Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol glutaredoxin dependent cellular pathways

Article abstract of DOI:10.1016/j.biochi.2014.07.027

In Trypanosoma cruzi, the modification of thiols by glutathionylation-deglutathionylation and its potential relation to protective, regulatory or signaling functions have been scarcely explored. Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating activity, having potential functionality to control intracellular homeostasis of protein and non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the contribution of the protein to increase the general resistance against oxidative damage and intracellular replication of the amastigote stage. Also, studies performed with epimastigotes overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests novel insights about the protein involvement in redox signaling.

Full text of DOI:10.1016/j.biochi.2014.07.027

Accepted Manuscript
Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol
glutaredoxin dependent cellular pathways
Vanina E. Márquez, Diego G. Arias, Maria L. Chiribao, Paula Faral, Carlos Robello,
Alberto A. Iglesias, Sergio A. Guerrero
PII:
S0300-9084(14)00218-1
10.1016/j.biochi.2014.07.027
BIOCHI 4515
DOI:
Reference:
To appear in: Biochimie
Received Date: 11 March 2014
Accepted Date: 29 July 2014
Please cite this article as: V.E. Márquez, D.G. Arias, M.L. Chiribao, P. Faral, C. Robello, A.A. Iglesias,
S.A. Guerrero, Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol
glutaredoxin dependent cellular pathways, Biochimie (2014), doi: 10.1016/j.biochi.2014.07.027.
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ACCEPTED MANUSCRIPT
Original research article
Redox metabolism in Trypanosoma cruzi. Biochemical characterization of dithiol glutaredoxin
dependent cellular pathways
1
1
2,3
3
2,3
Vanina E. Márquez *, Diego G. Arias *, Maria L. Chiribao , Paula Faral , Carlos Robello ,
1
1
Alberto A. Iglesias , Sergio A. Guerrero
1
Instituto de Agrobiotecnología del Litoral, Facultad de Bioquímica y Ciencias Biológicas,
CONICET-UNL, Santa Fe, Argentina.
2
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo,
Uruguay.
3
Unidad de Biología Molecular, Institut Pasteur, Montevideo, Uruguay.
*These authors contributed equally to this work.
Corresponding Author:
Sergio A. Guerrero, Laboratorio de Bioquímica Microbiana, Instituto de Agrobiotecnología del
Litoral, Facultad de Bioquímica y Ciencias Biológicas, CONICET-Universidad Nacional del
Litoral. Ciudad Universitaria - Paraje “El Pozo”, S3000ZAA Santa Fe, Argentina. Tel: 54 342 457
5
209 ext. 131. Email: sguerrer@fbcb.unl.edu.ar
Keywords: trypanosoma; redox metabolism; glutaredoxin; thioltransferase; deglutathionylation
1

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Abstract
In Trypanosoma cruzi, the modification of thiols by glutathionylation-deglutathionylation and its
potential relation to protective, regulatory or signaling functions have been scarcely explored.
Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating
activity, having potential functionality to control intracellular homeostasis of protein and
non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol
concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending
on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein
disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to
protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the
contribution of the protein to increase the general resistance against oxidative damage and
intracellular replication of the amastigote stage. Also, studies performed with epimastigotes
overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway
connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented
about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling
pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests
novel insights about the protein involvement in redox signaling.
2

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1
. Introduction
During the life cycle, Trypanosoma cruzi is exposed to both reactive oxygen (ROS) and nitrogen
RNS) species. These dangerous species are generated endogenously as by-products of the parasite
(
aerobic metabolism, a phenomena reported for many other organisms, or exogenously generated
from the host immune system [1]. Both ROS and RNS can affect the cellular redox balance,
consequently altering other major cell functions. In biological systems, thiols have an essential
function in scavenging ROS and RNS either spontaneously or catalytically. Thiol redox state is a
mediator in metabolic, transcriptional and signaling cell processes, supporting cellular functions
under basal physiological and/or oxidative (or nitrosative) stress conditions [2]. Glutathionylation is
one of the most common thiol modification mechanisms inside the cell, which mainly operates
during oxidative stress but also physiologically by mediating redox signals exerted by ROS [2, 3].
Glutaredoxin (Grx), an oxidoreductase with deglutathionylating activity, has a relevant role in
intracellular homeostasis of protein thiols. Grx has a strong preference for glutathione mixed
disulfides [4] and its cellular functionality has been mainly related to regulation of enzyme activity
by glutathionylation-deglutathionylation [2, 4, 5].
The cellular redox status can influence cell viability through regulation of cellular proliferation,
growth arrest or programmed cell death (PCD) [6]. Depending on diverse factors (type of organism,
tissue or apoptotic stimulus), Grxs can act linked to pro-apoptotic or anti-apoptotic pathways [5].
PCD was previously described in protists [7], including T. cruzi [8, 9]. Although the biological
significance of PCD in protozoan is still unclear, it was postulated that apoptosis-like cell death can
function as a way for improving infectivity [8, 10]. In trypanosomatids, most of the molecular
effectors that would be involved in the sequence of apoptosis-like events were not elucidated. In
these microorganisms, different cellular actions were found related to the apoptosis-like cell death
induced by human serum complement. Such cell processes include: i) decrease of T(SH) and GSH
2
levels [8], ii) ROS production [8], iii) release of cytochrome c from mitochondria to cytosol [8] and
iv) accumulation of calcium into mitochondria [8, 11].
In trypanosomatids, trypanothione [T(SH) ] dependent mechanism involved in redox homeostasis
2
and ROS/RNS detoxification has been relatively well characterized [12, 13]. Conversely, even
when these parasites contain free glutathione, the modification of thiols by glutathionylation-
deglutathionylation and its potential relation to protective, regulatory or signaling functions have
been poorly explored. In fact, glutathionylation was exclusively studied as a mechanism for
protecting several enzymes of the antioxidant network in T. brucei [14]. A dithiolic glutaredoxin in
3

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T. cruzi (TcrGrx) was previously characterized by our group [15], we reported on the identification
of two genes encoding dithiolic Grx in the T. cruzi genome (Tc00.1047053506475.116 and
Tc00.1047053511431.40) having a high identity between them and coding for two identical
proteins (TcrGrx). At the moment, this is the unique dithiol Grx reported in T. cruzi. Afterwards,
Ceylan and co-workers [16] advanced in the study of two glutaredoxins in African trypanosomes,
one of them (namely Grx2) sharing a high identity percentage (higher than 80%) with the dithiolic
Grx previously reported in T. cruzi [15]. However, the characterization of Grx in trypanosomatids,
concerning biochemistry, kinetics and cellular functionality is far from complete. In this work we
report on the many faceted biochemical properties of pure recombinant TcrGrx, showing its
relevance in maintaining intracellular physiological concentrations of GSH, and other low
molecular mass thiols. Additionally, we present information about the relevance of TcrGrx activity
(
glutathionylation-deglutathionylation) on the parasite vital processes.
2
2
. Experimental procedures
.1. Reagents
Trypanothione disulfide and glutathionyl-spermidine disulfide were acquired from Bachem
Torrance, CA, USA). DIEGSSG was from Cayman Chemical (Michigan, USA). All other reagents
(
and chemicals were of the highest quality commercially available from Sigma-Aldrich (St. Louis,
MO, USA) or similar.
2
.2. Expression and purification of TcrGrx and mutants TcrGrxC31S and TcrGrxC34S
TcrGrx was obtained as previously described [15]. Mutants of TcrGrx in active site cysteine
residues were obtained by site-directed mutagenesis on tcrgrx gene cloned in pRSET-A by PCR-
TM
driven using specifically designed primers, following Quick-Change
mutagenesis protocol.
Expression and purification of TcrGrxC31S and TcrGrxC34S was performed in the same way as
TcrGrx [15]. Purity of the recombinant protein was analyzed by SDS-PAGE [17]. Protein content
was determined by the method of Bradford [18].
2
.3. Gel filtration chromatography
The estimation of the molecular size of native TcrGrx was performed in a Superdex 200 (GE-
Healthcare) column equilibrated with 25 mM Tris-HCl pH 7.5, 1 mM EDTA and 100 mM NaCl.
The purified protein was analyzed chromatographically together with different molecular mass
4

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standards: bovine serum albumin (66 kDa), ovoalbumin (43 kDa), carbonic anhydrase (29 kDa) and
lysozyme (14 kDa).
2
.4. Activity assays
Thioltransferase activities were measured spectrophotometrically by following consumption of
NADPH at 340 nm and 30 °C, by means of coupled assays that guarantee the regeneration of
reduced TcrGrx, using a Multiskan Ascent one-channel vertical light path filter photometer
(
Thermo Electron Co.). The standard reaction medium (50
ꢀ
L final volume) contained
1
00 mM Tris-HCl pH 7.5, 2 mM EDTA and 300 µM NADPH, and the different coupled reactions
2
were analyzed after the specific additions. T(SH) -dependent reduction of GSSG or CySS or
S-nitrosothiols by TcrGrx was performed in a reaction mixture containing 100 µM TS2,
µM TcrTR, 15-1000 µM GSSG or CySS or GSNO or CySNO and different concentrations
0.5-5 µM) of TcrGrx. GSH-dependent reduction of TS , (Gsp) or HEDS by TcrGrx was
1
(
2
2
performed at different concentrations (0.25-5 ꢀM) of TcrGrx and 3mM GSH, as previously reported
[
15, 19]. The ability of TcrGrx for transferring electrons to TcrcTXNPx, TcrmTXNPx and
TcrGPxI, was determined by measuring the reduction of t-bOOH [20]. The capacity of TcrGrx for
transferring electrons to TcrMSRAs was determined by measuring the L-Met(S)SO reduction [21].
GSH-dependent reduction of dehydroascorbate (DHA) by TcrGrx was performed as previously
reported [22].
Reduction of the two interchain disulfide bonds of insulin catalyzed by recombinant TcrGrx was
analyzed in an assay adapted from the previously described by Holmgren [23]. The ability of
TcrGrx to participate in redox regulation of enzymatic activities was assayed using
TcrUDP-GlcPPase as a target, assay for UDP-GlcPPase activity was performed according to
Fusari et al [24]. Briefly, the formation of PPi from UTP and Glc-1P was monitored at 37 ºC in a
reaction mixture containing 100 mM MOPS pH 8.0, 1mM MgCl , 1 mM UTP, 1 mM Glc-1P and
2
1
U pyrophosphatase. Pure recombinant TcrUDP-GlcPPase was reduced in presence of 1 mM DTT
for 30 min at 30 °C, the excess of reducer agent was removed by desalting in an ultrafiltration
device (Microcon, Millipore). Pre-reduced TcrUDP-GlcPPase was inactivated by oxidative
treatment with 2 mM diamide or 10 mM GSSG in standard reaction medium. The excess was
removed as mentioned previously. Oxidized TcrUDP-GlcPPase was then incubated under the same
conditions with 10 mM GSH and variable concentrations of TcrGrx. At different times, aliquots
were withdrawn and assayed for UDP-GlcPPase activity.
5

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All kinetic parameters are the mean of at least three independent sets of data, which were
reproduced within ±10 %.
2
.5. Glutathionylation of TcrUDP-GlcPPase with dieosinediglutathione (DIEGSSG)
Glutathionylation of TcrUDP-GlcPPase was evaluated using DIEGSSG, following a protocol
adapted from previously reported [25]. TcrUDP-GlcPPase (11 M) was incubated with DIEGSSG
33 M) in 0.1 M phosphate buffer pH 7 for 30 min at 37 °C. The excess was removed by
ꢀ
(
ꢀ
ultrafiltration in spin tubes, and diafiltered against 0.1 M phosphate buffer pH 7.
Deglutathionylation was performed in a reaction containing 0.5 mM DTT, or 0.1 mM GSH,
-1
4 UꢁmL glutathione reductase, 1 mM NADPH, with or without 10 ꢀM TcrGrx depending on the
experiment. Proteins in the reaction mixtures were subjected to non-reducing SDS-PAGE. Before
staining with Coomassie blue the gel was scanned in Typhoon 9400 (GE Healthcare). The
DIEGSSG has low fluorescence, having the eosin-GSH molecules higher fluorescence intensity.
Thus, glutathionylated proteins are labeled with the fluorescent signal corresponding to eosin
isothiocyanate (
λexc = 518 nm; λem = 545 nm).
2
.6. Glutathione-Grx redox equilibrium
Redox equilibrium assays were carried out incubating the oxidized protein (10 to 40 µM) for 4 h at
0 ºC in a reaction mixture containing 100 mM Tris-HCl pH 7.5, 2 mM EDTA and 1 mM GSH.
3
After incubation, 5 % (w/v) TCA was added to separate proteins from the reaction mixture. In an
aliquot of the reaction mixture the remaining GSH concentration was estimated using DTNB
reagent [26]. The GSSG generated was determined by a kinetic method as previously described
[
22].
2
.7. Measurement of thiol pKa by UV absorption
The pH-dependent nucleophilic cysteine ionization was followed by the absorption of the thiolate
anion at 240 nm [27, 28]. The proteins were analyzed at 25 °C in a pH range from 2 to 11. Spectra
of 1-5 ꢀM TcrGrx or TcrGrxC34S oxidized or reduced (with 10 mM DTT for 10 min at room
temperature), were recorded between 200 and 340 nm. The spectra were measured against buffer
solution in a stoppered quartz cuvette in a Boeco S-22 UV-Vis spectrophotometer and the
absorbance was converted into molar extinction coefficient. For a single thiolate group a value
–
1
–1
between 4 and 6 mM cm was used, according to previous reports [22, 29].
2
.8. Determination of thiol pKa of nucleophilic cysteine by DTNB reduction assay
6

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To determine the pKa value of cysteinyl thiol groups the reaction rate as a function of pH was
measured. We used the mutant in the Cys34, according to a previous reported strategy [30]. The
rate of DTNB reduction by TcrGrxC34S was determined by incubation of reduced protein (20 µM)
with 500 µM DTNB in 50 µl of the reaction media at different pH values, at 25 °C. The rates of
DTNB reduction were measured by monitoring the absorbance at 405 nm. The apparent second
order rate constants were calculated and plotted against pH.
2
.9. T. cruzi cultures, pTEX constructs and transfection procedures
T. cruzi Dm28c strain was cultured in LIT medium supplemented with 10 % foetal bovine serum at
8 °C. Plasmid constructs for T. cruzi transfections were based on pTEX vector [31], using
2
restriction sites in the multicloning site (EcoRI or BamHI and XhoI). T cruzi epimastigotes were
transformed with the constructs pTEX (empty vector), pTEX/GFP, pTEX/TcrGrx, pTEX/RFP-
TcrGrx and pTEX/TcrGrx-RFP. Transfection procedures were performed as described by Piñeyro
and co-workers [32].
2
.10. Peroxide sensitivity and induction of apoptotic - like cell death
Suspensions of logarithmic-phase T. cruzi (transfected stably with pTEX, pTEX/GFP or
6
-1
-1
pTEX/TcrGrx) epimastigotes at 2x10 mL were prepared in fresh LIT (containing 2 g
ꢁL glucose)
+
10 % SFB medium and transferred in aliquots of 1 mL to 48 well plates. Glucose oxidase was
added to different wells to obtain 0-0.1 mM final concentrations. Samples were taken at different
times of incubation (15, 45, 180 and 240 min) under oxidative condition to evaluate epimastigote
viability [33]. Experimental data refer to the analysis of three independent experiments, which were
reproduced within ±10 %.
For apoptotic-like cell death induction assay, logarithmic-phase epimastigotes were collected by
centrifugation and resuspended in PBS (control condition) or PBS 10-15 % human fresh serum, at a
7
-1
density of 1x10 parasites
separated at different times for control and apoptotic-like phenotype quantification and TUNEL
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay using ApopTag®
ꢁmL . Aliquots of 1mL were dispensed in 48 wells plate. Samples were
(
Plus Fluorescein In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA), following
manufacturer instructions.
For caspase-3-like activity quantification, control or HFS treated epimastigotes suspensions
2
(
10 mL) were prepared in the same way, incubated in 25 cm T culture flask, for a period of 6 h.
7

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Caspase-like activity in extracts containing 50
ꢀ
g protein were measured using Caspase 3 assay kit,
colorimetric (Sigma, St. Louis, MO, USA), following the instructions of the manufacturer.
2
.11. Cell infection and invasion assay
Infection of HeLa cells [34] was performed following a protocol adapted from [35]. Briefly, HeLa
5
cells (2.5x10 cells/well) were cultured in 12 well plates, on DMEM 10 % SFB in 5 % CO
2
atmosphere chamber at 37 °C. Semiconfluent cells were infected with T. cruzi trypomastigotes
pTEX or pTEX/TcrGrx) at a ratio 5:1 (trypomastigote:cell). Non internalized trypomastigotes were
removed 2 h later by washes with PBS, and fresh DMEM was added. The plates were incubated in
% CO atmosphere chamber at 37 °C. At 0 and 48 h post-infection, cultures were sampled and
(
5
2
subjected to nuclear staining with DAPI. Cells and intracellular parasite counts were performed
from epifluorecence microphotographs of several microscopy fields with CellNote application
(
http://cellnote.up.pt/). Experimental data are the mean of three independent experiments.
2
.12. Western blotting
7
Protein extracts for western blotting were prepared resuspending 1x10 washed parasites directly in
SDS-PAGE sample buffer, boiling 10 minutes. Protein in SDS-PAGE gels were blotted onto
nitrocellulose membrane. The membrane was blocked with 5 % skimmed milk in PBS,
subsequently incubated with primary antibody at 4 °C overnight, and then incubated with
horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h. Detection was carried out
using Supersignal detection kit (Pierce, Rockford, IL, USA ).
2
.13. Localization studies
Digitonin differential membrane permeabilization protocol was performed as described in [36]. A
8
pellet containing 10 parasites was treated successively with increasing concentrations of digitonin
-
1
(
0-4 mgꢁmL ) in extraction buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA and 300 mM
saccharose pH 7.5). Protein fractions in the supernatants were subjected to SDS-PAGE and western
blot analysis. Polyclonal sera raised against TcrcTXNPx, TcrmTXNPx, TcrAPX, TcrGlcK, TbcytC
were used as cytoplasmic, mitochondrial matrix, endoplasmic reticulum, glycosomal and
mitochondrial intermembrane space markers, respectively.
Indirect inmunofluorescence localization approach was adapted from Wilkinson and co-wokers
(
2008) [37]. Epimastigotes stably transformed with pTEX/RFP-TcrGrx and pTEX/TcrGrx-RFP
were settled in silanized slides and fixed with 4 % (w/v) formaldehyde, and then permeabilized 10
8

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min with 0.2 % (v/v) Triton-X100 at room temperature. Permeabilised parasites were blocked with
2
% (w/v) BSA and washed with PBS 1X. Afterwards, slides were incubated for 1 h at room
temperature with the specific localization markers: anti-TcrmTXNPx for mitochondria, anti-
TcrcTXNPx for cytoplasm, anti-TbrcytC for inner-mitochondrial membrane space or anti-TbrBip
for endoplasmic reticulum. After washes, the slides were incubated with goat anti-rabbit DyLight
4
88 (Pierce, Rockford, IL, USA) secondary antibodies. Finally, after washes slides were mounted
with antifade mounting solution plus DAPI, and visualized under a confocal microscope.
3
3
. Results
.1. Monomeric TcrGrx disulfide is reduced by glutathione, glutathionylspermidine and
trypanothione
We have reported recently [15] the identification of two genes encoding two identical proteins,
having identical amino acid sequence (TcrGrx). We have described a procedure to recombinantly
produce TcrGrx at high purity degree, based on one of the mentioned sequences. The functional
protein was found to have a monomeric structure, as it eluted in a single peak corresponding to a
molecular mass of ~16 kDa from a Superdex 200 column, which agrees with the expected size for
the recombinant protein expressed as a fusion to a histidine-tag. On the other hand, the
electrophoretic profiles of TcrGrx treated with different oxidants or reducing agents in non-reducing
SDS-PAGE showed a similar mobility, thus indicating that TcrGrx would not form covalent
oligomers after redox modification. Our findings agree with previous reports on Grxs from other
sources [4, 38].
T. cruzi has two TXNs (TXNI and TXNII) actively involved in redox metabolism that can use
various low molecular mass-thiols [22]. To explore in detail the functionality of TcrGrx, we sought
for similar properties in using the thiol compounds T(SH)2, GSH, Gsp-SH, and dihydrolipoamide.
An increasing oxidation rate of NADPH directly proportional to TcrGrx concentration was
observed in kinetic studies performed with these thiols, except for dihydrolipoamide. The reduction
of TcrGrx by T(SH) followed second-order kinetics. The second order rate constant (k´) thus
2
2
calculated was about 2-fold lower than that value determined in the case of T(SH) -dependent
reduction of TcrTXNI (Table 1). Differently, third-order kinetics were observed for TcrGrx
reduction by monothiols (GSH or Gsp-SH), in agreement with the behaviour observed for other
members of the TRX-family [22, 39, 40]. When initial velocities were determined at various GSH
or Gsp-SH concentrations two kinetic phases were observed for the reduction of TcrGrx depending
on monothiol concentration (more details in Supporting information, Fig. S1). Data were fitted to
9

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the kinetic model corresponding to the overall range of GSH concentrations [39], to obtain the
parameters corresponding to each kinetic phase (Table 1). These parameters support that GSH and
Gsp-SH are oxidized by TcrGrxwith similar catalytic performance.
On the other hand, no activity was detected when T(SH) , GSH or Gsp-SH were replaced by
2
dihydrolipoamide, indicating a putative glutathionyl-moiety dependence for using reducing
substrates.
3
.2. Reduction of non-protein substrates by TcrGrx, TcrGrxC31S and TcrGrxC34S
The active site of TcrGrx is formed by the motif CEYC in the positions 31 to 34 of the protein. To
obtain further experimental support about the participation of cysteine residues in the catalytic
mechanism, two TcrGrx mutants were generated. The resulting TcrGrxs having the respective
active sites CEYS (TcrGrxC34S) and SEYC (TcrGrxC31S) were kinetically studied.
TcrGrx-dependent reduction of non-protein disulfides, such as GSSG and CySS, were kinetically
analyzed using enzymatic coupled systems. Second-order rate constants (k´) thus obtained (Table 2)
indicated a better catalytic performance for TcrGrx in reducing GSSG than CySS, with a difference
of two orders of magnitude. Moreover, second order rate constants for GSSG-reduction catalyzed
by TcrGrx resulted about 2000-fold higher than that corresponding to the spontaneous GSSG
-1
-1
reduction by T(SH) (10 M ·s ) [22] at pH 7.5 and 30 ºC. As shown in Table 2, the kinetic
2
behaviour of the mutant TcrGrxC34S for this reaction was similar to the wild type protein, which
suggests that Cys 34 is not critical for the ability of TcrGrx to reduce GSSG. These results agree
with previous reports determined for other dithiol Grxs [30, 41]. TcrGrxC31S showed no reductase
activity, indicating the essential role of the Cys 31 residue for this protein functionality.
Table 2 also provides additional information about TcrGrx. DHA reductase activity reported in
Grxs having an active site motif CPYC [4, 42], was found for TcrGrx. T(SH) dependent reduction
2
−
1
−1
of DHA was characterized by a second order rate constant of 22 M ·s (at pH 6.5) [43].
TcrGrx-catalyzed reduction of DHA by GSH at pH 7.5, showed a rate constant two orders of
magnitude higher than the spontaneous reaction (Table 2). Concerning the second-order rate
constants values detailed in Table 2 for GSSG, CySS and DHA reduction by TcrGrx, they are in
good agreement with those rates reported for TXNs from T. cruzi (Table 2), T. brucei [28] and
Crithidia fasciculata [44]. We also analyzed the ability of TcrGrx and TcrGrxC34S to reduce S-
nitrosyl derivatives such as GSNO and CySNO (see Table 2), determining that both proteins
increased by three orders of magnitude the rate constant of the direct reaction between GSNO and
1
0

ACCEPTED MANUSCRIPT
-1
-1
T(SH) (k´
= 2 M ·s at pH 7.5 and 30 ºC, [22]). Conversely reduction of CySNO was not
GSNO
2
catalyzed by TcrGrx.
.3. TcrGrx is a more effective reductant of glutathionylated substrates than protein
3
disulfides
In trypanosomatids, reduction of hydroperoxides can be catalyzed by TXNPxs [45] and GPxs [46].
After hydroperoxides reduction, TXN is the main reducer of the disulfide bonds formed in
TcrTXNPxs and TcrGPxs [20, 47]. We evaluated TcrGrx capacity to perform the reduction of the
disulfide in TcrPxs in standard coupled reactions. TcrGrx neither catalyzed the reduction of
TcrTXNPxs (cytosolic or mitochondrial) nor of TcrGPxI (Table 2), which is in agreement with the
kinetic properties reported for Grx2 from T. brucei [16]. In a similar way, in assays of disulfide
reduction in TcrMSRAs, we evidenced that TcrGrx could not replace TcrTXN (Table 2), whose
functionality has been previously probed [21]. On the other hand, TcrGrx was not effective to
reduce insulin in standard protein disulfide assays performed in the presence of either GSH or
2
T(SH) , as previously reported for other Grxs, such as human Grx1 and Grx2 [42] .
On the other hand, we recently characterized, the redox regulation of an
UDP-glucose pyrophosphorylase from Entamoeba histolytica (EhiUDP-GlcPPase) [48], revealing
the presence of several reactive cysteines relevant for the enzyme activity. We identified in the T.
cruzi genome project (www.tritrypdb.org) a nucleotide sequence (Tc00.1047053506359.60) coding
for a TcrUDP-GlcPPase with high identity to EhiUDP-GlcPPase. The recombinant TcrUDP-
GlcPPase was generated for enzymatic analysis (data not shown), and it exhibited kinetics and
redox regulation behaviour similar to that reported for EhiUDP-GlcPPase [48]. Thus, TcrUDP-
GlcPPase was inactivated by more than 95% by 30 min incubation with 2 mM diamide, after which
the activity was recovered to 82% of the initial value by 5 min treatment with 25 µM TcrTXNI.
Noteworthy, TcrGrx was not effective for recovering activity even after 30 min incubation. When
the oxidation of TcrUDP-GlcPPase was performed with GSSG, a significant loss of activity was
observed, suggesting that TcrUDP-GlcPPase was glutathionylated.
To further explore glutathionylation of TcrUDP-GlcPPase we performed the oxidation with
DIEGSSG (dieosinediglutathione). This glutathione derivative has low fluorescence due to
quenching phenomena, having the monomeric form high fluorescence signal. It is used to
fluorescently label cysteine residues when they are glutathionylated. As shown in Fig. 1A, after
treatment with DIEGSSG the band corresponding to TcrUDP-GlcPPase becomes labelled with the
1
1

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fluorescent glutathione. No fluorescence was detected in the lane corresponding to
TcrUDP-GlcPPase alone, the reaction mixture without the protein or DIEGSSG reagent. On the
other hand, in Fig. 1B it is shown that the signal corresponding to TcrUDP-GlcPPase labelled
disappear after treatment with DTT, and the level of the signal diminishes with GSH, or even more
with GSH plus TcrGrx treatments. In terms of enzyme activity, as shown in Fig. 1C, the loss of
activity after GSSG oxidation was effectively reverted by GSH in a TcrGrx mediated (and
concentration-dependent) reaction. Hence, TcrGrx showed a restriction towards mixed disulfide
between TcrUDP-GlcPPase and GSH.
31
3
.4. Physicochemical properties determinations: redox potential of TcrGrx and pKa for Cys
Redox potential of TcrGrx was determined using a method based in the equilibrium with GSH.
Different ratios of oxidized TcrGrx and GSH were incubated for 6 h, after which the remaining
GSH concentration was estimated using DTNB reagent [26] and the GSSG generated was
determined separately by a kinetic method based on NADPH-dependent reduction of GSSG by GR,
using a GSSG calibration curve. The reduced and oxidized fractions of TcrGrx were estimated
analyzing ratios of GSH:GSSG and, with these data, the equilibrium constants for TcrGrx reduction
by GSH was determined [49]. Afterwards, the redox potential of TcrGrx was calculated from the
R·T
z·F




[
TcrGRX
]
·
[
]
GSSG
]
]




Nernst equation:
. Analysis of different
Eº7.5 TcrGRX = Eº7.5 GSSG/GSH
+
·ln
2
[
TcrGRX -S
·
[
GSH
2
reaction mixtures resulted in a standard redox potential at pH 7.5 of −203 ± 11 mV for TcrGrx.
The obtained data were confirmed by means of Haldane relations as described by Arias et al. [22].
The apparent equilibrium constant (Keq app) for the reaction: 2 GSH + TcrGrxOx ↔ GSSG +
TcrGrx , K
is calculated as the ratio between the third-order kinetic constant (direct reaction)
eq app
Red
and the second-order kinetic constant (inverse reaction). Accordingly, using the Nernst equation and
the Keq app, a global standard redox potential at pH 7.5 for TcrGrx of -180 ± 10 mV was obtained.
Consequently, considering the experimental error, values obtained by both methods differ in less
than 10%. For the couple GSSG/GSH a value of −270 mV at pH 7.5 was used for all calculations
[
6].
For several thiol disulfide oxidoreductases, the first cysteine thiol found in the active site (CXXC) is
the nucleophile responsible for the reactivity [50]. In order to obtain a better knowledge about the
molecular basis of TcrGrx properties, the pKa value of Cys31 was determined (Fig. 2) using two
independent methods: i) measurement at 240 nm of the thiol ionization states of Cys31 (pKa = 6.8 ±
1
2

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0
.2, Fig. 2A) and ii) pH dependence highlighted of the reduction rate of DTNB by TcrGrxC34S
(
pKa = 6.6 ± 0.1, Fig. 2B). In both cases, the pKa value was consistent with the inflection point
observed in the pH dependent TcrGrx activity profile (Supporting information Fig. S2), suggesting
a dependence of the disulfide reductase activity with the ionization state of redox active cysteines. It
is worth noting that the pKa value for the nucleophilic Cys of TcrGrx is higher than the pKa values
(
average 3.0-4.0) reported for classic Grxs [30].
3
.5. TcrGrx is expressed in the different developmental stages of T. cruzi. Subcellular
localization
To further explore on the functional role of TcrGrx we performed studies evaluating the relevance
of the protein for the physiology of the parasite. We sought to establish the intracellular localization
of TcrGrx and also analyse if levels of the active protein are related with the parasite resistance to
oxidative stress as well as with its virulence. It is known that the complex life cycle of T. cruzi
includes several developmental stages in vertebrates and invertebrates host. These stages have
different morphology, expression patterns and biochemical properties [51, 52]. We investigated the
expression of TcrGrx in different morphologic stages of T. cruzi by using immunologic and
fluorescent techniques. In all morphological stages analyzed (epimastigote, cell-derived
trypomastigote and amastigote), a protein band of the expected size (ca. 15 kDa) was recognized by
polyclonal antiserum produced in rabbit against pure recombinant TcrGrx (Supporting information
Fig. S3).
We obtained epimastigotes overexpressing TcrGrx fused to red fluorescent protein (RFP) at either
the N-terminus (RFP-TcrGrx) or the C-terminus (TcrGrx-RFP), which were subjected to
co-immunolocalization and confocal microscopy (Fig. 3). Specific antibodies against proteins of
known localizations were applied as compartment markers: anti-TcrmTXNPx for mitochondria
[
[
45], anti-TcrcTXNPx for cytoplasm [45], anti-TbrcytC for inner-mitochondrial membrane space
53] or anti-TbrBip for endoplasmic reticulum [54]. The same pattern of fluorescence was observed
for RFP-TcrGrx and TcrGrx-RFP epimastigotes (Supporting information Fig. S4). A control assay
was performed using anti-TcrGrx to confirm the same localization for TcrGrx, RFP-TcrGrx and/or
TcrGrx-RFP. We found fluorescence signal co-localization only when antibodies against
TcrcTXNPx were used, which suggests a cytosolic localization for TcrGrx. Furthermore, RFP
fusion (either at the N- or the C-terminus) also gave fluorescence in the nuclear compartment (Fig.
3
). It is worth mentioning that the nucleotide sequences coding for TcrGrx (GeneIDs:
Tc00.1047053506475.116 and Tc00.1047053511431.40) did not include any sequence coding for
1
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an import signal to nucleus (or any other compartment signal). It could be presumed that TcrGrx
would be translocated to the nucleus following a cellular mechanism (interaction with other
proteins) already described for mammalian Grx1 [55]. On the other hand, to confirm the cytosolic
localization, epimastigotes of T. cruzi were successively subjected to lysis with increasing
concentrations of digitonin [36]. Analysis of soluble fractions by western blot revealed that TcrGrx
was present in the protein fraction released from epimastigotes at a digitonin concentration of
-1
0
.05 mg
ꢁmL , the fraction were TcrcTXNPx was also detected (Supporting information Fig. S5).
3.6. TcrGrx is involved in the resistance to oxidative stress and in-host replication in cell
cultures
We evaluated if TcrGrx overexpression in T. cruzi could help to cope with an oxidative
environment. Thus, T. cruzi cells stably transfected with pTEX (empty vector), pTEX/GFP or
pTEX/TcrGrx were challenged by means of glucose oxidase. The addition of glucose oxidase
directly to the culture medium, which contains glucose, resulted in a continuous generation of H O .
2
2
Under basal condition, all three T. cruzi cell lines showed similar proliferation kinetics (Supporting
information Fig. S6), which indicates that overexpression of GFP and TcrGrx has no detrimental
effect on epimastigote proliferation. It came up also from kinetics under basal condition (Supporting
information Fig. S6) that the overexpression of Grx did not affect the method of measure of
proliferation and viability (based in the reduction of rezasurin [33]). The overexpression of TcrGrx
was tested by western blot assay using specific antibody anti TcrGrx (Supporting information Fig.
S6- inset). Under oxidative stress conditions, epimastigote forms overexpressing TcrGrx showed
significantly higher viability percentages (p< 0.05, Fig. 4C), when compared with cultures of
epimastigotes overexpressing GFP or parasites transfected only with pTEX vector (Fig. 4). This
result supports the contribution of TcrGrx to the general resistance against oxidative damage, even
when in vitro kinetic study (see section 3.2. & Table 2) indicates that it was unable to couple to
TcrTXNPxs or TcrGPxI for detoxifying peroxides.
The involvement of TcrGrx in the infectivity of T. cruzi was also studied. The infection assay of
non-phagocytic HeLa cells was performed with TcrGrx overexpressing cell-derived T. cruzi
trypomastigotes, or control parasites transformed only with the empty vector (pTEX). After 2 h of
interaction between HeLa cells and cell-derived trypomastigotes, a counting of internalized
amastigotes was completed. The analysis in terms of infected cell percentages did not show
differences in the infectivity of both TcrGrx overexpressing trypomastigotes and the pTEX-
transfected trypomastigotes (Fig. 5). We considered relevant to evaluate if TcrGrx had an effect on
1
4

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intracellular amastigotes replication. Thus, amastigotes overexpressing TcrGrx and the
corresponding control were counted inside the cells at 48 h post-infection. We observed a slightly
higher number of amastigotes per cell in cultures infected with the TcrGrx overexpressing parasites
(
Fig.5), the observed difference is significant at the level of 95% (t-test statistics). Our results
suggest that TcrGrx would be not essential in the process of internalization of trypomastigotes into
the host cell, but point a clue about the relevance of TcrGrx in the survival of parasites once they
are inside the host cell.
3.7. TcrGrx is linked to apoptosis-like cell death in T. cruzi
We searched for other putative physiological functions for TcrGrx by quantifying the magnitude of
apoptotic-like cell death events generated after stimulus. Cultures of T. cruzi epimastigotes (stably
transfected with pTEX, pTEX/GFP or pTEX/TcrGrx) were stimulated to apoptosis by addition of
human fresh serum (HFS) as a complement source. Different parameters (previously established as
phenotypes characteristic for this kind of programmed cell death) were analyzed: i) morphological
changes in epimastigote forms of the parasite (immediate response after stimulus) [9], ii) increased
caspase-like activity (medium time elapsed from stimulus) [8], or iii) TUNEL labeling of apoptotic
nuclei (long time response after stimulus) [8]. In our hands, 15 min after addition of 10 % HFS into
axenic cultures of TcrGrx overexpressing epimastigotes, the percentage of cells with an altered
morphology (spheroid shaped cells that have lost motility) was increased significantly. In all time
intervals studied, it was observed a higher percentage of apoptotic epimastigotes in cultures
overexpressing TcrGrx than in the case of cultures of epimastigotes transfected with both
pTEX/GFP and pTEX alone (Fig. 6A). This phenomenon was increased even more in cultures
supplemented with 15 % HFS (Fig. 6A). The differences observed were significant at a level of
9
5% (ANOVA-test). HFS-treated epimastigotes displayed TUNEL-labelled positive nuclei, being
the levels of TUNEL-positive cells higher in cultures of parasites overexpressing TcrGrx than in
cultures of the microorganism transfected with pTEX empty vector. These differences (significant
at a level of 90%) persisting on time (Fig. 6B), suggest that apoptotic-like cell death occurrence in
T. cruzi could be regulated in a reaction catalyzed by TcrGrx. Furthermore, we analyzed, in the
above mentioned parasites lines, the levels of caspase-like activity after 3 h from apoptosis
induction (Fig. 6C). The highest activity was found in epimastigotes overexpressing TcrGrx. As
shown in Fig. 6C, significant differences (90 % of confidence) were found between epimastigotes
overexpressing TcrGrx before and after apoptosis induction. Such behaviour was not observed in
epimastigote cells transfected with pTEX empty vector were tested.
1
5

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4
. Discussion
The ability of T. cruzi to cope with oxidative stress is essential for survival during its infection to
mammalian tissues [56-58]. Currently, there is a general understanding about the mechanisms
responsible for this antioxidant resistance, but the biochemical and kinetic characterization of
several actors of the redox metabolism is still incomplete. Similar to Grxs in T. brucei [16], TcrGrx
was unable to transfer reducing equivalents to 2-Cys peroxiredoxins (both cytoplasmatic and
mitochondrial) or glycosomal GPxI. Recombinant TcrGrx exhibited the ability to accept reducing
2
equivalents from GSH, Gsp-SH or T(SH) ; being, in its reduced status, able to reduce low
molecular mass disulfides, such as GSSG or CySS. Our results suggest that GSH-dependent
reduction of DHA can be catalyzed by TcrGrx, which could be an alternative pathway for keeping
ascorbate under its reduced form in the parasite.
Moreover, the redox regulation of enzymes activities or cellular signaling has been scarcely
elucidated in trypanosomatids. We present evidence suggesting that TcrGrx is functional for in vitro
modifying the activity of UDP-GlcPPase (we have previously demonstrated this activity on a
glyceraldehyde-3-Pdehydrogenase from Triticum aestivun [15]) through glutathionylation/
deglutathionylation reactions, which could be highly relevant for protecting key sulfhydryl groups
in this and other proteins of the parasite. TcrGrx showed a strong specificity for GSH-mixed
disulfides, a property already reported for Grxs from other organisms, such as E. coli, S. cerevisiae,
or mammalian cells [38, 59-61]. Additionally, it exhibited differential capacity for catalyzing
2
T(SH) dependent reduction of S-nitrosothiols, with specificity for GSNO. The capacity of TcrGrx
for reducing GSNO could work as a part of an antioxidant system against stress generated by RNS.
Possibly, GSH would react with exogenous NO, producing GSNO, which would be then
regenerated by T(SH) /TcrGrx or GSH/TcrGrx systems to yield GSH and HNO (it reacts with O to
2
2
-
generate NO ). Similar mechanisms were proposed for GSNO detoxification in P. falciparum and
2
mammalian cells [62, 63]. The present work provides results to support the hypothesis of an active
participation of T(SH) /TcrGrx or GSH/TcrGrx redox pairs in the regulation of intracellular levels
2
of GSNO from T. cruzi living under oxidative stress condition. Fig 7 summarizes the reaction
proposed for TcrGrx as a component of the redox network operating in T. cruzi.
The standard redox potential value of TcrGrx (average value of −190 mV at pH 7.5)
thermodynamically justifies the capacity of this redox protein to use T(SH)2 or Gsp-SH
(Eº′ of −242 mV [28]), and GSH (Eº′ of −240 mV [26]) as reducing substrates. It is coherent with a
flow of reducing equivalents transported through a trypanothione-dependent system. The estimated
1
6

ACCEPTED MANUSCRIPT
pKa value for nucleophilic cysteine of TcrGrx (pKa = 6.6 at 25 °C) is atypical regarding “classical”
Grxs. It was previously demonstrated that residues of the dipeptide between cysteine residues in the
active site play an important role in several physicochemical properties of proteins in the
thioredoxin family [28, 64]. The presence of a proline residue in this dipeptide is a feature of
“classical” Grx [4] that is determinant for the tertiary structure of the protein and the consequent
reactivity of residues around cysteines in the active site, influencing their electrostatic interactions
with thiols [64]. The absence of this proline residue between Cys 31 and Cys 34 in TcrGrx, could be
a reason for the atypical pKa value for the nucleophilic cysteine. Nevertheless, at the intracellular
pH (about 7.0), more than 50 % of the Cys 31 is in the thiolate state, as required for reactivity in
thiol-disulfide oxidoreductases [65]. Comparing with the nucleophilicity of cysteines from other
relevant thiol-disulfide oxidoreductases in the parasite a lower pKa value than TcrTXNI (pKa=7.0)
or TcrTXNII (pKa=7.3) [22], was observed for TcrGrx.
By means of specific antibodies designed against TcrGrx, we evidenced the occurrence of Grx in
cellular extracts from T. cruzi epimastigote, trypomastigote and amastigote stages. The protein
showed cellular localization in cytoplasm and probably ocurrs also in the nucleus. Probably, it
performs deglutathionylation in different molecular targets in both cell compartments, a hypothesis
that would be matter of future studies. We analyzed, through in vivo assays, the role of TcrGrx in
the protection of the parasite living under oxidative conditions. This task was previously described
for Grxs from others organisms, for example mammalian Grx2 [66] and yeast Grxs [67]. RNAi-
based experiences are usually exploited for exploring gene function in several cell types.
Unfortunately RNAi machinery is not present in T. cruzi [68], being knockout experiments in this
parasite extremely laborious and practically unreliable in several cases as a consequence of the
physiologic characteristic of T. cruzi [69]. Overexpression of the gene of interest is frequently the
viable alternative for studying gene function in T. cruzi. In our experiments, the overexpression of
TcrGrx did not affect the normal proliferation parameters of the cultures. However, under
conditions of oxidative stress, parasites overexpressing TcrGrx showed an improved resistance,
which suggests that the protein could play a role in the response of T. cruzi to hostile (oxidative)
environments. It is tempting to speculate that TcrGrx could help to maintain both DHA and GSH
intracellular (or nuclear) levels. Moreover, one of the possible mechanism for glutathionylation is
catalyzed by Grxs [70], for example, human Grx2 which can catalyze thiol oxidation and
glutathionylation of proteins in the mitochondrial membrane [71]. It is possible that
glutathionylation catalyzed by TcrGrx collaborates with sulfhydryl homeostasis, preventing
irreversible oxidation of proteins [72].
1
7

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We have also verified that the overexpression of TcrGrx slightly improves T. cruzi survival inside
the cell host. The evaluation of T. cruzi multiplication level inside infected cells has been previously
used as indicator of the relevance of antioxidant molecules, like TcrGrx, for the parasite viability in
an oxidative environment [32, 58]. Based in our experimental data, TcrGrx could be also involved
in a cellular pathway that connects the apoptotic-like stimulus, and the final phenotype of apoptotic-
like death. As observed in other studies [5], several components of apoptosis signaling pathways
could be subject of regulation by Grxs, resulting in augmented or decreased levels of apoptosis,
depending mainly on apoptotic stimulus and cells type. There is a considerable variability in the
responses observed in different systems, it is clear that Grx became important for regulating cell
signaling even when components subjected to regulation can vary in different contexts (such as the
cell type, tissue or cellular injury). In our experiments we observed responses that suggest different
roles for TcrGrx as a result of the varied challenges evaluated. In trypanosomatids, the
understanding of the apoptotic signaling pathways is still not completed, but different pathways are
being outlined depending on the inducer (summarized in [73]). In view of these antecedents for
PCD in trypanosmomatids, it will be necessary to identify TcrGrx targets for establishing the actual
function played by TcrGrx in cell death in T. cruzi.
5
. Conclusions
Antioxidants defences are essential for T. cruzi to face the oxidative environment they will find
after host cells infection. Our work provides information about the biochemical and enzymatic
properties of TcrGrx as well as on its contribution to the parasite antioxidant defence. In T. cruzi,
TcrGrx is an oxidoreductase probably involved in reduction of glutathione, dehydroascorbic acid
and mixed disulfides, such as glutathionylated proteins. Novel information reported herein proposes
the participation of this Grx in redox signaling pathways in T. cruzi, probably through
glutathionylation-deglutathionylation mechanisms. Our results support a functional relationship
between TcrGrx and programmed cell death in T. cruzi. The involvement of TcrGrx in several
parasite physiological processes suggests novel insights about the protein involvement in redox
signaling.
Acknowledgments
We thank Dr. Andre Schneider (University of Berne, Switzerland) for T. brucei
α-cytochrome c
antibody, Dr. James Bangs (University of Wisconsin, USA) for T. brucei -BiP antibody and Dr.
α
Juan J. Cazzulo (Universidad Nacional de San Martin, Argentina) for T. cruzi (amastigote,
trypomastigote and epimastigote) crude extracts. This work was supported by grants from UNL
1
8

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(
CAI+D Orientados & Redes), CONICET (PIP 112-2011-0100439), and ANPCyT (PICT´12 2439).
VEM is a fellow from CONICET. DGA, AAI and SAG are investigator career members from
CONICET. The authors declare that there are no conflicts of interest.
1
9

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List of Abbreviations
Cys: cysteine
CySNO: s-nitrosocysteine
CySS: cystine
DHA: dehydroascorbic acid
DIEGSSG: dieosinediglutathione
DTNB: 5,5'-dithiobis-(2-nitrobenzoic acid)
GFP: green fluorescent protein
Grx: glutaredoxin
Grx-S : glutaredoxin, disulfide form
2
Grx-SSR: glutaredoxin mixed disulfide
GSH: glutathione, reduced form
GSNO: S-nitrosoglutathione
Gsp-SH: glutathionylspermidine, reduced form
(
Gsp) : glutathionylspermidine, oxidized form
2
GSSG: glutathione, oxidized form
HEDS: 2-hydroxyethyl disulfide
HFS: human fresh serum
IMS: intermembrane mitochondrial space
PCD: programmed cell death
PDI: protein disulfide isomerase
RFP: red fluorescent protein
RNS: reactive nitrogen species
ROS: reactive oxygen species
RSH: monothiol
T(SH) : trypanothione, reduced form
2
TcrAPx: ascorbate peroxidase from T.cruzi
TcrGPxI: glutathione peroxidase from T.cruzi
TcrGrx: glutaredoxin from T.cruzi
3
3
1
4
TcrGrxC31S: glutaredoxin from T. cruzi, mutant in Cys
TcrGrxC34S: glutaredoxin from T. cruzi, mutant in Cys
TcrMSRAs: methionine sulfoxide reductases A from T.cruzi
TcrTXNI: tryparedoxin I from T. cruzi
2
0

ACCEPTED MANUSCRIPT
TcrTR: trypanothione reductase from T. cruzi
TcrUDP-GlcPPase: UDP-glucose pyrophosphorylase from T. cruzi
TR: Trypanothione reductase
Trx: thioredoxin
TS : trypanothione, oxidized form
2
TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling
TXN: tryparedoxin
TXNPx: tryparedoxin peroxidase
2
1

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