The PfNF-YB transcription factor is a downstream target of

melatonin and cAMP signalling in the human malaria parasite

Plasmodium falciparum
Introduction
Plasmodium falciparum causes the most severe form of
malaria and leads to over 1.5 million people deaths
worldwide (World Health Organization, WHO) each year.
The malaria parasite is an unicellular eukaryote that spends
most of its life in intact human hepatocytes and red blood
cells (RBC) [1, 2]. In the latter cells, in particular,
Plasmodium multiplies and maturates towards the forms
ready to invade other erythrocytes. To reach its develop-
ment, Plasmodium dierentiates into ring, trophozoite and
schizont forms. Cell cycle control, signalling pathways and
parasite antigenic versatility are important elds for
investigating new ways to arrest parasite growth.
In an eort to understand how the parasite development
is orchestrated, our group demonstrated that melatonin, a
tryptophan-derived metabolite, increases parasitemia in
P. falciparum and P. chabaudi [3, 4]. A complex down-
stream signalling cascade is activated by the indoleamine,
thus inducing a rise in the second messengers IP
3
, Ca
2+
and
cyclic AMP [5, 6]. cAMP signalling has an important role in
Plasmodium biological processes by activating PKA. In
addition, PKA also modulates anion conductance and
vesicle tracking in P. falciparum [7, 8]. The involvement
of Ca
2+
in the regulation of long-term cell adaptation
through its ability to control gene expression is considered a
hallmark in cell biology (for review, see [9]). In vertebrates,
melatonin has also been shown to regulate transcription
factors, phosphorylation of cAMP-responsive element
binding protein activity and c-Fos expression [10]. In
addition, melatonin signalling downregulates the expres-
sion of Rex-1 and Oct4 transcription factors in murine
embryonic stem cells [11].
The nding that melatonin controls Plasmodium cell
cycle and regulates transcription factor expression in
vertebrates led us to postulate that a melatonin-induced
signalling cascade may control the expression of cell cycle
genes in Plasmodium. Several investigations have been
performed to identify genes encoding transcriptional regu-
lators in the genome of P. falciparum [12]. So far, only few
transcription factors have been well characterized in
P. falciparum. These are Myb1 protein [13], high-mobility
group box (HMGB) proteins and Apetala2 (AP2) [14].
Gene expression analysis of malaria parasite revealed that
transcription is generally monocistronic and develop-
mentally regulated [1520].
Abstract: Plasmodium falciparum causes the most severe form of malaria and
is responsible for the majority of deaths worldwide. The mechanism of cell
cycle control within intra-erythrocytic stages has been examined as a
potential means of a promising way to identifying how to stop parasite
development in red blood cells. Our group determined that melatonin
increases parasitemia in P. falciparum and P. chabaudi through a complex
signalling cascade. In vertebrates, melatonin controls the expression of
transcription factors, leading us to postulate rather that the indoleamine
would aect PfNF-YB expression in human malaria parasites. We show here
that PfNF-YB transcription factor is highly expressed and colocalized in the
nucleus in mature parasites during intra-erythrocytic stages, thus suggesting
an important role in cell division. Moreover, we demonstrate for the rst
time that melatonin and cAMP modulate the PfNF-YB transcription factor
expression in P. falciparum at erythrocytic stages. In addition, PfNF-YB is
found to be more ubiquitinated in the presence of melatonin. Finally, the
proteasome inhibitor bortezomib is able to modulate PfNF-YB expression as
well. Taken together, our dada reinforce the role played by melatonin in the
cell cycle control of P. falciparum and point this indolamine as a target to
develop new antimalarial drugs.
Wa nia R. Lima
1
, Miriam Moraes
1
,
Eduardo Alves
1,2
, Mauro F.
Azevedo
3
, Dario O. Passos
1
and
Ce lia R. S. Garcia
1
1
Departamento de Fisiologia, Instituto de
Biocie ncias, Universidade de Sa o Paulo, Sao
Paulo, Brazil;
2
Departamento de Parasitologia,
Instituto de Cie ncias Biome dicas,
Universidade de Sao Paulo, Sa o Paulo, Brazil;
3
Macfarlane Burnet Institute for Medical
Research and Public Health, Melbourne,
Victoria, Australia
Key words: cAMP, malaria, melatonin,
signalling, transcription factor
Address reprint requests to Celia Regina da
Silva Garcia, Rua do Mata o, travessa 14, n.
321. Cidade Universita ria, Sa o Paulo, CEP
05508-090, Brazil.
E-mail: cgarcia@usp.br
Received April 17, 2012;
Accepted June 1, 2012.
J P I
1 0 2 1
B
Dispatch: 16.6.12 Journal: JPI CE: Nivetha Raj
Journal Name Manuscript No. Author Received: No. of pages: 9 PE: Vigneswari
J. Pineal Res. 2012
Doi:10.1111/j.1600-079X.2012.01021.x
2012 John Wiley & Sons A/S
Journal of Pineal Research
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On the other hand, it has been shown that polyubiquitin
gene transcript and ubiquitin-conjugated proteins are
upregulated by heat shock protein in trophozoite and
schizont [21]. Recently, the ubiquitin proteasome system
(UPS) and the unusual kinase 7 (Pfpk7) were reported to be
central to the downstream molecular mechanism of mela-
tonin action in P. falciparum cell cycle [22]. The UPS
regulates the turnover of several proteins implicated in the
cell cycle control. Cyclins, transcription factors and protein
kinases are regulated during the cell cycle events by UPS
mainly in the mitosis phase [23, 24]. The question then
arises whether melatonin controls the cell cycle by regulat-
ing the expression of transcription factors in P. falciparum.
After a detailed in silico analysis of some transcription
regulators involved in the cell cycle of P. falciparum, we
decided to study the CCAAT-box-binding DNA protein B
transcription factor (Pf11_0477). The transcription factor
Pf11_0477 was rst described by Silvestrini et al. [25], who
demonstrated that the mRNA length is about 1 kb and it is
highly expressed in asexual schizont stage and sexual
gametocyte stage. The CCAAT-box-binding DNA protein
is evolutionary conserved from yeast to human. It is also
named NF-Y, CBP, a-CP1 or CP1, and was rst identied
as a MHC class II promoterbinding proteins [26]. NF-Y is
well described in other species such as mouse, chicken and
yeast [2729]. It is an ubiquitous heteromeric protein
composed of three subunits, NF-YA, NF-YB and NF-YC,
all required for the DNA binding activity [30].
Our data show for the rst time that the malarial
transcription factor, PF111_0477, here named PfNF-YB, is
activated by an external signal, melatonin, and by the
second messenger, cAMP. Moreover, PfNF-YB is post-
translational modied by ubiquitination and is modulated
by a proteasome inhibitor. These ndings provide infor-
mation to help understand the signalling transduction
pathways in the lethal Plasmodium falciparum.
Materials and methods
Culture of Plasmodium falciparum
Plasmodium falciparum strain 3D7 parasites were cultured in
asks at 37C and 5% haematocrit in RPMI 1640 medium
supplemented with 10% human plasma, gassed with 90% N
2
,
5% O
2
and 5% CO
2
[31]. High concentration of ring stage
parasites culture was centrifugedfor 5 minat 1200 rpm 1 at room
temperature. Thesupernatantwasdiscarded, andtheparasitized
erythrocyteswereincubatedfor5 minwith8%sorbitol solution.
After incubation, the erythrocytes were centrifuged again,
washed twice with complete RMPI mediumand added back to
the normal culture as previously described [32].
Immunoblot analysis
Parasites isolated from highly synchronous P. falciparum
culture were incubated with lysis buer (12.5 mm TrisHCl,
150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate,
1% BSA, 1 mm PMSF, pH 7.5) for 1 hr on ice or sonicated
thrice with the same lysis buer without detergent. The
lysate was centrifuged, and the supernatant was collected.
The proteins were separated in 812% SDS-PAGE gel,
blotted into polyvinylidene uoride (PVDF) membrane and
blocked with saline solution 5% milk. The membranes were
incubated overnight with primary antibodies in appropri-
ated dilutions at 4C, washed with TBSTween and
incubated with peroxidase-conjugated secondary antibody
for 1 hr at room temperature. The membranes were washed
again and developed using a chemiluminescent substrate
luminol (ECL; Amersham-Pharmacia Biotech 2 ).
Antibodies
A rabbit polyclonal anti-PfNF-YB antibody was developed
by Proteimax (Sa o Paulo, Brazil). A rabbit polyclonal
b-actin antibody (2033), mouse monoclonal b-tubulin
antibody (T4026) and rabbit polyclonal anti-ubiquitin
(U5379) were ordered from Sigma (St. Louis, MO, USA).
Secondary Alexa 488, 564 and 594 antibodies were obtained
from Molecular Probes (Eugene, OR, USA).
Immunoprecipitation assay
To immunoprecipitate PfNF-YB, parasites lysates were
incubated at 4C with anti-PfNF-YB antibody in a total
volume of 2 mL lysis buer diluted four times with PBS 3 .
Protein A-Sepharose beads (50 lL of 50% solution; Phar-
macia) were added to the samples. After further incubation
for 1 hr at 4C, beads were washed ve times (1 mL each)
in ice-cold lysis buer. Immunoprecipitated proteins were
eluted by heating at 95C for 5 min in Laemmli sample
buer (50 mm TrisHCl, pH 6.8, 2% SDS, 0.001%
bromophenol blue, 10% glycerol, 100 mm dithioerythritol).
Ubiquitin-protein conjugates are known to be deubiquiti-
nated by an NEM-sensitive pathway [33]. To minimize the
protein deubiquitination during cell lysis, lysis buer
included 5 mm of the ubiquitin isopeptidase inhibitor
NEM [34].
Immunouorescence analysis
Experiments were performed with synchronous parasites,
and the culture was washed twice in PBS. Briey, the cells
were xed in 4% paraformaldehyde/0.0075% glutaralde-
hyde for 30 min, permeabilized with saponin and blocked
with 3% BSA/PBS for 30 min following the protocol
established by Tonkin et al. [35]. Anti-PfNF-YB antibody
was diluted at 1:500. Fluorophore-conjugated secondary
antibodies were used at the following dilutions: AlexaFluor
594 goat anti-rabbit (1:1000), AlexaFluor 488 goat anti-
rabbit (1:1000). We used TOPRO-3 to identify the nuclei
and WGA Alexa 564 as membrane marker. The slides were
analysed on a Carl Zeiss LSM 510 Confocal Laser
Scanning Microscope.
Melatonin, 6-Bnz-cAMP sodium and bortezomib
treatments
Cultures of parasitized erythrocytes previously synchro-
nous with sorbitol were treated with melatonin (nal
concentration of 100 nm; [32]), cAMP analogue, 6-Bnz-
cAMP sodium (10 lm) or Bortezomib (50, 100 and 200 nm)
and incubated at 37C for 6 hr.
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RNA extraction, cDNA synthesis and real-time PCR
Parasitized erythrocytes were lysed using Trizol LS (Invi-
trogen 4 ) according to the manufacturers instructions. The
cDNA synthesis was carried out with 300 ng of random
primers and 500 ng of total RNA previously treated with
DNAse (Invitrogen), using the Superscript II Kit (Invitro-
gen) as described in the manufacturers instructions. The
relative quantitation of gene expression was assessed by
SYBER green quantitative real-time PCR (qPCR) in a 7300
Real Time PCR System (ABI 5 ) under the following condi-
tions: 50C for 2 min, 95C for 10 min, 40 cycles of 95C
for 15 s; 55C for 30 s; and 60C for 30 s. The eciency of
amplication for each gene was previously determined
using dilutions of cDNA or gDNA. Genes outside the
range 90110% were discarded from the analysis. For each
reaction were used 28 ng of cDNA, oligonucleotides
(800 nm nal concentration) and Max Mix PCR (ABI)
(1 nal concentration). The mRNA was amplied using
the following primers. PF11_0477: 5-ACAAGCGAGG-
CTAGTGACAG-3, 5-TTCAGATATTCGGTTAATGG-
TTC-3; PFL1310C: 5-CAATTCATGGGGATAAGCA
G-3,5-CTTGAAGCTACATCGGTTGC-3; PF07_0029,
5-TCCGCAAACATGGAAAGAAT-3, 5-ATTGGGTG-
ACGAGCATTGAT-3; PFL0465c: 5-AATTCTGTTTC
CGATTTCAGC-3, 5-TAACGTTGTGCCTTCTAAAC-
G-3; PFI1475w: 5-ACAACTGAAGATGGGGGTCA-3,
5-TTTTGGTGGTGATGGTTGTG-3; MAL-18S: 5-AA
CACAAGGAAGTTTAAGGCAACAA-3, 5-GCGTGC-
AGCCTAGTTCA-3. The ribosomal 18S gene was
amplied and used to normalize the quantitation results
as endogenous control. The reactions were carried out in
triplicates, and the expression values shown represent the
relative amplication of each cDNA sample compared
with the control. The signicance of each relative gene
expression was determined by the Students t-test.
Flow cytometry analysis
Infected erythrocytes with parasitemia up to 5% and
haematocrit of 2% haematocrit were incubated with 50,
100 and 200 nm bortezomib. Parasites were xed and
permeabilized as described by Schuck et al. [36]. The
parasites cell cycle development was analysed by ow
cytometry (FACSCalibur 6 ), and plots were acquired using
CELLQuest software (Becton & Dickison 7 ).
Results
The PlasmoDB database of Plasmodium mRNA expression
represents an integrative source of genes that had their
expression modulated during the intra-erythrocytic cycle
(http://www.plasmodb.org 8 ). Therefore, we used the Plas-
moDB 9 database to select some of these transcripts and
investigate their expression after melatonin treatment of
P. falciparum. The selected candidates have also been
associated with the cell cycle control and parasite invasion
process. Then, searching for downstream regulators of
Plasmodium cycle associated with melatonin hormone, we
performed quantitative RT-PCR experiments to detect
changes at the transcriptional level in trophozoite parasites
(24 hr postinvasion, hpi) treated with 100 nm melatonin for
6 hr. Real-time RT-PCR was performed for ve selected
mRNA [putative DNA helicase 60 (PFL1310c), heat shock
protein 90 (PF07_0029), CCAAT-box-binding DNA pro-
tein subunit B (PF11_0477, also called here as PfNF-YB),
merozoite surface protein 1 (PFI14765w) and zinc nger
transcription factor (PFL0465c)] in three independent
assays. The results showed that melatonin treatment
signicantly upregulated the expression of three mRNA
(PFL1310c, PF07_0029 and PfNF-YB), but it did not
change the expression of PFI14765w and PFL0465c
(Table 1).
To address whether PfNF-YB protein expression could
also be changed in the presence of melatonin in trophozoite
form, we rst customized a synthetic rabbit polyclonal
antibody against the C-terminus portion of PfNF-YB
(Fig. S1). Total lysates of trophozoite from higly synchro-
nous cultures at 24 hpi and treated with melatonin were
used to carry out immunoblot analysis. Western blot results
revealed an increase in PfNF-YB protein expression in the
presence of melatonin in comparison with control samples
(Fig. 1A). Moreover, two bands were observed: a stronger
band with around 40 kDa and a much weaker band with
approximately 42 kDa. The detection of a 42-kDa band
suggests that PfNF-YB might undergo post-translational
modication. Both isoforms exhibit an increased
abundance in the presence of melatonin.
Given that the anti-PfNF-YB antibody showed to be
highly specic to the PfNF-YB protein in the immunoblot
analysis, we next decided to investigate the localization of
this transcription factor in P. falciparum. Thus, confocal
microscopy was carried out to acquire the distribution of
PfNF-YB protein throughout the parasite intra-erythro-
cytic development (Fig. 1B). PfNF-YB was detected in the
cytoplasm and nucleus of ring, trophozoite and schizont
stages. A colocalization in the nucleus of schizont enriched
with merozoites was observed, although not all merozoite
nuclei were stained. No staining was detected in the red
blood cells, conrming the specicity of the antibody
against the PfNF-YB protein of Plamodium.
Table 1. List of genes modulated via melatonin signalling veried
by RT-PCR
Gene ID
a
Description Mean
b
S.E.M. P value
PFL1310c DNA
helicase 60
1.63 0.20 0.02
PF07_0029 Heat shock
protein 90
1.98 0.17 0.01
PF11_0477 CCAAT-box
DNA binding
protein subunit B
2.46 0.06 1E-04
PFI1475w Merozoite
surface protein 1
1.41 0.16 0.18
PFL0465c Zinc nger
transcription factor
1.44 0.16 0.22
a
Gene ID and description are in agreement with PlasmoDB
annotation.
b
Mean represents the mean fold change values between melatonin-
treated and control samples.
Statistical analysis was carried out using two-tailed unpaired t-test.
Melatonin and transcription factor expression in P. falciparum
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cAMP is a second messenger essential for a signalling
cascade that leads to gene regulation through several
transcription factors. Therefore, we investigated whether
cAMP pathway modulates the PfNF-YB expression along
the three intra-erythrocytic stages. Synchronous parasites at
ring, trophozoite and schizont stages were treated for 6 hr
with 20 lm 6-Bnz-cAMP, a membrane permeable and
selective cAMP-dependent protein kinase A (PKA) activa-
tor, and the abundance of PfNF-Y protein and mRNA was
estimated by immunoblot analysis and quantitative
RT-PCR, respectively. The treatment with 6-Bnz-cAMP
produced an opposite eect on the control of PfNF-Y
expression in ring form compared with mature trophozoite
(Fig. 2). An increase in the abundance of both PfNF-YB
mRNA and protein was induced by the cAMP pathway
activation in mature trophozoite, whereas this activation
decreased the PfNF-YB expression in the immature form,
the ring stage. By contrast, no change was observed in the
PfNF-YB expression at mRNA and protein level in
schizont parasites treated with the cAMP analogue.
The CBF/NF-Y proteins are post-translationally mod-
ied by phosphorylation, acetylation, ubiquitination and
glycosylation in mammals [37, 38]. It is also known that
melatonin induces augmentation of mRNA encoding for
UPS system members in P. falciparum [22]. This led us to
wonder whether PfNF-YB could be a target of ubiquiti-
nation in the human parasite. First, we veried whether
PfNF-YB undergoes ubiquitination and further whether
melatonin would change the PfNF-YB ubiquitination
level. Using anti-PfNF-YB-specic antibody, we have
immunoprecipitated PfNF-YB from RBC lysates parasit-
ized with mature trophozoite (34 hpi) after treating the
parasites with 100 nm melatonin for 6 hr. Next, the
immunoprecipitates were submitted to SDS-PAGE and
hybridized with an anti-ubiquitin antibody. Indeed, the
results revealed that PfNF-YB is ubiquitinated (Fig. 3).
Furthermore, we also observed that PfNF-YB protein is
most intensely detected in the sample treated with mela-
tonin, indicating that the hormone stimulates the ubiqui-
tination of PfNF-YB. In 10 the control sample containing
only beads, the antibody was omitted and no signal was
(A)
(B)
Fig. 1. 12 Immunoblot analysis of PfNF-YB protein in melatonin-
treated parasites and its localization in nontreated Plasmodium
falciparum. (A) Western blot of total lysates from trophozoites
treated with 100 nm melatonin for 6 hr and nontreated controls.
Membranes were hybridized against the anti-PfNF-YB-specic
antibody. The anti-b-tubulin antibody was used as a normalizer of
protein loading. PfNF-YB protein resulted to be more abundant in
melatonin-treated parasites than in nontreated controls. (B) The
cellular localization of PfNF-YB was examined in ring, trophozoite
and schizont stages of intra-erythrocytic cycle. PfNF-YB was
localized at both cytoplasm and nucleus. Immunouorescence was
carried out with the anti-PfNF-YB antibody (green), and nuclear
DNA was stained with TOPRO-3 (blue) and the RBC membrane
bordering with WGA (red). The confocal images exhibited a
specic staining to parasites and displayed localization in both
cytoplasm and nucleus in all stages. Scale bar = 5 lm.
(A)
(B)
Fig. 2. 13 PfNF-YB expression regulation by cAMP signalling. Par-
asites were treated with 10 lm 6-Bnz-cAMP for 6 hr, and total
RNA samples were puried and submitted to real-time RT-PCR.
18S mRNA was quantied to normalize the PfNF-YB mRNA
levels. (A) The graphics reveal a dierential regulation of PfNF-YB
mRNA stimulated by the cAMP analogue in ring (6 hr) and
mature trophozoite (30 hr). No signicant change was observed in
6-Bnz-cAMP-treated schizonts. (B) Western blot assay conrmed
the quantitative RT-PCR results. PfNF-YB protein was less
detected in ring. By contrast, PfNF-YB was increased in mature
trophozoite, and no change was observed in schizont treated with
6-Bnz-cAMP compared with nontreated controls. Anti-b-tubulin
antibody was used as a normalizer of protein loading. Statistical
analysis was carried out by t-test. *P < 0.05.
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detected, conrming the specicity and stringency of the
immunoprecipitation assay.
Protein ubiquitination is an upstream event that essen-
tially takes place before the proteasomal degradation.
Proteasome inhibitors such as MLN-273 and bortezomib
(BTZ) block Plasmodium development [39, 40]. BTZ was
reported to impair the intra-erythrocytic cycle of P. falci-
parum, at early trophozoite, 24 hpi [40]. As we have
demonstrated that the melatonin treatment increases the
ubiquitination state of PfNF-YB, we treated parasites with
BTZ to verify whether the proteasomal degradation of
PfNF-YB protein would be inhibited after proteasome
blockage. We rst estimated the PfNF-YB protein content
in samples treated with BTZ throughout intra-erythrocytic
cycle. Surprisingly, the immunoblot results revealed that
PfNF-YB protein levels were much lower at ring and
trophozoite stages treated with BTZ in comparison with
control samples (Fig. 4A). In addition, BTZ only slightly
increased PfNF-YB protein abundance in schizont form,
suggesting that its degradation was slightly avoided. These
data give rise to the hypothesis that there could be a strong
transcriptional regulation of PfNF-YB promoted by BTZ
expression. To test this hypothesis, we quantied the
mRNA amount for each parasite stage after the BTZ
treatment. The quantitative RT-PCR results showed a
signicant decrease in the abundance of PfNF-YB mRNA
in the three stages, but with a remarkable decrease in
schizont, 3.0- and 3.5-fold at early and mature forms,
respectively (Fig. 4B). It seems to be that the expected
accumulation of PfNF-YB protein after proteasomal
blockage was disguised by the repression of PfNF-YB
mRNA expression. This points to a balance between
transcriptional regulation and proteasomal protein
degradation strictly regulated in the presence of BTZ.
To examine the eect of proteasome inhibition on the
development of the intra-erythrocytic schizont form and
merozoite invasion, we treated highly synchronized P. fal-
ciparum (3D7 strain) cultures at mature schizont stage
(44 hpi) with BTZ during 6 hr to prevent excessive toxicity.
The experimental procedure was minimally modied from
Reynolds et al. [40], basically concerning to a shorter
treatment time. The drug was added at dierent concen-
trations, 50, 100 and 200 nm, and the parasitemia and
invasion were analysed by light microscopy and quantied
by ow cytometry. The microscopy images showed that
mature schizonts treated with 100 nm BTZ displayed
unreleased merozoites whereas schizonts treated with
DMSO (control) completely released their merozoites
(Fig. 5A). Bortezomib also decreased the parasitemia by
half (4%) through the impairment of schizont develop-
ment when all concentrations were compared with controls
(8%; Fig. 5B). The histogram plots showed that 100 nm
BTZ stopped the parasite cycle at mature schizont given
that a single peak representing multinucleated schizonts
was generated (Fig. 5C). By contrast, in the control
Fig. 3. 14 Immunoprecipitation of ubiquitinated PfNF-YB in Plas-
modium falciparum. Trophozoites nontreated and treated with
100 nm melatonin (MLT) for 6 hr were lysed, and the samples were
submitted to immunoprecipitation with anti-PfNF-YB antibody.
Immunoprecipitates were separated by SDS-PAGE, and immu-
noblots were hybridized against an anti-ubiquitin antibody. Non-
conjugated beads were used as the assay control (beads only).
Results showed that PfNF-YB is ubiquitinated, and melatonin
treatment induced higher ubiquitination of PfNF-YB protein in
treated compared with nontreated parasites.
(A)
(B)
Fig. 4. 15 Ubiquitin proteasome system (UPS) inhibition in Plasmodium falciparum. Intra-erythrocytic forms of ring (12 hpi), trophozoite
(30 hpi), and early (40 hpi) and mature (44 hpi) schizonts were treated with 100 nm bortezomib (BTZ) for 6 hR to inhibit UPS machinery.
(A) The PfNF-YB protein level was analysed by immunoblot. Images showed a decrease in PfNF-YB abundance in ring and trophozoite
forms whereas no changes were observed in early and mature schizonts compared with the control (Ctl). Anti-b-actin antibody was used as a
normalizer of protein loading. (B) Quantitative RT-PCR showed that PfNF-YB mRNA expression was decreased by BTZ treatment in
comparison with control samples for all parasite forms. For ring and trophozoite, PfNF-YB expression was about 2.0- and 2.5-fold,
respectively, lower than that in the controls. In early and mature schizonts, there was a signicant diminution, about 3.0- and 4.0-fold,
respectively. 18S mRNA was quantied to normalize the PfNF-YB mRNA levels. Statistical analysis was carried out by t-test. *P < 0.05.
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samples, we observed two high peaks indicating the
mononucleated rings and multinucleated schizonts, respec-
tively. Taken together, these results showed that protea-
some inhibition causes impairment of the developmental
progression at late schizont stage. Moreover, merozoite
egress was prevented to occur from mature schizonts,
demonstrating that BTZ blocks P. falciparum invasion.
Discussion
Studies of mutagenesis carried out by dierent groups
clearly showed that CCAAT sequence in the promoter
region is required for recognition and binding of NF-Y
subunit B transcription factor (NF-YB) to DNA [26, 29, 41,
42]. NF-YB binds to CCAAT motif in several promoters of
mammalian genes during G
2
/M phase of cell cycle, such as
topoisomerase IIa, cyclin B1, CDC25C, E2F, CDC2 and
kinase thymidine to modulate their expression [4345]. On
the other hand, further investigations showing whether the
expression of PfNF-YB, the NF-YB transcription factor
homologue from the human malaria parasite, can be
regulated during intra-erythrocytic stages are required.
Despite the scarcity of data demonstrating the regulation
of NF-YB expression by changes in cytosolic calcium levels,
a recent report suggests a modulation of NF-Y activity
during depletion of intracellular calcium in human NIH
3T3 cells [46]. In the present work, we described for the rst
time that melatonin and cAMP can modulate PfNF-YB
transcription factor expression in intra-erythrocytic stages
of P. falciparum. Mature trophozoite and schizont stages
are characterized by an increase in metabolism, cellular
pathway and DNA duplication [47]. Signal transduction via
melatonin in humans promotes transcriptional upregula-
tion of several transcription factors, that is, homeo box A4
(HOXA4), forkhead box O1A (FOXO1A), transcription
elongation factor B (SIII), polypeptide 3 (TCEB3) and
peroxisome proliferative activated receptor delta (PPARD)
[48]. Conversely, melatonin downregulates PHD nger
protein 15 (PHF15) and zinc nger protein 33a (ZNF33A)
in human peripheral blood mononuclear cells [48]. Here, we
documented that the melatonin signalling upregulates
PfNF-YB transcription factor expression by twofold in
trophozoite stage of P. falciparum.
The promoter sequence of several cAMP-responsive
genes exhibits elements that mediate transcriptional activa-
tion in response to increased levels of intracellular cAMP
[4951]. In P. falciparum, the PfNF-YB regulators are
linked to cAMP/PKA signalling, leading to its downregu-
lation in ring and upregulation in trophozoite forms. This
tight regulation of PfNF-YB via cAMP is modulated by
PKA. Indeed, cAMP response induces the catalytic subunit
PKA translocation to the nucleus where PKA modulates a
variety of transcriptional regulators. The catalytic subunit
gene of cAMP-dependent protein kinase (Pfpka-c) exists as
a single copy in P. falciparum. Pfpka-c has been described
as being expressed at high level in the pathogenic sexual
stages and in the asexual parasites. PfPKA activity can be
readily detected in schizonts and the parasite enzyme can be
(A)
(B)
(C)
Fig. 5. 16 Parasitemia and parasite invasion
analysis in Plasmodium falciparum under
ubiquitin proteasome system (UPS) inac-
tivation. (A) Highly synchronized cultures
of mature schizonts (44 hpi) were incu-
bated with 100 nm bortezomib (BTZ) for
6 hR. Light microscopy images revealed
that the UPS inhibition impaired the
merozoite egress from mature schizonts
whereas in DMSO-treated cultures, there
was a clear RBC re-invasion after the
complete merozoite release. (B) Synchro-
nous schizonts (44 hpi) were treated with
50, 100 and 200 nm BTZ for 6 hr and
submitted to ow cytometry analysis to
quantify parasitemia percentage. Control
cultures were treated with DMSO solvent.
YOYO-1 uorescent dye was used to stain
the DNA. The results showed that the
parasitemia was twofold reduced approx-
imately in schizonts after UPS inhibition
by the three BTZ concentrations.
(C) Histogram plots from ow cytometry
analysis show only one population repre-
senting mature schizonts after 6 hr of BTZ
treatment. By comparison, two popula-
tions, ring and mature schizonts, were
observed for nondrug and DMSO-treated
controls. Statistical analysis was carried
out by t-test. *P < 0.05. Scale
bar = 5 lm.
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stimulated by cAMP [52]. These data may explain the
dierent modulation of PfNF-YB expression by cAMP
signalling throughout the intr-erythrocytic stages.
Protein ubiquitination strongly increases as a result of
polyubiquitin gene transcription upregulation in trophozoite
to schizont forms of P. falciparum [21]. In fact, our data
showedthat PfNF-YBis ubiquitinatedinmature trophozoite
(34 hpi) where the UPS is more active. Our results demon-
stratedthat melatoninincreases PfNF-Ybexpression(Fig 2).
Moreover, we show that the ubiquitination of PfNF-YB
protein is augmented in melatonin-treated parasites. Never-
theless, we cannot arm that this increase in PfNF-YB
ubiquitination is attributed to the melatonin-induced poly-
ubiquitination or protein upregulation, or both.
The impairment of merozoite egress from mature schizont
treated with BTZ can be a result of diverse events such as
modication of a regulatory signal for cell proliferation,
transcription, DNA repair, intracellular tracking, endocy-
tosis and/or signal transduction[24], as aconsequence of UPS
inhibitionbyBTZ. The dierent levels of reductioninmRNA
PfNF-YBabundance throughout intra-erythrocytic stages in
the presence of BTZ may be generated by two ways: (i) by
aecting the proteasome-mediated degradation of PfNF-YB
repressor and (ii) by global inhibition of acid nucleic
synthesis. Interestingly, only inthe schizont form, the protein
abundance does not decrease, indicating that PfNF-YB
protein levels are, to some extent, regulated by UPS. These
data led us to postulate that the mechanism of PfNF-YB
regulation by UPS may involve dierent pathways in ring/
trophozoite stages when compared with schizont stage, thus
demonstrating the complexity of PfNF-YB modulation and
its distinct role in P. falciparum cell cycle control.
Melatonin hormone concentration changes because of the
circadian rhythm in host. This variability is a key signal that
controls the synchronous maturation of Plasmodium in vivo
[3]. As malaria parasite senses plasma melatonin concentra-
tion, inhibition or suppression of nocturnal melatonin is
suggested as one of the ways to prevent growth and
development of malarial parasites [53]. Srinivasan and
collaborators [54] have recently reviewed Plasmodiumsignal-
ling cascade by melatonin and suggest that this is a potential
target against malariaparasites. Our results support the claim
that melatonin acts in a set of signalling pathways in the
cytoplasm and reaches the nucleus of the parasite where it
leads to alterations in gene control. Moreover, melatonin
signalling promotes changes in the PfNF-YB activity by
ubiquitin post-translational modication.
In conclusion, our data clarify how melatonin and cAMP
signalling can act in favour of P. falciparum survival based
on the nding that cAMP and melatonin promote a
downstream response, modulating the expression of this
transcription factor probably by the ubiquitination process.
As PfNF-YB transcription factor is involved in cell cycle
division and transcriptional regulation, these data suggest a
mechanism by which melatonin signalling induces the
production of schizont forms, increasing parasitemia, and
modulates the transcription of genes related to the ubiquitin
proteasome system. Then, how the parasite orchestrates the
signalling to trigger PfNF-YB activation is a fundamental
question to dissect the biology of the human malaria
parasite. Therefore, melatonin antagonist or agonists can
be considered as promising targets for generating new
antimalarial drugs.
Acknowledgements
We thank Fundac a o de Amparo a` Pesquisa do Estado de
Sa o Paulo (FAPESP), INCT/INBEQMeDI and Pronex
Malaria for funding C.R.S.G., W.R.L., E.A., D.O.P and
M.S.M. Our many thanks to Hemocentro Hospital do
Servidor Pu blico for providing us blood and plasma.
Conict of interest
The authors declare that they have no conict of interest.
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Supporting Information
Additional supporting Information may be found in the
online version of this article:
Figure S1. NF-YB alignment of partial amino acid
sequence across various species. The sequences included in
the conserved domain that bind to CCAAT sequence in
promoter regions (blue), and the peptides used to
synthesized the anti-PfNF-YBantibody (red). The sequences
were acquired from GenBank for Homo sapiens
(AAA59930.1) and Mus musculus (CAA39024.1) and from
PlasmoDB database P. knowlesi (PKH_094400), P. vivax
(PVX_092925), P berghei (PBANKA_090260), P. yoelii
(PY05259), P. chabaudi (PCHAS_070380) and P. falcipa-
rum (PF11_0477).
As a service to our authors and readers, this journal
provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be re-organized
for online delivery, but are not copy-edited or typeset.
Technical support issues arising from supporting informa-
tion (other than missing les) should be addressed to the
authors.
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Click on the Add stamp icon in the Annotations
section.
Select the stamp you want to use. (The Approved
stamp is usually available directly in the menu that
appears).
Click on the proof where you'd like the stamp to
appear. (Where a proof is to be approved as it is,
this would normally be on the first page).
7. Drawing Markups Tools for drawing shapes, lines and freeform
annotations on proofs and commenting on these marks.
Allows shapes, lines and freeform annotations to be drawn on proofs and for
comment to be made on these marks..

How to use it
Click on one of the shapes in the Drawing
Markups section.
Click on the proof at the relevant point and
draw the selected shape with the cursor.
To add a comment to the drawn shape,
move the cursor over the shape until an
arrowhead appears.
Double click on the shape and type any
text in the red box that appears.