substrate preferences and metal compound inhibition of cytosolic
and mitochondrial human thioredoxin reductases
Oliver Rackham1, Anne-Marie J. Shearwood1, Ross Thyer1, Elyshia McNamara1, Stefan
M.K. Davies1, Bernard A. Callus2, Antonio Miranda-Vizuete3, Susan J. Berners-Price4,
Qing Cheng5, Elias S. J. Arner5 and Aleksandra Filipovska1+.
1
Western Australian Institute for Medical Research, Centre for Medical Research and
2
School for Biomedical, Biomolecular and Chemical Sciences, University of Western
Australia, Australia,
3
Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del
Rocío/CSIC/Universidad de Sevilla, E-41013 Sevilla, Spain.
4
Institute for Glycomics, Gold Coast Campus, Griffith University,Queensland, QLD
4222, Australia,
5
Division of Biochemistry, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, SE-171 77 Stockholm, Sweden,
+
Address correspondence to: Aleksandra Filipovska, Western Australian Institute for
Medical Research, Level 5, Medical Research Foundation Building, Rear 50 Murray
Street, Perth, Western Australia 6000, Australia. Tel: +61 8 9224 0330, Fax: +61 8 9224
0322; E-mail: afilipov@waimr.uwa.edu.au
1
ABSTRACT
Key components of the mammalian thioredoxin system are the cytosolic and
mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and thioredoxins (Trx1 and
Trx2), that are important for antioxidant defense and redox regulation of cell function.
TrxR1 and TrxR2 are two selenoproteins generally considered to be comparable with
each other in properties, but functionally separated through their different compartments.
To compare their properties side-by-side, we expressed recombinant human TrxR1 and
TrxR2 and determined their substrate specificities and inhibition by metal compounds.
TrxR2 clearly preferred its endogenous substrate Trx2 over Trx1, while TrxR1
efficiently reduced both Trx1 and Trx2. TrxR2 displayed strikingly lower activity with
DTNB, lipoamide and the quinone substrate juglone as compared to TrxR1, and, in
contrast to the latter, could not reduce lipoic acid. However, Sec-deficient two-amino
acid truncated TrxR2 was almost as efficient as full-length TrxR2 in reduction of
DTNB. We found that the gold(I) compound auranofin efficiently inhibited both fulllength TrxR1 and TrxR2 and truncated TrxR2. In contrast, some newly synthesized
gold(I) compounds as well as cisplatin inhibited only full-length TrxR1 or TrxR2, but
not truncated TrxR2. Surprisingly, one gold(I) compound, [Au(d2pype)2]Cl, was a
TrxR1-specific inhibitor while another, [(iPr2Im)2Au]Cl, only inhibited TrxR2. These
compounds also inhibited TrxR activity in cells, but their cytotoxicity involved different
mitochondrial pathways, not always dependent on the proapoptotic proteins Bax and
Bak. In conclusion, this study reveals significant differences between human TrxR1 and
TrxR2 in substrate specificities and metal compound inhibition, which may be exploited
for development of specific TrxR1- or TrxR2-targeting drugs.
Keywords: thioredoxin reductase, auranofin, gold(I) compounds, thioredoxin
2
Introduction
The thioredoxin system consists of isoenzymes of thioredoxin reductase (TrxR) and
thioredoxin (Trx) that are responsible for a wide range of cellular functions, including
redox regulation, antioxidant defence and synthesis of deoxyribonucleotides [1, 2]. The
major cytosolic forms of TrxR and Trx are known as TrxR1 and Trx1 while those
present in mitochondria are known as TrxR2 and Trx2 [1]; the four proteins are encoded
by distinct genes that are all essential as their respective deletions are embryonically
lethal in mice [3-6]. A third TrxR isoenzyme, thioredoxin glutathione reductase (TGR),
is mainly expressed in testes and seems to be involved in spermatogenesis [7, 8]. All
mammalian TrxR isoenzymes are selenoproteins with a redox active selenocysteine
(Sec) residue in their active sites and belong to the pyridine nucleotide-disulfide
oxidoreductase family of proteins that catalyze NADPH-dependent reduction of their
native Trx substrates [9-12]. They are homodimers containing a flavin adenine
dinucleotide (FAD) redox cofactor and a redox active disulfide within a conserved
CVNVGC motif in one subunit, which interacts with a redox active selenenylsulfide
/selenolthiol motif at the C-terminus of the other subunit [9, 11-15]. In addition to Trx,
mammalian TrxR isoforms also accept a range of low molecular weight disulfidecontaining substrates including 5,5’ dithiobis-(2-nitrobenzoic acid) (DTNB), lipoic acid,
and lipoamide, as well as non-disulfide substrates such as selenite, quinone compounds
and ascorbic acid [1, 2, 9, 11, 13, 16, 17]. This broad substrate specificity of mammalian
TrxRs is usually attributed to two unique features of the enzymes, the easily accessible
C-terminal selenenylsulfide /selenolthiol active center and the ability of the N-terminal
FAD/-CVNVGC redox centre to directly reduce certain small substrate compounds
independent of the canonical Sec-containing active site [18-21].
3
Most of current knowledge about mammalian TrxR enzymes is based upon extensive
studies of the cytosolic TrxR1 purified from either bovine [22], rat [23] or human tissues
[10, 24, 25], as well as mouse and rat proteins expressed in recombinant form [13, 26].
Although the gene for mitochondrial TrxR2 has been cloned from a range of mammals
including rat, mouse, cow and human [24, 27-30] and was first purified from rat liver in
1999 [27], there is still limited knowledge about its exact role in mitochondria. Much of
what is known about the biochemical properties of TrxR2 stems from the work of
Bindoli and Rigobello with coworkers studying the purified enzyme from rat [31, 32],
and studies of semisynthetic variants produced by Hondal and coworkers [21, 33].
Moreover, the crystal structure of mouse TrxR2 has been solved [16] as well as that of
both rat [13, 17] and human TrxR1 [34]. Because of evident structural similarities
between TrxR2 and TrxR1, particularly regarding the redox active centers and broad
substrate specificities of both enzymes, it is generally thought that TrxR2 has similar
redox regulating and antioxidant function in mitochondria as TrxR1 would have in the
cytosol [13, 16, 17]. A functional analogy to this would be found in Drosophila
melanogaster where the mitochondrial and cytosolic forms of TrxR are encoded by the
same gene [35]. Although mammalian TrxR1 and TrxR2 have some substrates in
common, the different subcellular localization of the isoenzymes has likely given rise to
some differences, including specific protein substrates such as Trx2, glutaredoxin 2 and
cytochrome c for TrxR2 [36-38] and thioredoxin-related protein 14 for TrxR1 [39]. The
important question if TrxR2 may have some distinct biochemical features differing from
those of TrxR1 has not yet been well addressed, but clues for such differences can be
found in the literature. For example, it was recently reported that several TrxR-catalyzed
substrate reactions might be selenium-independent, which was found using TrxR2derived enzyme scaffolds [21, 40], while such activity with similar substrates were not
4
found using a TrxR1-derived protein [41, 42]. Furthermore, it was suggested recently
that TrxR2 might prefer Trx1 as a substrate [43], however, the substrate specificities of
human TrxR1 and TrxR2, with their natural Trx1 and Trx2 substrates or with other
small molecular weight compounds, has not been investigated previously in a direct
side-by-side comparison using well defined enzyme preparations. Therefore, here we
have compared the activities of pure human recombinant TrxR1 and TrxR2 using both
recombinant forms of their endogenous human Trx1 and Trx2 substrates, as well as the
small molecular dithiol compounds DTNB, lipoamide and lipoic acid and the quinone
substrate juglone. We found that human TrxR2 has different substrate affinities
compared to human TrxR1 and, moreover, that its N-terminal redox center can function
effectively in the absence of the Sec residue, as was shown before for mouse TrxR2
[21]. We also compared the inhibition pattern of human TrxR1 and TrxR2 with metalbased inhibitors, because such inhibition is considered to have significant biomedical
importance. Importantly, we found pronounced differences in inhibition between TrxR1
and TrxR2 using different recently synthesized gold(I) compounds. We also found that
these compounds inhibited total TrxR activity in cells and caused cell death via
mitochondria, but by different pathways, some of which required Bax and Bak while
others did not.
Materials and Methods
Materials
[(iPr2Im)2Au]Cl, [Au(d2pype)2]Cl and [Au(d2pypp)2]Cl were synthesized and used as
reported elsewhere [44-46]. Platinol (cisplatin) was from Mayne Pharm Pty Ltd. Native
4-16% polyacrylamide gels were from Invitrogen. 2',5'-ADP Sepharose was from GE
Healthcare. All other reagents used in this study were from Sigma including the gold(I)
5
compounds auranofin and aurothioglucose. Full length human TrxR1 was amplified
from a testes cDNA library (Clontech) by PCR with primers that introduced flanking
NcoI and BseAI sites. NcoI and BseAI digestion was used to remove the rat TrxR1
insert along with the engineered SECIS from pET-TRSter [47] and replace it with the
hTrxR1 PCR fragment. The engineered SECIS from pET-TRSter was reinserted
subsequently into the human TrxR1-encoding plasmid as a BseAI fragment. DNA
sequencing confirmed that the TrxR1 sequence was identical to the human TrxR1
isoform 2, Entrez Gene reference sequence NM_ 182729.1, where the second amino
acid, asparagine, was changed to asparatic acid due to the insertion of an NcoI restriction
site. Human TrxR2 was amplified from HeLa cell cDNA by PCR. The fragment
corresponding to amino acids 37-623 of TrxR2 followed by an engineered bacterial
SECIS [48] was flanked by NcoI and PacI sites. The PCR product was digested with
NcoI and PacI and inserted into NcoI and PacI cut pETDuet-1 (Novagen) to make pEThTrxR2. DNA sequencing showed that the hTrxR2 sequence was identical to the Entrez
Gene reference sequence NM_006440.3. Trx1 was made as described before [13]. Trx2
(amino acids 59-166, NP_036605.2) was cloned into pGEX-4T-2 and expressed as a
GST fusion protein in E.coli Rosetta2, purified using glutathione-sepharose (GE
Healthcare), eluted by thrombin cleavage according to the manufacturer’s instructions
and further purified by size exclusion chromatography in PBS.
Expression and purification of human recombinant TrxR1 and TrxR2
Recombinant human TrxR1 or TrxR2 were expressed in either E. coli ER2566 (New
England Biolabs) or Rosetta2 cells (Novagen), transformed with the corresponding pEThTrxR1 or pET-hTrxR2 plasmids, and in both cases the pSUABC plasmid [47]
according to the previously established protocol [48], in growth medium containing 10 g
NaCl, 10 g peptone, and 10 g yeast extract per liter water, supplemented with 0.5 mM
6
IPTG, 100 µg.ml-1 L-cysteine and 5 µM sodium selenite. Cells were harvested and
lysed for 1 h using lysozyme, snap frozen in liquid nitrogen and sonicated (six 15 s
pulses at setting 20 using a Sonifier 150, Branson). The initial purification on 2',5'-ADP
Sepharose (GE Healthcare Life sciences) and subsequent purification on PAO Sepharose
was performed according to Cheng et al. [48]. The full-length enzymes were further
purified by an ÄKTA-Explorer system (GE) using a Superdex 200 10/300 column (GE)
with a total bed volume of 24 ml. The two-amino acid Sec-deficient truncated human
TrxR2 was deliberately expressed as such in E.coli ER2566 cells by transformation with
a plasmid lacking a bacterial-type SECIS element and having a UAA replacing the
original UGA stop codon, thus expressing the enzyme without its last two amino acids
(resulting in a C-terminal -Gly-Cys-COOH instead of -Gly-Cys-Sec-Gly-COOH). This
enzyme was purified using 2′,5′-ADP Sepharose, followed by gel filtration on an AKTA
Purifier system using a Superdex 200 10/300 column (GE).
Mass spectrometry
MS analyses were performed on a 4000 Q-TRAP (Applied Biosystems, Foster City, CA,
USA) operating with an ion spray voltage of 5500 V, an ion source gas 1 at 30 and
scanning over a mass range from 600 to 2000 m/z and detection in Q3 scanning mode.
Initial m/z peaks were deconvoluted with the ‘Bayesian Protein Reconstruct’ tool to
provide the intact protein masses within the Analyst 1.5.1 software (Applied
Biosystems).
Gel electrophoresis
We used either 4-16% Bis-Tris native polyacrylamide gels or 10% Tris-Glycine SDS
denaturing polyacrylamide gels to resolve the enzymes for 1 h at 100 V. The gels were
7
stained with Coomassie Brilliant Blue R250 in 50% methanol and 7% acetic acid for 1 h
and destained for 3 h in 20% methanol and 7.5% acetic acid.
Protein concentration
Total TrxR concentration was determined by absorbance measurement at 463 nm (for
oxidized FAD) and calculated using the extinction coefficient for FAD of 11,300 M−1
cm−1 assuming one FAD per subunit [11]. The concentration of active enzyme was
calculated assuming two subunits per molecule for all TrxRs. Protein concentration
determined by the absorbance measurement at 463 nm was used to calculate the specific
activities of the enzymes. Protein concentration was also determined by the
bicinchoninic acid (BCA) assay [49] using bovine serum albumin (BSA) as a standard.
This was used to compare the protein concentration determined by the absorbance at 463
nm and to determine protein concentration of cell lysates.
TrxR activity measurements
The activity of TrxR was determined using the DTNB reduction assay to calculate units
and specific activity, as described previously [50]. The rate of DTNB reduction (0.0125
- 6.4 mM) by different TrxR preparations (14.5 – 90 nM) in the presence of 150 µM
NADPH in 50 mM Tris-HCl, 1 mM EDTA, pH 7.0 or pH 8.0 buffer was monitored
following an increase in absorbance at 412 nm in a final volume of 500 µl. The amount
of product formed was determined from the extinction coefficient 13,600 M−1 cm−1 of
the thionitrobenzoate (TNB−) anion, considering that 2 mol TNB− are formed per 1 mol
NADPH, with one unit corresponding to the oxidation of 1 µmol NADPH per minute.
The oxidation of NADPH by TrxR preparations using racemic (R,S)-lipoic acid or
lipoamide as substrates was measured as a decrease in absorbance at 340 nm. A mixture
8
of TrxR (14.5 – 90 nM) and 150 µM NADPH in 50 mM Tris-HCl, 1 mM EDTA, pH 8.0
buffer was incubated with either lipoic acid (0.4 – 6.4 mM) or lipoamide (0.5 - 10 mM)
in a total volume of 500 µl. Similarly, the oxidation of NADPH by TrxR (14.5 – 90 nM)
using the quinone substrate juglone (0.625 – 80 µM) was measured at 340 nm. The
decrease in absorbance was monitored on a Perkin Elmer Lambda 30 spectrophotometer
and the rate of NADPH consumption was determined using the extinction coefficient of
6200 M-1 cm-1.
Insulin assay
The activity of TrxRs with Trxs as substrates was determined using the coupled insulin
assay according to published procedures [50, 51]. A constant amount of TrxR (14.5 – 90
nM, final assay concentration) and 100 µM NADPH in 50 mM Tris-HCl, 1 mM EDTA,
pH 7.0 or 8.0 buffer was pre-incubated at room temperature for 5 min. To determine the
enzyme activity, the incubation mixture was then added to a cuvette containing 0.16 mM
insulin, 100 µM NADPH, and 0.45-14.4 µM of either Trx1 or Trx2 in a total volume of
500 µl. The decrease in absorbance was recorded at 340 nm with a reference containing
the same mixture but without the addition of Trx. TrxR activity was recorded as the
initial linear decrease in absorbance at 340 nm to determine the rate of NADPH
consumption using the extinction coefficient of 6200 M-1 cm-1.
TrxR inhibition assay
Inhibition of recombinant TrxRs with increasing concentrations of the metal compounds
were measured by the DTNB assay adapted for microtiter plates. TrxRs (100 nM) were
reduced with 150 µM NADPH in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Metal compounds at varying concentrations were mixed with the reduced TrxRs in a
total volume of 50 µl and incubated for 30 min at room temperature in a microtiter plate.
9
At the end of the incubation, 250 µl of TE buffer containing 150 µM NADPH and 2.5
mM DTNB was added. The NADPH-dependent TrxR catalyzed reduction of DTNB was
monitored immediately at 30˚C for 3 min and determined as the linear increase in
absorbance at 412 nm using a BioTek ELx808 absorbance microplate reader. Data were
expressed as a percent of control TrxR activity that was not incubated with metal
compounds.
Cell culture and cell death assay
Factor dependent myeloid (FDM) mouse wild type and Bax/Bak-/- cell lines (a kind gift
from A/Prof Paul Ekert from Murdoch Children's Research Institute and Royal
Children's Hospital, Melbourne) [52] were cultured at 37˚C under humidified 95%
air/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) without phenol red,
containing Earle’s balanced salt solution and supplemented with 2 mM Glutamax,
penicillin (100 U.ml-1), streptomycin (100 µg.ml-1), 10% heat inactivated fetal calf
serum (FCS), 0.5 ng.ml-1 interleukin-3 (IL-3). To test for toxicity, cells were grown to
90% confluence and incubated for 24 h with their growth medium containing increasing
concentrations of the inhibitors. The cells were collected (1,000 g for 5 min), gently
resuspended in 0.5 ml binding buffer (10 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 2.5
mM CaCl2, 1 mM MgCl2, 4% BSA) containing 10 µl propidium iodide (30 µg/ml) and
incubated for 15 min at room temperature in the dark. Cell death was quantified using a
Becton Dickinson FACS Scan flow cytometer. The data is expressed as percent of cells
grown in the absence of inhibitors.
Mitochondrial isolation
Mitochondria were prepared from 3 x 106 FDM wild type and Bax/Bak-/- cells treated
with 50 µM of each compound for 8 h in their growth medium. Cells were sedimented
10
(150 g for 5 min at 4 °C), washed in solution A (100 mM sucrose, 1 mM EGTA, 20 mM
MOPS, pH 7.4), incubated on ice for 3 min in 100 µl solution B (100 mM sucrose, 1
mM EGTA, 20 mM MOPS, 10 mM triethanolamine, 0.1 mg/ml digitonin, pH 7.4) and
disrupted using a 1 ml homogenizer. The nuclei were sedimented (1,000 g for 5 min at 4
°C), the pellet washed and the combined supernatants were centrifuged (10,000 g for 10
min at 4 °C). The mitochondrial pellet was washed once with STE and cytosolic and
mitochondrial protein concentrations were determined by the BCA assay using BSA as a
standard.
TrxR activity in lysates
Factor dependent myeloid (FDM) wild type and Bax/Bak-/- cell lines were incubated in
their growth medium with either 5 µM or 50 µM concentrations of the metal compounds
auranofin,
aurothioglucose,
cisplatin,
[Au(d2pype)2]Cl,
[(iPr2Im)2Au]Cl
and
[Au(d2pypp)2]Cl. The cells were collected, suspended in 100 µl cell extraction buffer
(50 mM Tris, pH 7.6, 2 mM EDTA, 0.5 mM phenyl methyl sulfonyl fluoride and 0.5%
Igepal CA-630) and lysed by three cycles of rapid freezing and thawing. The cell lysates
were clarified by centrifugation (16,000 g for 15 min at 4˚C) and the supernatant was
used to measure total TrxR activity by the end-point Trx-dependent insulin reduction
method [53]. Briefly, 5 µg of cell lysate, 1 µg mitochondrial or 1 µg cytosolic lysate was
incubated with 20 µM recombinant human Trx1 or Trx2 (for mitochondrial lysates) in
the presence of 297 µM insulin, 1.3 mM NADPH, 85 mM Hepes buffer, pH 7.6, and 13
mM EDTA for 40 min at 37˚C, in a total volume of 50 µl. The reaction was stopped by
the addition of 200 µl of 7.2 M guanidine-HCl in 0.2 M Tris-HCl, pH 8.0 containing 1
mM DTNB. The thioredoxin-dependent thiols of the reduced insulin were determined by
measuring the absorbance at 412 nm using a BioTek ELx808 absorbance microplate
reader, with the background absorbance reference for each sample containing the same
11
components except Trx1 or Trx2. Data were expressed as a percent of TrxR activity in
cells treated with the metal compounds compared to control, untreated cells.
Statistical analysis
Data were analyzed and Km and Kcat values were calculated using Prism Graph Pad 5.0.
Statistical analyses were performed using a 2-tailed Student’s t test.
Results
Identification of the molecular masses of the human TrxR1, TrxR2 and the truncated
form of TrxR2
To enable this side-by-side comparisons of TrxR1 and TrxR2, first we expressed the
human enzymes, as well as the truncated version of TrxR2 (TrxR2) missing the last
two amino acids of the selenenylsulfide/selenolthiol redox active center, in E.coli as
recombinant proteins. This was enabled by the engineering of a bacterial-like SECIS
element in the open reading frame and by overexpressing the bacterial SelA, SelB and
SelC genes, as described previously for expression of rat TrxR1 [47]. We purified all of
the proteins to near homogeneity following established protocols, which resulted in
preparations with overall yields and specific activities shown in Table 1. The increase in
the specific activities of the enzymes following PAO Sepharose purification suggested
that this purification step was necessary to enrich for the full-length TrxRs, as was also
found earlier [41]. Analyzing the purity and molecular weights of the different TrxR
preparations by mass spectrometry showed that the TrxR1 molecular mass was 54769
Da (Fig 1A), the TrxR2 was 53117 Da (Fig 1B) and the truncated hTrxR2 was 52907
Da, confirming that it was missing the Sec and Gly amino acids at its C-terminus (Fig
1C). All recombinant enzymes had the expected molecular weights and this analysis also
12
confirmed the purity of the preparations. The apparent complete absence of UGAtruncated species in the full-length TrxR preparations indicated that the PAO affinity
purification step effectively separated these proteins from truncated variants arising from
premature (Sec-encoding) UGA termination, which was surprising in view of previous
findings that some UGA-truncated subunits of the rat TrxR1 always co-purified with the
full-length enzyme [41]. One explanation could be that truncated forms of the human
isoenzymes were highly unstable, which also would be in agreement with our
observations that these proteins easily precipitated upon storage (not shown). All
preparations analysed in this study were therefore used within 10 days of preparation
while for long-term use the enzymes were stored in 20% glycerol, which increased their
stability.
Human thioredoxin reductases form dimers and tetramers
We used native polyacrylamide gel electrophoresis to determine if the human TrxR
proteins differed in their tendency to form dimeric, tetrameric or oligomeric complexes.
We found that human TrxR1 existed as both a dimer and tetramer (Fig 2A), and in
addition it formed higher molecular weight complexes as previously found for the rat
TrxR1 [41]. The full length TrxR2 and TrxR2 were predominantly dimeric and higher
oligomers were virtually absent (Fig 2A). To analyze if disulfides, selenenylsulfides or
diselenides were involved in the formation of these dimers or tetramers, we treated the
proteins with high concentrations of the reducing agent dithiothreitol (DTT) before
analyzing them on either native or denaturing gels. We found that DTT resolved the
majority of the tetramers of all the TrxRs (Fig 2A). The higher molecular weight
complexes formed by the rat TrxR1 were, however, reduced predominantly to both
tetrameric and dimeric forms, indicating the remaining tetramers were not disulfide- or
selenenylsulfide-linked dimers of the protein as they were highly resistant to DTT (Fig
13
2A). In the denaturing gels (Fig 2B), monomers were mainly seen in the absence of DTT
for both TrxR1 and TrxR2, suggesting that the earlier identified intersubunit disulfide in
the dimer of mouse TrxR2 [16] is either not formed in human TrxR2 or is rather labile.
Importantly, however, we also observed that some dimeric species of the different TrxR
preparations persisted in the denaturing gels even in the presence of 100 mM DTT,
suggesting that these were not only disulfide or selenenylsulfide-linked dimers (Fig 2B).
The exact nature of the different oligomeric forms of mammalian TrxR is yet unknown,
but these are regularly seen in mammalian cell extracts and the propensity to form these
species is apparently an inherent feature of these proteins. Future investigations using
tryptic digests of the enzymes followed by mass spectrometry could potentially identify
residues involved in formation of these dimers or tetramers.
Activity of the human TrxR isoenzymes using human Trx1 and Trx2 as substrates
Previous studies have shown that different mammalian TrxR isoenzymes can effectively
use E.coli Trx, human Trx1 and rat Trx2 [22, 23, 43]. Here, we investigated the
efficiency of human TrxR1 and TrxR2 with their cognate substrates, human Trx1 and
Trx2, at both pH 7 and pH 8 to mimic the pH of their respective subcellular
compartment microenvironments. We found that the Km of TrxR1 was similar for both
Trx1 and Trx2 at pH 7, while at pH 8 the Km of TrxR1 was lower for Trx1 compared to
Trx2. As illustrated by the catalytic efficiencies, however, TrxR1 may clearly use both
Trx1 and Trx2 as substrates (Table 2). In contrast, the Km values of TrxR2 for Trx2 were
significantly lower than for Trx1 at both pH 7 and 8, and the catalytic efficiency using
Trx2 as substrate was about 10-fold higher than using Trx1, thus showing that TrxR2 is
clearly more effective at using its endogenous substrate Trx2 compared to Trx1 (Table
2). Not surprisingly, we did not observe any reduction of either Trx substrate using
TrxR2, indicating that the Sec residue of TrxR2 is required for Trx reduction.
14
Activity of human TrxR isoenzymes using low molecular weight disulfide substrates
Determining the activity with the model substrate DTNB we found that TrxR1, at both
pH 7 and pH 8, had about 10-fold higher catalytic efficiency and about twice the kcat for
this substrate as compared to TrxR2 (Table 3). However, we also found that the
truncated variant, TrxR2missing the Sec residue still reduced DTNB almost as
efficiently as full-length TrxR2 (Table 3), in contrast to the two-amino acid truncated rat
TrxR1 that displayed only about 5-10% activity compared to the full-length enzyme [42,
47].
We also investigated if the human TrxR isoenzymes could reduce the small molecular
weight disulfide compounds lipoamide and lipoic acid, which are known substrates for
rat TrxR1 and mouse TrxR2 (25, 60). We found that the Km of human TrxR1 using
lipoamide was lower than the corresponding Km of TrxR2 and the catalytic efficiency
was 5-fold higher, showing that human TrxR1 reduces lipoamide more effectively than
TrxR2 (Table 3). TrxR2 reduced lipoamide with comparable efficiency to the fulllength TrxR2. Surprisingly, we found that although TrxR2 reduced lipoamide it could
not reduce lipoic acid (Table 3), in contrast to the rat TrxR1, which has been shown to
reduce both lipoic acid and lipoamide [54], similarly to the human TrxR1 (Table 3).
Activity of human TrxR isoenzymes with the quinone substrate juglone
We investigated if the human enzymes could reduce the quinone substrate juglone, that
was previously found to be efficiently reduced by both the selenolthiol motif and in a
Sec-independent redox cycling manner by rat TrxR1 [26, 55]. We found that human
TrxR1 could indeed reduce juglone and was significantly more efficient than TrxR2,
mainly due to a lower Km than that seen with TrxR2 (Table 4). Highly efficient redox
cycling with juglone has been shown before for the truncated TrxR1 [55, 56] and we
15
found that TrxR2 could also reduce juglone at a similar extent as full-length TrxR2
(Table 4).
Metal-based compounds have different inhibitory effects on human TrxR1 and TrxR2
Gold(I) compounds such as auranofin and aurothioglucose have been identified as
highly effective inhibitors of selenoprotein thioredoxin reductases in vitro, as well as in
cells in the case of auranofin [57-60]. Therefore we analyzed if these compounds could
inhibit human TrxR1 and TrxR2 to the same extent. Indeed, we found that
concentrations above 5 µM of either auranofin or aurothioglucose effectively inhibited
both of the full-length enzymes (Fig 3A and 3B). Furthermore, the DTNB reductase
activity of the TrxR2 enzyme was also inhibited by auoranofin and aurothioglucose at
these concentrations, indicating that the N-terminal redox center of this enzyme was also
inhibited (Fig 3A and 3B). Thus, auranofin and aurothioglucose are not selectively
inhibiting the Sec-containing active site motif of mammalian TrxRs. Notably, at lower
concentrations (ranging from 0.1 to 1 µM) auranofin and aurothioglucose were here
found to be more specific inhibitors for TrxR1 as compared to TrxR2.
Cisplatin has long been used as a chemotherapeutic agent, based on its ability to bind to
DNA and lead to cell death [61]. In addition, cisplatin has also been shown to effectively
inhibit rat and bovine TrxR1 (13, 65, 66) and when TrxR1 was knocked down in human
A549 cancer cells by siRNA these became more resistant to cisplatin [53]. Therefore,
we analysed the effects of cisplatin on the human recombinant TrxR enzymes and found
that the activities of both were inhibited at similar levels by cisplatin using
concentrations above 10 µM (Fig 3C). Inhibition of the TrxR2 DTNB reductase
activity using cisplatin was very limited, suggesting that the Sec-containing motif was
the prime target of cisplatin (Fig 3C).
16
Bis-chelated gold(I) phosphine compounds have been shown to exhibit significant
anticancer activity [62]. Recently we also found that pyridyl phosphine derivatives were
selectively toxic to tumorigenic cells but not non-tumorigenic cells and could also
inhibit in vitro activity of rat TrxR1 and total TrxR activity in cells [44, 46, 62, 63].
Therefore here we investigated if the observed reduction of TrxR activity in human cells
was likely to have been mainly due to inhibition of TrxR1, TrxR2, or both enzymes. We
found that [Au(d2pype)2]Cl, previously shown to have anticancer activity [64], indeed
was a potent inhibitor of both TrxR1 and TrxR2 (Fig 3D). Although the
[Au(d2pype)2]Cl complex was a more effective inhibitor of TrxR1, the inhibition of
TrxR2 was likely selective for the Sec-containing active site because the DTNB
reductase activity of hTrxR2 was unaffected at concentrations up to 50 µM (Fig 3D).
We also investigated the inhibitory effects of the related gold complex [Au(d2pypp)2]Cl
on the enzymes and found that it was a potent inhibitor of TrxR1 but, surprisingly,
above 5 µM concentrations this compound was a significantly less potent inhibitor of
TrxR2 (Fig 3E). The inhibition by this compound also seemed to be limited to the Sec
active site of TrxR2 because the activity of TrxR2 was not affected (Fig 3E).
Recently, we reported that a new series of gold(I) N-heterocyclic carbene compounds
exhibited
anticancer
activity
[45,
65]
and
the
gold
complex
bis(1,3-di-
isopropylimidazol-2-ylidene)gold(I) chloride ([(iPr2Im)2Au]Cl) was selectively reactive
towards Sec compared to Cys [45]. Consequently, we also observed that this compound
specifically inhibited TrxR activity in cells but not the activity of glutathione reductase
[45]. Here, surprisingly, we found that at high concentrations [(iPr2Im)2Au]Cl inhibited
TrxR2 but not TrxR1 or the truncated TrxR2 (Fig 3F). These findings collectively
showed that TrxR1 and TrxR2, although both likely inhibited by targeting of their Sec
residues, displayed markedly different rates of inhibition between the pyridyl phosphine
17
compounds [Au(d2pype)2]Cl and [Au(d2pypp)2]Cl, and the carbene compound
[(iPr2Im)2Au]Cl, suggesting that specific TrxR1- or TrxR2-targeting metal inhibitors can
be developed for potential specific clinical use.
Effects of metal-based TrxR inhibitors on cell viability in wild type and Bax/Bak-/- cells
There is evidence in the literature to suggest that the molecular mechanisms of antitumor
activity of different classes of linear and lipophilic gold(I) compounds may be different,
although these classes of compounds all seem to cause cell death via mitochondrial
pathways [64, 66]. Also cisplatin has been suggested to involve mitochondrial
dysfunction at the onset or progression of cell death [67], although additional cytosolic
or endoplasmic reticulum-related events of oxidative stress are also likely involved [68].
Since all of these metal-based compounds inhibit mammalian forms of TrxR, to
different extents (see above), we investigated if their mechanisms of action at the
cellular level will always depend upon Bax and Bak and thus likely involve opening of
the Bax/Bak pore in the mitochondrial outer membrane. To test this we used wild-type
and Bax/Bak-/- cell lines, where the latter are resistant to apoptotic stimuli involving the
formation of the Bax/Bak pore [52]. We treated these cell lines with increasing
concentrations of selected gold(I) compounds or cisplatin and measured cell death by
flow cytometry. This showed that at 2.5 µM concentration auranofin induced cell death
dependent on the Bax/Bak pore, however at higher concentrations auranofin did not
require the pore to induce cell death (Fig 4A). The [Au(d2pype)2]Cl and
[Au(d2pypp)2]Cl compounds induced cell death independent of Bax and Bak (Fig 4D
and E). Aurothioglucose was not toxic to either of the cell lines (Fig 4B), even after a 72
h incubation with up to 100 µM concentrations of aurothioglucose with the cell lines
(data not shown). Very high concentrations of up to 50 µM cisplatin after a 24 h
incubation were not toxic to the Bax/Bak-/- cells while the wild-type cells showed ~50%
18
cell death at about 15 µM, thus suggesting that cisplatin leads to a cell death that
requires Bax/Bak pore formation (Fig 4C). The Bax/Bak-/- cells also survived at
concentrations up to 20 µM of [(iPr2Im)2Au] Cl whereas this compound was highly
toxic to the wild type cells with approximately 90% cell death at 1 µM, suggesting that
the mechanism of cytotoxicity of [(iPr2Im)2Au] Cl also involved the Bax/Bak pore,
although very high concentrations could lead to cell death independent of Bax/Bak (Fig
4F).
Effects of metal-based inhibitors on TrxR activities in wild type and Bax/Bak-/- cells
We also investigated if the different metal compounds inhibited TrxR activity in cells.
Although aurothioglucose effectively inhibited the activity of the recombinant TrxR
enzymes (Figure 3), we observed limited inhibition of TrxR activity in both cell lines
(Fig 5A and 5B). The lack of cytotoxicity (Fig 4B) and low cellular TrxR inhibition (Fig
5A-D) even at high µM concentrations of aurothioglucose, suggests that poor cellular
uptake may be an explanation for its lack of effect in these cells. We found that both 5
µM and 50 µM concentrations of the other five metal compounds effectively inhibited
TrxR activity in both cell lines after 5 h (Fig 5A and 5B). The inhibition of TrxR activity
was greater after an 8 h incubation with the five compounds, particularly with 50 µM
concentration of the compounds (Fig 5C and 5D) indicating that the inhibition of TrxR
activity in cells is time and concentration dependent. We incubated FDM cells with 50
µM of the metal compounds and separated them into cytosolic and mitochondrial
fractions to investigate the effects of these compounds on the endogenous TrxR1 and
TrxR2 activity. We found similar levels of enzyme inhibition in both cell lines (Fig 6).
Auranofin was a significantly more effective inhibitor of both enzymes compared to
cisplatin whereas aurothioglucose had limited inhibitory effects most likely due to its
poor cellular uptake. The lipophilic gold compounds were more effective inhibitors of
19
TrxR2, most likely as a result of their accumulation inside mitochondria [45, 46, 65].
These findings collectively suggest that the inhibition of TrxRs may contribute to
cytotoxicity and, furthermore, that the observed cell death triggered by metal-based
compounds may involve different mitochondrial pathways not always dependent on the
presence of Bax and Bak.
Discussion
In this study we made a side-by-side comparison of substrate and inhibition specificities
of pure recombinant human TrxR2 and TrxR1 and found significant differences between
the two human enzymes that were previously not observed and are beyond those
determined solely by their different subcellular compartments. The divergent
sensitivities of TrxR1 and TrxR2 to different inhibitors should underpin inhibitorspecific differences in cytotoxicity profiles of metal-based anticancer drugs that target
cellular TrxR as part of their molecular mechanisms for toxicity.
Although we used established protocols for recombinant expression of TrxRs [48], this
is the first study comparing the two human isoenzymes and the first to express fulllength recombinant human TrxR2. Earlier attempts to express recombinant human
TrxR1 gave much lower yields than expressing rat TrxR1, which was explained by
higher frequency of rare codon usage in the human gene and a higher instability of the
human enzyme [69, 70], which we also noted. We were surprised for the near-complete
enrichment of the full-length enzymes following PAO Sepharose purification,
considering that earlier studies found rat TrxR1 to be purified as heterodimers of
truncated and full-length subunits still displaying a specific activity of 40 U/mg or more
[41]. That suggests that homodimeric full length rat TrxR1 would have significantly
higher specific activity than 40 U/mg [41], while we found that homogeneously full-
20
length human TrxR1 and TrxR2 had specific activities of about 40 U/mg in the DTNB
model assay, which is the commonly denoted inherent activity of mammalian TrxR [50].
Human TrxR2 had a clear preference for its native Trx2 substrate and its catalytic
efficiency was higher at pH 8, indicating that the mitochondrial localization of TrxR2,
where the pH is generally one unit higher compared to the cytosol, has tailored its
differences in affinity and activity for its natural substrate Trx2. Importantly, the
differences in catalytic efficiency between the mitochondrial and cytosolic TrxRs as
found here should have implications for their intracellular functions. It is clear that
TrxR1 can reduce the mitochondrial Trx2 equally well as its native Trx1, as observed
before for Trxs from other species [43], but the question remains whether TrxR1 would
ever have the possibility to reduce Trx2 in vivo considering the different subcellular
compartments of the two proteins.
Human TrxR1 clearly reduced DTNB and lipoamide more efficiently than TrxR2, while
the selenenylsulfide active center motif of TrxR2 was not essential for their reduction as
shown before for mouse TrxR2 [21], but in contrast to that study, we found that human
TrxR2 could not reduce lipoic acid. Although lipoic acid is a substrate for human TrxR1
we still found that its efficiency was two-fold lower compared to that for lipoamide.
Endogenous lipoic acid is a covalently bound dithiol cofactor for the α-keto acid
dehydrogenase enzyme complexes, such as pyruvate dehydrogenase and α-ketoglutarate
dehydrogenase [71] and is negatively charged compared to the neutral charge of
lipoamide. This charge difference should play an important role in substrate
discrimination for human TrxR2 and in its reduced efficiency of lipoic acid reduction, as
compared to the lipoamide reduction by human TrxR1. It may be thus that in humans
lipoic acid is not reduced by TrxR2 and that other enzymes such as lipoamide
dehydrogenase and glutathione reductase catalyze this reaction [72].
21
We found that human TrxR1 had a three-fold higher affinity for the quinone substrate
juglone compared to TrxR2 and the TrxR2 suggesting that the N-terminal motif alone
can reduce juglone. Juglone can be rapidly reduced by the selenenylsulfide/ selenolthiol
active center of rat TrxR1, which can result in the formation of a nucleophilic arylating
juglone derivative that can target selenocysteine and irreversibly inactivate this motif.
Nevertheless, juglone may continue to efficiently redox cycle directly with the Nterminal CVNVGC/FAD motif of rat TrxR1 and thus show high activity [13, 26, 73].
Our kinetic data indicate that the affinity of TrxR2 for disulfide substrates other than its
endogenous Trx2 substrate is significantly lower than the affinity of TrxR1 for these
types of substrates. Because of its mitochondrial localization it may obviously still be
that TrxR2 is more efficient at using endogenous mitochondria localized substrates such
as Trx2, Grx2 and cytochrome c [37, 38] and there may also be other mitochondria
specific substrates for which TrxR2 has high affinity yet to be discovered. Our results
suggest that the functions of the mitochondrial TrxR2 and cytosolic TrxR1 are less
overlapping than generally believed, also when considering their native biochemical and
kinetic properties. The previously reported crystal structure of mitochondrial TrxR2
indeed indicates that there are distinct differences in the positioning of residues around
the redox active centers of TrxR2 as compared to TrxR1, which may contribute to or be
responsible for the divergent reduction of different substrates as well as their affinities
[16]. From our data we conclude that, (i) TrxR1 has a broader substrate specificity
compared to TrxR2, (ii) TrxR2 has greater affinity for its endogenous substrates that are
different to those for TrxR1, which may reflect other functional requirements within the
mitochondrial compartment, and (iii) hTrxR2 is catalytically more efficient at the higher
endogenous pH of the mitochondrial matrix.
22
Several emerging anticancer therapies identify TrxR as a target for drug development
[74, 75] as altered activities of the Trx system proteins have been observed in several
human diseases [18, 76]. Many therapeutically used compounds have been identified as
TrxR inhibitors [18, 26, 73, 76-78], including gold(I) compounds that have also shown
early promise as anticancer drugs [45, 46, 60, 74, 77, 79, 80]. Auranofin is probably the
most effective inhibitor of mammalian TrxR found to date [60, 81] and here we show
that at micromolar concentrations it is as an effective inhibitor of both the N-terminal
dithiol and C-terminal selenolthiol redox centres of human TrxRs. This indicates that
auranofin has high affinity not only for selenocysteines but thiolates as well, suggesting
that auranofin has a limited specificity for selenocysteines. The finding that the related
compound, aurothioglucose also inhibited all three enzymes was in contrast to a recent
finding that it is a better inhibitor of the N-terminal redox center compared to the Cterminal selenenylsulfide/selenolthiol active center of the mouse TrxR2 [21]. These
findings may reflect differences between the human and mouse TrxR2 enzymes.
Interestingly, at nanomolar concentrations auranofin and aurothioglucose were more
specific inhibitors of TrxR1 compared to TrxR2 and less effective at inhibiting TrxR2,
suggesting that the selenolthiol motif of TrxR is inhibited in preference to the dithiol
motif. Also, the active site microenvironment around the C-terminal selenolthiol motif
of the reduced enzyme must finetune its susceptibility to different inhibitors, as
illustrated by the differences in inhibition between TrxR1 and TrxR2. Thus, the
tetrapeptide C-terminal active site of TrxR should not only be regarded as an easily
accessible Sec-presenting motif targeted indiscriminately by electrophiles, which is a
rather simplified view often found in the literature. Cisplatin inhibited the activity of
TrxR1 and TrxR2, but not TrxR2, suggesting that its mechanism of inhibition likely
involves coordination of platinum to the Sec-containing redox center. This was
23
suggested to generate selenium compromised thioredoxin reductase-derived apoptotic
proteins (SecTRAPs) that may induce cell death by a gain of function [56]. The bischelated Au(I) pyridyl phosphine compounds were inspired by auranofin, but designed
to lower their thiol reactivities while improving the selectivity for Sec [62, 63].
Consequently we found that the bis-chelated gold(I) compounds [Au(d2pype)2]Cl and
[Au(d2pypp)2]Cl were effective inhibitors of the selenenylsulfide/ selenolthiol active
center of the TrxRs, thus potentially also yielding cytotoxic SecTRAP proteins.
However, such proteins have not been shown to form from TrxR2 yet.
The cationic gold(I) compounds could inhibit both TrxR1 and TrxR2 in vitro, however
because they are lipophilic and are consequently accumulated inside mitochondria as a
result of the high mitochondrial membrane potential (m), we observed their specific
inhibition of TrxR2 in cells and conclude that previously observed cellular TrxR
inhibition [46] also likely involved inhibition of TrxR2. Surprisingly, the
[Au(d2pype)2]Cl and [Au(d2pypp)2]Cl compounds were significantly better inhibitors
for TrxR1 in vitro whereas [(iPr2Im)2Au]Cl inhibited only TrxR2. This indicates not
only that there is a difference in the inhibition mechanism of these compounds, but also
that the active centres of the cytosolic and mitochondrial TrxR isoenzymes may have
different affinities for these compounds. We are currently attempting to investigate the
mechanism of binding of these gold(I) compounds by co-crystallizing them with the
purified TrxRs.
At low concentration auranofin led to Bax/Bak dependent cell death likely by
preferentially reacting with selenoproteins such as TrxR2 whose inhibition by auranofin
has been shown to lead to peroxiredoxin-3 oxidation and Bax/Bax dependent apoptosis
[57]. High concentrations of auranofin caused cell death independent of Bax/Bak, most
likely by binding both thiols and selenols, causing changes in the mitochondrial thiol
24
redox pool that have been shown to cause apoptosis by inducing mitochondrial
permeability transition (MPT) [60, 81]. Therefore the mechanism of action of auranofin
via the Bax/Bak pore is concentration dependent and may vary between cell types.
Nevertheless, the high thiol reactivity of auranofin is likely to limit its anticancer activity
in vivo as its toxicity to cultured cancer cells was reduced 10-fold in the presence of
serum proteins, where the loss of activity was attributed to binding to extracellular
protein thiols [82]. Although aurothioglucose was an effective inhibitor of recombinant
TrxRs, its inhibition of cellular TrxRs was limited and it was not toxic to the two cell
lines, most likely because polymeric gold(I) thiolates do not readily enter cells and
consequently have very low cytotoxicity and lack anticancer activity [83]. In contrast,
cell death induced by cisplatin was found to be entirely dependent of the presence of
Bax and Bak, suggesting that TrxR inhibition and/or DNA binding could lead to
Bax/Bak dependent cell death. Although the exact mechanism of cisplatin-induced cell
death needs to be investigated further, the combined ability of cisplatin to form DNA
lesions and inhibit TrxR activity make it an effective chemotherapeutic agent, and may
contribute to its success as a clinically used anticancer drug.
The lipophilic cations [Au(d2pypp)2]Cl and in particular [Au(d2pype)2]Cl were more
TrxR1-specific inhibitors and caused cell death that was independent of the Bax and Bak
proteins. We have previously shown that these compounds cause cell death via
mitochondria [46] and this may be a result of their high lipophilicity, that can cause
generalized permeabilization of the mitochondrial membrane or induce the MPT that
dissipates the m. In addition, a small amount of these compounds may remain in the
cytoplasm, as we observed some inhibition of TrxR1 activity that may further contribute
to the onset of cell death independent of Bax and Bak. In contrast to these compounds,
the [(iPr2Im)2Au]Cl compound led to Bax/Bak dependent cell death at lower
25
concentrations. We have shown previously that [(iPr2Im)2Au]Cl is a moderately
lipophilic cation that selectively accumulates in the mitochondrial matrix driven by the
m [45] and here we showed that it is a TrxR2-specific inhibitor. We suggest that the
mechanism of action of this compound involves mitochondrial uptake followed by
specific inhibition of the TrxR2 that leads to apoptosis via mitochondria which requires
the formation of the Bax/Bak pore. However, at higher concentrations this compound
can cause cell death independent of the Bax/Bak pore, much like [Au(d2pype)2]Cl and
[Au(d2pypp)2]Cl, most likely as a result of its lipophilicity. An alternative explanation
may be that the compounds are irreversibly modifying the Sec active site and thereby
forming SecTRAPs that induce cell death. Cell death induced by different SecTRAPs
may be caused by different mechanisms that may not always require the Bax and Bak
proteins. The molecular mechanisms by which the inhibition of TrxRs leads to cell death
and whether it requires Bax/Bak pore formation must evidently be specifically studied
and resolved for every individual TrxR-targeting drug and for each specific cell type.
In conclusion, we have shown that the human cytosolic TrxR1 and mitochondrial TrxR2
have different affinities for low molecular-weight disulfide substrates and metal-based
inhibitors. In combination with cell death assays in Bax/Bak double knockout cells our
data enable models to be proposed for the mechanisms of action of these compounds
that take into account the physiologically relevant cellular locations of TrxR1 and
TrxR2, their compound-specific targeting profiles and the patterns of inhibition
determined by different kinetic properties of TrxR1 and TrxR2.
Acknowledgements
The work was supported by The Australian Research Council (Future Fellowships
FT0991008 to AF, FT0991113 to OR and Discovery Grant DP0986318), Karolinska
26
Institutet, The Swedish Research Council (Medicine) and The Swedish Cancer Society.
Mass spectrometry analyses were performed in facilities provided by the Lotterywest
State Biomedical Facility-Proteomics node, WAIMR. We thank Associate Professor
Paul Ekert for kindly giving us the FDM cells. The gold pyridyl phosphine compounds
were synthesized by Coby Sutcliffe and Anthony Humphreys in Prof Sue BernersPrice’s research group and the carbene compound was synthesized by James Hickey in
A/Prof Murray Baker’s and Prof Sue Berners-Price’s groups. The synthesis of these
compounds was published previously and we thank them for providing them for this
study.
Abbreviations
The abbreviations used are: DTNB, dithionitrobenzoic acid; DTT, dithiothreitol;
MALDI-TOF,
matrix-assisted
laser
desorption/ionization
time-of-flight,
TrxR,
thioredoxin reductase; TrxR1, cytosolic thioredoxin reductase; TrxR2, mitochondrial
thioredoxin reductase; Trx1, cytosolic thioredoxin; Trx2, mitochondrial thioredoxin;
PAO, phenylarsine oxide; auranofin, 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranosatoS-triethylphosphine
gold(I);
[Au(d2pype)2]Cl,
Bis[1,2-
bis(dipyridylphosphino)ethane]gold(I) chloride; [Au(d2pypp)2]Cl, Bis[1,3-bis(di-2pyridylphosphino)propane]gold(I)
chloride;
[(iPr2Im)2Au]Cl,
bis(1,3-di-
isopropylimidazol-2-ylidene)gold(I) chloride.
27
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33
Figure legends
Table 1. Thioredoxin reductase activities and protein amounts after 2',5' ADP Sepharose
and PAO Sepharose purifications.
a
The activity from two independent preparations of TrxR1 and TrxR2 are represented as
(1) and (2)
b
TrxR2 was only purified on 2',5' ADP Sepharose and as it is missing the Sec residue
its purification on PAO Sepharose was not applicable (N/A).
Fig 1. Mass spectrometry analysis of human thioredoxin reductases. Samples from PAO
purified hTrxR1 (A) and hTrxR2 (B) and 2',5' ADP Sepharose purified hTrxR2 (C)
were prepared and analysed by mass spectrometry as described in the methods section.
Deconvoluted zero-charged ions corresponding to the average mass are shown. Peak of
54769 Da for the full-length hTrxR1, a peak of 53117 Da for the full-length hTrxR2 and
a peak of 52907 for the truncated hTrxR2 correlate with their respective theoretical
masses.
Fig 2. Analysis of human thioredoxin reductases by native and denaturing
polyacrylamide gel electrophoresis. Recombinant full-length rTrxR1, hTrxR1, hTrxR2
and truncated hTrxR2 were resolved on 4-16% native gels (A) or on denaturing 10%
SDS Tris-glycine gels (B) in the presence or absence of 100 mM DTT.
Table 2. Kinetic analysis of human thioredoxin reductases using human thioredoxin 1
and thioredoxin 2 as substrates.
a
The activity of TrxR2 using Trx1 and Trx2 as substrates was not detected (ND).
34
Table 3. Kinetic analysis of human thioredoxin reductases using small molecular weight
disulfide compounds, DTNB, lipoamide and lipoic acid as substrates.
a
The activity of TrxR2 and TrxR2 using lipoic acid as substrate was not detected
(ND).
Table 4. Kinetic analysis of human thioredoxin reductases using the quinone substrate
juglone as a substrate.
Fig 3. Inhibition of hTrxR1, hTrxR2 and hTrxR2 activities using five different metal
compounds. In vitro inhibition of hTrxR activities with increasing concentrations of the
metal compounds were measured by the DTNB assay. Recombinant TrxRs (100 nM)
were reduced with 150 µM NADPH in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH
8.0). Varying concentrations of the metal compounds auranofin (A), aurothioglucose
(B), cisplatin (C), [Au(d2pype)2]Cl (D), [(iPr2Im)2Au]Cl (E) and [Au(d2pypp)2]Cl (F)
were mixed with the reduced TrxRs and incubated for 30 min at room temperature. The
NADPH-dependent TrxR catalyzed reduction of DTNB was monitored immediately
after the addition of TE buffer containing 150 µM NADPH and 2.5 mM DTNB at 30˚C
for 3 min, and determined as the linear increase in absorbance at 412 nm. Data are
expressed as percent of control TrxR activity that was not incubated with metal
compounds mean ± SEM from 3 independent experiments. *, p < 0.05 of TrxR2
compared to TrxR1 and †, p < 0.05 of TrxR2 compared to TrxR2, by a 2-tailed
Student’s t test.
Fig 4. Effects of metal compounds on factor dependent myeloid (FDM) wild type and
Bax/Bak-/- cells. FDM wild type and Bax/Bak-/- cell lines were grown to 90% confluence
and incubated for 24 h in their growth medium containing increasing concentrations of
auranofin (A), aurothioglucose (B), cisplatin (C), [Au(d2pype)2]Cl (D), [(iPr2Im)2Au]Cl
35
(E) and [Au(d2pypp)2]Cl (F). To test for toxicity, cells were collected, gently
resuspended in 0.5 ml binding buffer containing 10 µl propidium iodide (30 µg/ml) and
cell death was quantitated by flow cytometry. Data are expressed as mean ± SEM from 3
independent experiments. *, p < 0.05 of FDM Bax/Bak-/- cells compared to wild type
cells by a 2-tailed Student’s t test.
Fig 5. Inhibition of total thioredoxin reductase activity in cells. Factor dependent
myeloid (FDM) wild type and Bax/Bak-/- cells were incubated with the metal
compounds auranofin, aurothioglucose, cisplatin, [Au(d2pype)2]Cl, [(iPr2Im)2Au]Cl and
[Au(d2pypp)2]Cl at 5 µM (A) or 50 µM (B) concentrations for 5 h and with 5 µM (C)
or 50 µM (D) concentrations for 8 h. The cells were collected, suspended in 100 µl cell
extraction buffer (50 mM Tris, pH 7.6, 2 mM EDTA, 0.5 mM phenyl methyl sulfonyl
fluoride and 0.5% Igepal Ca-630) and lysed by three cycles of rapid freezing and
thawing. Total cellular TrxR activity was measured by the end-point Trx-dependent
insulin reduction method using 5 µg of the cell lysate that was clarified by centrifugation
(16,000 g for 5 min at 4˚C). Data are expressed as mean ± SEM from 3 independent
experiments.
Fig 6. Inhibition of TrxR1 and TrxR2 activity in cells. Factor dependent myeloid (FDM)
wild type (A) and Bax/Bak-/- (B) cells were incubated for 8 h in their growth medium
with 50 µM concentration of the metal compounds auranofin, aurothioglucose, cisplatin,
[Au(d2pype)2]Cl, [(iPr2Im)2Au]Cl and [Au(d2pypp)2]Cl. The cells were separated into
mitochondrial and cytosolic fractions and TrxR1 or TrxR2 activity was measured by the
end-point Trx-dependent insulin reduction method using 1 µg of lysate that was clarified
by centrifugation (16,000 g for 5 min at 4˚C). Data are expressed as mean ± SEM from 2
independent experiments. *, p < 0.05 of TrxR2 compared to TrxR1 by a 2-tailed
Student’s t test.
36