Novel Cyclic AMP binding proteins in Trypanosoma cruzi
Adriana V. Jägera# Javier G. De Gaudenzia#, Bárbara Mc Cormackb#, Jésica G. Mildb,c, Sergio
Pantanod, Daniel L. Altschulere*, Martin M. Edreirab,c,e*
#
These authors contributed equally to this work
*
Corresponding authors
a
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, UNSAM-
CONICET , Buenos Aires , Argentina.
b
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de
Buenos Aires, Ciudad de Buenos Aires, Argentina.
c
INGEBI-CONICET, Ciudad de Buenos Aires, Argentina.
d
Institut Pasteur, Montevideo, Uruguay.
e
Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh,
Pittsburgh, PA, USA.
Corresponding Author: Martin M. Edreira, Intendente Guiraldes 2160, Departamento de Química
Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellon 2,
Ciudad Universitaria, 1428 Ciudad de Buenos Aires, Argentina. Tel.: 54 11 4576 3300 ext 425
email: [email protected]
Daniel L. Altschuler, 200 Lothrop St., Pittsburgh, PA 15213, USA. Tel.: 1 412 648 8692. email:
[email protected]
Keywords: Trypanosoma cruzi, cAMP signalling, cAMP binding proteins, cAMP novel effectors.
Abstract
Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In
the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite’s
life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known
about cAMP’s mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A
(PKA) has been cloned and characterised in this parasite; however experimental evidence
indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new
cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a
combination of search methods 27 proteins with putative cAMP binding domains (CBDs) were
identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, consistent
with the conservation of a nucleotide-binding domain, and all the sequences segregated into two
main branches: one containing kinases-like proteins and the other gathering hypothetical proteins
with different or no apparent function. Comparative modelling of the strongest candidates
provides support for the hypothesis that these proteins may give rise to structurally viable
cyclic nucleotide binding domains. To gain structural insights on the topology of T. cruzi’s CBDs
we generated comparative models of the strongest candidates using PKA-RIα-cAMP as template.
Pull-down and nucleotide displacement assays validated experimentally these sequences as bona
fide cAMP-binding proteins, thus confirming the expression of new putative PKA-independent
cAMP effectors in T. cruzi.
1. Introduction
Chagas disease is a potentially life-threatening disease caused by the protozoan parasite,
Trypanosoma cruzi. Endemic to the Americas, it represents a serious health threat among people
living in poor rural populations in Latin America. In addition to an estimated 10 million infected
people and an alarming 25,000 deaths per year, at least 55,600 individuals from endemic areas
annually require etiological treatment against T. cruzi infection (WHO, 2007). Moreover, this
regional issue is now becoming global due to migration of infected people to developed countries
(Schmunis, 2007). There is currently no vaccine, and treatment is limited to two old anti-parasitic
drugs (nifurtimox and benznidazol) that have important limitations that include variable efficacy,
long treatment courses and toxicity (Bahia et al., 2014). For this reason, there is an urgent need for
new therapies, which requires the identification of new potential targets for the design of novel
drugs for anti-trypanosomal therapy.
T. cruzi has a complex life cycle involving four developmental stages that alternate between the
insect blood-sucking vector and the mammalian host. In the insect gut, a proliferative non-infective
form of the parasite (epimastigote) differentiates into a non-replicative infective form (metacyclic
trypomastigote). Metacyclic trypomastigotes gain access to the mammalian host through feces
contamination at the insect bite wound and infect a wide range of cells. Upon invasion of the host
cell, trypomastigotes rapidly differentiate into amastigotes, an intracellular form that first replicates
and then differentiates back into trypomastigotes. Trypomastigotes are released into the
bloodstream, spread and infect other tissues, and can be picked up by the bug vector when the
insect feeds from the host. The molecular mechanisms involved in the stage specific
transformations occurring in the T. cruzi life cycle are not completely understood (Clayton, 2010).
Several studies suggested the involvement of cAMP in proliferation/differentiation (Seebeck et al.,
2004). Early reports demonstrated a negative role for cAMP in epimastigote proliferation (RangelAldao et al., 1987; Rangel-Aldao et al., 1988); high cAMP levels blocked DNA synthesis (Oliveira
et al., 1984) and consistent with this observation, known parasite mitogens were able to decrease
cAMP levels (Oliveira et al., 1993). However, inhibition of protein kinase A (PKA, the only known T.
cruzi cAMP effector to-date) by genetic (PKI expression) as well as pharmacologic (H89)
approaches led to epimastigote death, instead of the expected rescue (Bao et al., 2008). Early
evidence also indicated a potential role for cAMP in T. cruzi differentiation: the passage from
epimastigote to metacyclic stage was accompanied by an increase in cAMP levels, an effect
mimicked by cAMP analogs, inhibition of PDE, and a peptide from hindguts of the insect vector
that activates adenylyl cyclase activity in T. cruzi (Fraidenraich et al., 1993; Gonzales-Perdomo et
al., 1988; Rangel-Aldao et al., 1988).
Similarly, Trypanosoma brucei, the etiological agent of the Human African Trypanosomiasis or
sleeping sickness, has a multi-stage life cycle that involves morphological changes as it is
transmitted among the mammalian host and the insect vector, usually the tsetse fly. In vitro
differentiation of T. brucei from replicating long slender bloodstream forms to non-dividing short
stumpy forms was induced by a soluble factor, SIF (stumpy inducing factor), which could stimulate
an immediate two- to threefold elevation of intracellular cAMP. Additionally, SIF effects could be
mimic by membrane-permeable cAMP analogues or the PDE inhibitor etazolate (Vassella et al.,
1997). More recently, the relevance of cAMP-mediated signalling in T. brucei was demonstrated by
the pharmacological validation of trypanosomal PDEs as drug targets (de Koning et al., 2012),
where inhibition of PDE by CpdA caused a dramatic increase of intracellular cAMP, immediately
stalling cell proliferation and triggering cell death within 3 days. Furthermore, in searching for
resistance to the T. brucei PDE inhibitor Gould et al. (Gould et al., 2013) identified a group of
kinetoplastid unique proteins (cAMP Response Proteins (CARPs)) as mediators of the cAMP
signalling involved in cell death. These results clearly show the significance of the cAMP cascade
as a novel target for antiparasitic drug development.
Although, much attention is focused on the putative machinery involved in synthesis (i.e. cyclase)
and degradation (i.e. phosphodiesterase) of cAMP (Gould and de Koning, 2011), the role of cAMP
effector pathways is currently unknown.
In mammalian cells, cAMP-dependent pathways are transduced by two ubiquitously expressed
intracellular effectors, the classic PKA and the more recently discovered Exchange protein directly
activated by cAMP (Epac), as well as cAMP gated-ion channels in specific tissues (Cheng et al.,
2008). However, in the case of Trypanosomatids, while reported evidence indicates the presence
of PKA-independent pathways, the genome of the parasite does not appear to have Epac or ion
channels, suggesting a highly unusual cAMP signalling mechanism (El-Sayed et al., 2005). This
prompted us to search for proteins containing cyclic nucleotide binding domains (CBDs) as
potential new cAMP effectors. In silico studies using the predicted T. cruzi proteome lead to the
identification of 27 proteins with CBDs. The strongest in silico candidates were experimentally
validated as bona fide cAMP-binding proteins. Therefore, for the first time, we were able to identify
new PKA-independent cAMP effectors in T. cruzi as potential targets for future intervention.
2 Materials and Methods
2.1. Sequence search and phylogenetic analysis
In order to identify T. cruzi proteins containing cNMP domains, the parasite’s complete predicted
proteome was run against the Simple Modular Architecture Research Tool (SMART,
http://smart.embl-heidelberg.de/) and the Prosite database (http://prosite.expasy.org/). Potential
cAMP-binding proteins were identified using the following cNMP binding motifs: Pfam PF00027,
Smart SM00100, Prosite PS00888, PS00889 and PSPS50042. As an additional method, a
selection of cNMP-containing sequences from T. cruzi, Escherichia coli, and Homo sapiens were
used in BLAST searches against trypanosomatid databases (TriTrypDB v5.1). BLAST hits having
log E-values less than 1E-9 were used in further analysis against Hidden Markov Model profiles
(HMMER 3.0). The HMMER searches were continued until no potential proteins with cNMPs could
be found. The final list of putative CBS proteins was generated from hits that emerged in searches
using at least two different methods. Multiple sequence alignments were performed using DNAStar
package with the default alignment parameters. For the calculation of protein distances, the
neighbour-joining method was used. Precent divergences between all pairs of sequence from the
multiple alignments were calculated, and the NJ method was applied to the distance matrix. The
unrooted
tree
obtained
was
plotted
using
Treeview
software
(taxonomy.zoology.gla.ac.uk/rod/treeview.html).
2.2. Molecular modelling of T. cruzi CBDs
Human homologues with known 3D structure comprising the putative CBD of each sequence of
the T. cruzi’s strongest putative cAMP binding proteins were identified using the HHpred 2.0
program
(Soding
et
al.,
2005).
The
closest
human
homologue
of
TcCLB.508523.80/TcCLB.507035.110 (different size alleles) and TcCLB.510297.110 was the
regulatory domain of PKAIα, which has been solved by X-ray crystallography and annotated with
the PDB code 3PNA (Badireddy et al., 2011). Both sequences match the template with Evalues of 5E-23 and 5.3 E-23, respectively. Homology models based on this template were
constructed using Modeller v9.11 (Eswar et al., 2007). The coordinates of the bound cAMP were
taken from the original X-ray structure after a structural alignment performed with STAMP (Russell
and Barton, 1992). To optimize the orientation of the side chains around the ligand, the cAMP
bound models underwent energy minimization and 10 ns of molecular dynamics simulation using
the hybrid solvation approach of the SIRAH force field (www.sirahff.com), as reported in
(Gonzalez et al., 2013). The final minimized models contained only two and one residues
outside allowed regions of the Ramachandran plots for sequences TcCLB.508523.80
TcCLB.508523.80
and
TcCLB.510297.110,
respectively
(see
supplementary
figures
XXRamachPlotCAMBIAR NOMBRE? and XX RamachPlotCAMBIAR NOMBRE?. PDB files of
both models are provided as supplementary material).
2.3. Cloning into the Gateway system
Full-length coding sequences and the isolated CBD of the putative cAMP binding proteins were
amplified by PCR from 100ng of total genomic DNA of T. cruzi CL-Brener, using iProof High-
Fidelity DNA polymerase (Biorad) or FastStart High Fidelity Enzyme Blend (Roche) respectively.
For TcCLB.508523.80/TcCLB.507035.110 the following primers were used:
8523 fw (5’-CACCATGTACGGGACCTTTTTTAAAGGG-3’)
7035 fw (5’- ATGGTGCTCGCGTACGGTGGG-3’)
In both cases, the reverse primer was: rev (5’-TCAAATTGCATCACCCTTTACATGGCATTTCC-3’)
For full-length TcCLB.510297.110 the primers were as follows:
0297 fw (5’-ATGCCCATGACATCATTTTCGTCGATCTC -3’)
0297 rev (5’-CTATCCACTCCCTGTACTCAAAGGGC-3’)
TcCLB.510297.110 CBD primers:
0297 fw (5’-TTTCCCACATCGTTGGTACTTCG-3’
CBD 0297 rev (5’-TTACAACCAGAGTTCCCGTCGTTCTA-3’)
PCR products were subcloned into the vector pENTR/D-TOPO or pCR8/GW/TOPO TA
(Invitrogen) and subsequently transferred by LR reaction to pDEST17 (Invitrogen) as destination
vector. Within this vector, the ORF is N-terminally fused, to obtain a his-tagged fusion protein.
2.4. Recombinant protein expression
Plasmids pDEST17-CLB.508523.80, full length or CBD, were introduced into E. coli BL21. One
colony was inoculated in 5 ml of LB with ampicillin and grown at 37ºC overnight. The next day a
1:20 dilution was grown at 37ºC to an optical density of 0.8 (600 nm), and induced by adding 0.5
mM isopropylthiogalactoside (IPTG) for 2-3 hours. 50 ml of the cultured bacteria were harvested by
centrifugation and stored at -80ºC until needed.
2.5. cAMP binding assay
Protein samples were prepared on ice or at 4ºC. Bacterial pellet was resuspended in lysis buffer
(Tris-HCl/NaCl 50 mM pH: 7,4; lysozyme 0,25 mg/ml; protease inhibitor cocktail (Roche); βmercaptoethanol 10 mM (Sigma); N-Lauroylsarcosine sodium salt 0,6% (Sigma)) and sonicated for
4 pulses of 15 seconds each in ice. The extract was centrifuged at 4,500 rpm for 20 min at 4ºC. 2%
of Triton X-100 was added to the supernatant and incubated for 30 min at RT with gently rocking.
15% of glycerol was added to the supernatant and stored at -80ºC. For the binding assays, 1 ml of
the lysate, containing approximately 1 mg of recombinant protein, was incubated with cAMPconjugated agarose resin (A0144, Sigma) at 4ºC over-night with rocking. The resin was extensively
washed with washing buffer (Tris-HCl/NaCl 50 mM, pH 7.4) and eluted with SDS-cracking buffer at
60ºC for 10 min. For the nucleotide displacement experiment, 100 mM of the indicated nucleotide
(Sigma) was added to the binding reaction.
2.6. SDS-PAGE and immunoblot
The eluted protein was separated on a 12,5% SDS-polyacrylamide gel and transferred onto a
polyvinylidene difluoride membrane (PVDF). Membranes were incubated in blocking buffer (TBS
containing 5% milk and 0,5% Tween 20) for 1 hour at RT. An anti-His antibody (Genscript) was
used as primary antibody (4ºC ON). Horseradish peroxidase-conjugated goat anti-mouse IgG was
used as secondary antibody (2 hours at RT). ECL Western blotting detection reagents (Pierce
Thermo Scientific) were used for visualization of the reactive bands.
3. Results
3.1. Identification of putative cAMP binding proteins in T. cruzi’s genome
In order to identify new potential cAMP effectors in T. cruzi, we performed an in silico search for
putative cAMP binding proteins using the whole parasite predicted proteome. A combination of
searching methods allowed us to identify several proteins with putative CBDs: 70 hits were
obtained from the SMART server, 24 hits from Prosite, 25 from HMMER profiles searches and 31
using BLAST algorithm (see Material and Methods). In total, 80 different putative cNMP binding
proteins were obtained by at least one of the mentioned methods (Table S1). From these
sequences, 27 hits were found in at least in two databases, 24 in three databases and 18 proteins
were found by all search methods (Table S1 and figure S1). Our final list of candidates included
putative cAMP binding proteins that were hits in at least two different databases (Figure 1).
Multiple sequence alignment of T. cruzi CBD using bacterial CBD CAP_ED as reference, showed
conserved sequence motifs within the core of the domain (Figure S1). Phylogenetic analysis
showed that these proteins segregated into two main branches, one containing hypothetical
protein kinases (Figure 2A, green boxes) and the other branch including hypothetical proteins with
and without other associated domain. On the other hand, phylogenetic analysis of the CBDs
presented a more homogeneous distribution, consistent with an evolutionary conserved
nucleotide-binding domain (Figure 2B). Similar to other organisms, putative effectors share a
conserved CBD that would allow the recognition of the cyclic nucleotide by proteins with different
functional domains; such as a PAS fold, phosphoglycerate kinase domain and domain of unknown
function, among others (Figure 1).
3.2. cAMP Binding Proteins in T. cruzi
As a first step to achieve our goal of finding new cAMP effectors of the PKA-independent pathway
in T. cruzi, we analysed the strongest cAMP-binding proteins from the in silico list of candidates.
The proteins presenting the CBD with the lowest E-value, according to the Pfam database
(http://pfam.sanger.ac.uk), were: TcCLB.510297.110 (E-value: 2.3E-13), TcCLB.508523.80 (Evalue: 8E-13) and TcCLB.507035.110 (E-value: 1.5E-13). TcCLB.510297.110 codes for a
hypothetical 1691 amino acids long protein with a molecular weight of 190 KDa, that presented
high RNA expression in metacyclic trypomastigote stages (http://tritrypdb.org)(Minning et al.,
2009). It holds two CBDs, the studied one, comprising amino acids 269 to 390 and a second
one (residues 408-513) of higher E-value. In addition, TcCLB.510297.110 contains C2 domains
(Ca2+-dependent membrane-targeting domain found in proteins that bind phospholipids and are
involved in signal transduction or membrane trafficking) (Figure 1).
TcCLB.507035.110 and TcCLB.508523.80 are alleles that share an overall 99% identity with a
100% identity within the CBD. TcCLB.507035.110 encodes a 708 amino-acids long protein, with
high levels of mRNA expression in trypomastigotes and metacyclic trypomastigotes; while,
TcCLB.508523.80 codes for a 218 amino-acids shorter protein. TcCLB.507035.110 and
TcCLB.508523.80 contain two CBDs within the share sequence (residues: 233-345/372-489 and
residues: 5-117/144-261, respectively). No other known function or functional domain could be
identified in these proteins (Figure 1). Noteworthy, in searching for resistance to the T. brucei PDE
inhibitor CpdA using a genome-wide RNA interference library screening approach, Gould et al.
(Gould et al., 2013) identified a group of kinetoplastid unique proteins (CARPs), as cAMP sensors
(Gould et al., 2013). Although cAMP binding was not experimentally verified, the prediction of the
presence of CBDs in CARP1 clearly suggested an important role in cAMP signalling in T. brucei.
Significantly, sequence analysis shown that TcCLB.507035.110 and TcCLB.508523.80 are
orthologs of T. brucei Tb427tmp.01.7890, the gene which codes for the potential cAMP effector
identified in [20] (http://tritrypdb.org).
3.3. CBD features in T. cruzi
In general, the CBD is a small conserved region of around 120aa that includes both β strands and
helical elements. The structure of the cAMP-binding site is organized as an eight-stranded β barrel
that function as a pocket for the nucleotide. One of the important features of the domain is the
phosphate-binding cassette (PBC) constituted by the β strand 6, a short turn of helix, and the β
strand 7. Within the PBC sequence there are few conserved residues such as an arginine that
binds to the exocyclic phosphate of cAMP and a pair Glycine-Glutamate that binds the ribose.
Moreover, it has been shown that the arginine has co-evolved with a set of glycines located in
different loops across the domain, of particular importance, a glycine in a distal β2-β3 loop that
allosterically couples cAMP binding to distal regulatory sites (Berman et al., 2005; Kannan et al.,
2007), features that are all conserved in the studied candidates. A multiple sequence comparison
between CBDs identified in T. cruzi and other known cAMP effectors showed the presence of the
key conserved motifs (Figure 3A). Notably, most of the conserved residues showed to be critical for
the structure of the cAMP pocket or the PBC (Figure 3A).
3.4. Molecular modelling of T. cruzi’s CBDs
Aimed at gaining structural insights on the topology of T. cruzi’s CBDs, we generated
comparative models using mammalian cAMP-bound PKA-RIα as template. A well-conserved
overall organization of the cAMP-binding pocket was observed. The alignment of
TcCLB.508523.80 against the PKA-RIα template (XXXCHECK THIS Figure 3A) presented an
insertion comprised between amino acids 57 and 67. This generates a more elongated loop
between β strands 4 and 5 (Figure 4B), which is the region of highest sequence and length
variability in the CBD family (Canaves and Taylor, 2002). In fact the homologue PKA-RIIβ
presents also a similarly long loop, which is not resolved in the electronic density of the
PDB structure 1CX4 (Diller et al., 2001). In contrast, TcCLB.510297.110 aligned without gaps
in the modelled region (XXXCHECK THIS Figure 3A), resulting in a higher similarity between
the structural model and template (compare Figure 4A and C and supplementary movie).
Placing cAMP within the binding sites of both protein models (see Methods) suggests that
the binding of the second messenger might be stabilized by both polar and non-polar
interactions alike those found in the known crystallographic structures. In particular, the
most highly conserved residues discussed in the previous paragraph are found in good
position to interact with the ligand (Figure 4 and supplementary movie). Only Tyrosine 173
in PKA-RIα is conservatively substituted by a Histidine within the  barrel that constitutes
the cAMP binding site (Figure 4 A, C).
The main difference encountered within the binding site was observed upon optimization of
the H-bonding pattern in the presence of cAMP and was not obvious from the sequence
analysis. We found that Alanine at position 173 in PKA-RIα is replaced by Serine 42 and
Cisteine 334 in TcCLB.508523.80 and TcCLB.510297.110, respectively (see insets in Figure
4). In both structural models , these Serine/Cysteine establish and H-bond with an Oxygen
in the cyclic phosphate group, putatively increasing the affinity for the negatively charged
moiety.
3.5. Predicted CBDs can bind cAMP
To examine whether TcCLB.510297.110 and TcCLB.508523.80/TcCLB.507035.110 (100% identity
within the CBD) represent bona fide cAMP-binding proteins, the full-length version of these
proteins were expressed as His6-tag fusions (E. coli expression vector pDEST17, Invitrogen), and
purified from BL21/DE3 E. coli strain. cAMP binding was tested by affinity chromatography with
cAMP-conjugated agarose resin (Sigma-Aldrich). As shown in Figure 4, binding of bacterially
expressed
His-tagged
domains
to
cAMP-conjugated
resin
corroborated
the
prediction
(TcCLB.510297.110 not shown). Moreover, nucleotide-binding specificity was verified by
displacement with free cAMP, cGMP and AMP (Figure 4). Due to the size and biochemical
characteristics of TcCLB.510297.110, the full-length protein could not be expressed in a soluble
state. Although, the isolated His-tagged CBD was able to bind cAMP (not shown).
4. Discussion
Cyclic AMP is one of the most widely studied signalling molecules and has been implicated in a
wide range of biological functions (Sutherland, 1972). For many years, PKA was the only known
cAMP effector. Accordingly, it was believed that all cAMP stimuli were mediated by this kinase. It
was not until the identification of Epac as a new cAMP effector [13], that cAMP-mediated effects
would be classified into two categories: PKA-dependent and PKA-independent pathways, acting
independently, converging synergistically or presenting opposite effects depending on specific
cellular environments (Cheng et al., 2008). Similarly, in T. cruzi the only characterized cAMP
effector is PKA (Bao et al., 2008; Bao et al., 2009; Bao et al., 2010; Huang et al., 2006). In
addition, the absence of sequences for Epac in the T. cruzi genome strongly contributed to the idea
of an exclusive PKA-mediated pathway for cAMP signalling in this parasite. However, supporting
experimental evidence suggesting the existence of other biologically active cAMP effectors,
prompted us to search for new putative cAMP effectors in the parasite genome.
As a result of the in silico search, several hypothetical cAMP binding proteins were identified. We
decided to clone and characterize the CBDs that showed the lowest E-value, representing the
strongest putative cAMP-binding proteins. With this in mind, we analysed and biochemically
evaluated TcCLB.508523.80, TcCLB.507035.110 and TcCLB.510297.110 as potential new cAMP
effectors.
Sequence analysis and topology models revealed that the CBDs present in these proteins could
fold in a similar manner than other described effectors, creating a stable pocket that would be able
to accommodate the nucleotide. More importantly, we demonstrated for the first time that full-length
TcCLB.508523.80 and TcCLB.507035.110, as well as the isolated putative CBDs of the three
candidates, were capable of binding cAMP. Additionally, the protein-nucleotide interaction was
shown to be nucleotide-specific, since it was only displaced by free cAMP and not by cGMP or
AMP. These findings provide biochemical validation of the characterized proteins as potential
cAMP effectors in T. cruzi.
Steady-state transcriptome of the four life stages of T. cruzi using microarrays (Minning et al.,
2009)
demonstrated
an
up-regulation
in
the
expression
of
TcCLB.507035.110
and
TcCLB.510297.110 transcripts in infective non-proliferative trypomastigotes and metacyclics,
respectively. Preliminary RNAseq data confirm that TcCLB.507035.110 transcripts are elevated in
trypomastigotes as compared to intracellular amastigotes at 24 hours post-infection and show
higher expression in trypomastigotes over epimastigotes as well (B. Burleigh and N. El-Sayed,
personal communication). Although RNA expression does not always correlate with protein
abundance, these results encouraged us to hypothesize that TcCLB.507035.110 and
TcCLB.510297.110 might be involved in signalling during invasion. As a result of the host-parasite
interaction a bi-directional Ca2+ signalling is triggered (Burleigh and Woolsey, 2002). It could then
be assumed that the product of the gene TcCLB.510297.110 might be participating in the early
signalling events that initiate the invasion of the host cell and are mediated by cAMP and Ca2+.
A genome-wide RNAi screening in T. brucei recently identified four cAMP Responsive Proteins
(CARP 1-4) (Gould et al., 2013). Of particular interest is CARP1, an orthologous to
TcCLB.508523.80/TcCLB.507035.110,
which
is
specific
to
kinetoplastid
parasites
(http://tritrypdb.org/tritrypdb/). Its characterization suggested, in accordance with our data, that
CARP1 would be a primary cAMP sensor and might play a role in cAMP signalling in the parasite
(Gould et al., 2013).
Ongoing in vivo characterization of these newly described putative effectors are expected to
uncover new aspects of the cAMP biology in T. cruzi and provide new insights into the
mechanisms of pathogenesis involved in Chagas disease. Due to the fact that cAMP pathways in
T. cruzi would present some unique features (Laxman and Beavo, 2007) through divergent
effectors comparing to its human counterparts, we also expect to identify a new set of potential
targets for drug design.
Acknowledgments
The authors would like to thank Barbara Burleigh for her valuable comments on the manuscript
and, together with Najib El-Sayed, for sharing unpublished data. We wish to thank Soledad N.
Gonzalez for participating in the first steps of the project. Research reported in this publication was
supported by the FIC-NIH award number R03TW009001 (to DLA/MME) and UBACyT-GEF
20020110200024 (to MME). SP was partially funded by FOCEM (MERCOSUR Structural
Convergence Fund), COF 03/11.
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Figure Legends
Figure 1. Identification of putative cAMP binding proteins in T. cruzi.
27 proteins were
identified in the parasite genome using a combination of search methods: HMMER and BLAST for
local similarity and Smart and Prosite as databases for protein domains. Proteins were identified
by at least two of the previously mentioned techniques and listed according to the Pfam E-values,
in descending order. In black and grey the cNMP binding domains detected by Pfam database
(http://pfam.sanger.ac.uk) are shown. In yellow the cNMP binding domains according to SmartDB
(http://smart.embl-heidelberg.de/help/smart_about.shtml) are shown. Brackets on the left indicate
pairs of paralogues, due to the hybrid genotype of the T. cruzi CL Brener clone. Green IDs, protein
kinases. Red, orange and blue IDs: potential cAMP-binding proteins analysed in this study.
Figure 2. Phylogenetic analysis. A) Phylogenetic analysis of putative cAMP binding
proteins. Alignments of the 27 full-length sequences were performed with the ClustalW algorithm
using the DNAStar package. The unrooted tree was plotted with TreeView software (see Materials
and Methods). Green box, protein kinases. Red, orange and blue boxes: potential cAMP-binding
proteins analysed in this study. B) Phylogenetic analysis of CBDs. The key is as for Figure 2 A
but CBDs were used instead of full-length sequences. Green box, domains present in protein
kinases. Red, orange and blue boxes: potential CBDs analysed in this study.
XX CAMBIAR Figure 3. A) Analysis of CBD sequences. Multiple sequence alignment between
CBDs identified in T. cruzi and other known cAMP effectors. An orange arrow indicates the Serine
H-bonding the phosphate group in TcCLB.508523.80/TcCLB.507035.110. Homology models of
T. cruzi’s CBDs. Comparative model of CBD present in TcCLB.508523.80/TcCLB.507035.110
(red)(B) and TcCLB.510297.110 (blue)(C) spanning residues 1 to 103 (light colours) superimposed
to the X-ray structure 3PNA (dark colours) [15]. Unstructured regions are depicted in white or grey.
cAMP, which was used to superimpose both structures is presented in thick sticks. Residues with
at least one atom within a distance of 0.5 nm of cAMP are shown as balls-and-sticks for T. cruzi
CBD and black thin sticks for 3PNA. An orange arrow indicates the Serine H-bonding the
phosphate group.
Figure 4. Structural models of T.Cruzi’s cAMP binding proteins.
A) Cartoon representation of the X-ray structure of PKA-RIα (PDB id:3PNA). cAMP is shown
as sticks, while the most conserved residues in Figure 3 are presented as balls and sticks
in pale colors (XXXOJO CON ESTO YO ESTOY MIRANDO LA FIGURA 3 VIEJA. LOS
AMINOACIDOS QUE SE MUESTRAN SON LOS QUE TENIAN BARRITAS ROJAS O
NARANJAS). The inset on the top shows a different perspective on the binding site. For the
sake of visual clarity, only the excluded volume of the ligand is indicated as
semitransparent
grey
surface.
B
and
C)
Idem
to A for
TcCLB.508523.80
and
TcCLB.510297.110, respectively. The label in the top insets indicates the position the
tyrosine substituted by histidine in TcCLB.510297.110. The insets in the bottom depict the
specific interaction between cAMP and alanine 210 in PKA-RIα (A), serine 42 in
TcCLB.508523.80 (B) and cysteine 334 in TcCLB.510297.110 (C). In B, the extra loop between
β strands 4 and 5 is highlighted with a dashed red oval. In B and C the hydrogen atoms of
S42 and C334 are explicitly included to show the formation of a Hydrogen bond with the
phosphate moiety.
XXFigure 5. cAMP-binding assay. Binding of bacterially expressed His-tagged full-length
TcCLB.508523.80 protein to cAMP-coupled to agarose beads. Input: protein bound to the resin in
the absence of nucleotide. Western blot probed with anti-His antibody shows displacement of
bound His-tagged protein from cAMP-agarose in the presence of competing cAMP but not cGMP
or AMP.
Caption to Supplementary Movie: Comparison between the X-ray structure of PKA-RIα (orange,
left) and the models of TcCLB.508523.80 (yellow, middle) and TcCLB.510297.110 (green,
right). Coloring and representations as in Figure 4. Only the excluded volume of cAMP is
shown as semitransparent surface.