In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.


Introduction
People all over the world are affected by leishmaniasis and American trypanosomiasis, two neglected tropical diseases that infect over 6 and 12 million people, respectively [1][2][3]. Several issues, including a lack of safe/optional drugs, parasite resistance, and ineffective insect vector control, have caused researchers to race to develop more effective treatments for diseases [4], increasing the current alternative studies with promising results [5][6][7]. These vector-borne diseases are caused by protozoan parasites of the Trypanosomatidae family, Leishmania spp. (Lspp) and Trypanosoma cruzi (Tc), which have unique characteristics. Insect vectors release infective parasite forms during the blood meal in humans, where they face harsh extracellular conditions and quickly invade cells to avoid extracellular immune response [8,9]. It is well established that these intracellular parasites infect different cells, activating the release of Ca 2+ from intracellular stores [10,11] from both parasite and host cells, stimulating different signaling pathways that promote critical interactions. Ca 2+ -dependent phosphatases have thus emerged as critical regulator molecules [12] for intracellular trypanosomatids.
In the case of the A1 catalytic and CaN regulatory subunits in Homo sapiens, the sequences NP_000935 and NP_000936 (National Center for Biotechnology Information (NCBI) were used. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; (1988)-[cited 6 January 2021]. Available from https://www. ncbi.nlm.nih.gov/ (accessed on 01 January 2021)).

Obtaining Consensus Sequences and Multiple Alignment of CaN
The consensus amino acid sequences of the catalytic A1, A2, and CaN regulatory subunits of T. cruzi and Leishmania spp. were obtained using the EMBOSS Cons tool (https: //www.ebi.ac.uk/Tools/msa/emboss_cons/ (accessed on 30 May 2020)) (Supplementary File 1) [40]. The multiple alignment of trypanosomatids and H. sapiens was performed in the MEGA7 software [41] using the default ClustalW parameters. The alignments obtained were viewed in the SnapGene Viewer software (version 5.1.3, from GSL Biotech; available at snapgene.com).

Evaluation of Conserved Domains, Determination of Physicochemical Parameters, and Hydrophobicity Profiles
The identification of the conserved domains of the sequences under study was carried out through the Conserved Domains Database (CDD) [42][43][44] and ExPASy ScanProsite (https://prosite.expasy.org/scanprosite/ (accessed on 30 May 2020)) [45]. The domain structure was represented by PROSITE 'MyDomains' image creator tool [46].

In Silico Identification of Protein-Protein Interactions of CaN Regulatory Subunits
The identification of interactions of the CaNB subunits of T. cruzi (accession number ABO14295) and L. major (accession number LmjF. 21.1630) was carried out in the search tool for the recovery of interacting genes/proteins, STRING V.11.0 (https://string-db. org/ (accessed on 11 June 2020)) [49]. The resources used were experimental results and databases, with a value of 0.7 (high confidence) established as the minimum interaction score [49].

Prediction of the Subcellular Localization of CaN in Pathogenic Intracellular Trypanosomatids
The subcellular location of the CaN subunits in T. cruzi and Leishmania spp. was carried out in WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 11 June 2020)) [51] with Fungi selected as the type of organism; in addition, the CELLO2GO tool was also used (http: //cello.life.nctu.edu.tw/cello2go (accessed on 11 June 2020)) by selecting Eukaryotes [52].

Prediction of Entry to the Non-Classical Secretory Pathway of CaN in Pathogenic Intracellular Trypanosomatids
The SecretomeP 2.0 Server tool (http://www.cbs.dtu.dk/services/SecretomeP/ (accessed on 11 June 2020)) was used to predict the entry of the different CaN subunits into the non-classical secretory pathway using the prediction for mammalian sequences [53] with a threshold of 0.6.

Prediction of Cleavage by Calpains in CaN Catalytic Subunits of Pathogenic Intracellular Trypanosomatids
The prediction of potential calpain cleavage sites was determined using GPS-CDD (version 1.0 available at http://ccd.biocuckoo.org/ (accessed on 7 July 2020)) with a high threshold with a cut-off of 0.654 [54]. As a reference sequence, the catalytic subunit alpha isoform from H. sapiens (accession number NP_000935) was used.

Prediction of Phosphorylation Sites in the CaM-Binding Domain (CaM-BD) of CaN Catalytic Subunits from Leishmania Spp.
Predicted phosphorylation sites were evaluated using NetPhos 3.1, a web server (http://www.cbs.dtu.dk/services/NetPhos/ (accessed on 27 July 2020)) [55,56]. Sites with a threshold >0.5 were included in the results.  (Table 1). This reflects a diverse structural degree, evidenced in the domain structure of the deduced amino acid sequences under study (Figure 1), when compared with the catalytic sequence of human CaN with its three domains (binding to CaNBm, binding to CaM, and to AID). The TcCaNA1 and TcCaNA2 subunits possess the CaNB-binding (CaNB-BD) and catalytic domain, in the same way as LsppCaNA2 from Leishmania spp. However, LsppCaNA1 and LsppCaNA1_var possess all the domains present in human CaN. Interestingly, CaNA2 are those that have a higher pI (isoelectric point) than their A1 counterparts (4.83-5.61), with pI of 8.13 for TcCaNA2 and 6.37 for LsppCaNA2. The potential calpain cleaved sites determined in GPS-CDD (from more sites to fewer sites) are as follows; 29 for LsppCaNA1, 12 for TcCaNA2, 9 for LsppCaNA1_var, 5 for LsppCaNA2, and 3 for TcCaNA1. HsCaNAα has 19 sites ( Figure 1).

Results
In relation specifically to the CaNB-BD domain, the hydrophobicity profile in Leishmania spp. is more similar to the human subunit (HsCaNAα) than in the catalytic subunits of T. cruzi, presenting the highest hydrophobic profile in LsppCaNA2 ( Figure 2F), when compared with the other catalytic subunits of CaN ( Figure 2). When CaNB-BD is analyzed among all sequences, we observe that there are two mainly hydrophobic regions that go from amino acid M347 to amino acid S373 (Figure 3, highlighted in squares). These hydrophobic regions are partially conserved, the first one (region 1, on the left) with a higher degree of conservation in relation to HsCaNAα. Particularly, in the sequence MDVFTWSLPFV (region 1) of CaNB-BD, the amino acid V 349 changes in every catalytic subunit of intracellular trypanosomatids, with only one residue (L 369 ) being conserved in the second hydrophobic region (region 2, EMLVNVLNICS) of HsCaNAα, except in TcCaNA1 ( Figure 3).   In the case of the CaM-BD domain in HsCaNAα (ITSFEEAKGLDRINERMPPRRDA), it is only present in Leishmania in the LsppCaNA1 and LsppCaNA1_var subunits with a level of identity ranging from 42-45%. Regarding the autoinhibitory domain (AID), the level of identity is considerably lower, being 17-22% in the LsppCaNA1 and LsppCaNA1_var subunits (Figure 4), lacking the important residues for the autoinhibitory function, D488 and A489 (dotted line box). The analyzed domain structure suggests that, in the case of Leishmania CaNA1, they would have the complete domain structure; however, the role of AID in these subunits is unknown.   Figure 5B). These sites can be potentially phosphorylated by protein kinase A (PKA) or by an unspecified kinase (unsp) in HsCaNAα and LsppCaNA1 (or also for LsppCaNA1_var), also including in the latter the potential action of PKC or casein kinase II (Table 2).

CaN Regulatory Subunits of Obligate Intracellular Trypanosomatids Differ in Their
Calcium-Binding Domains from Their Human Counterpart and Myristoylation Potential, but Preserve Some Canonical EF-Loops and the Docking Site for Immunophilin-Immunosuppressive Drug Complexes As has been described [57], HsCaNB-1 and LsppCaNB (based on the sequences obtained L. major) have four EF hand motifs, while TcCaNB has only three. HsCaNB-1 and LsppCaNB have two low loops and two high affinity loops for Ca 2+ , while TcCaNB only has one of each type ( Figure 6 and Table S1). The complete loops, based on the HsCaNB-1 sequence, meet the condition of having the amino acid organization distributed as follows: Moreover, the TcCaNB and LsppCaNB subunits have a hydrophobic groove for the union of the immunophilin-immunosuppressive drug complexes, highlighting the conservation of this region that goes from M 119 to L 124 (hydrophobic groove), corresponding to HsCaNB-1, being much more hydrophobic in TcCaNB and LsppCaNB than in HsCaNB-1, determined by the grand average of hydropathicity index (GRAVY), which is used to represent the hydrophobicity value of a peptide; positive GRAVY values indicate hydrophobic and negative values mean hydrophilic ( Figure 7).  . CaNB sequence alignment of the human CaNB (HsCaNB-1) against the different obligate intracellular pathogen trypanosomatids (TcCaNB and LsppCaNB). Conserved residues are in black with * (100% conservation), in black with: (>70% conservation), and in black with · (between 50 and 70% conservation). The consensus is shown with a threshold of 50%.
Regarding the potential myristoylation in the intracellular trypanosomatid CaNB subunits, only TcCaNB has a glycine (G) in the second position in the N-term in the same way as the HsCaNB-1 subunit. Curiously, despite the fact that the domain structures between HsCaNB-1 and LsppCaNB are similar to each other, CaNB in Leishmania spp. lacks G in its N-terminal region to be myristoylated ( Figures 6 and 7, Figure S1).
When a Myristoylator (trained to predict myristoylation in the N-terminal end of the amino acid chain) was used to analyze possible myristoylation, only HsCaNB-1 was predicted to be potentially myristoylated with a score of 0.98984294 (high confidence), while TcCaNB was predicted to be non-myristoylated with a score of −0.146144 (Table S2).

CaN Regulatory Subunits of Obligate Intracellular Trypanosomatids Interact Only with Their Catalytic Monomer and Related Immunophilins
A PPI network was generated for each of the regulatory subunits; the red nodes represent the proteins of interest and the rest represent those with which it interacts ( Figure 9); the score of each node represents the evidence of the interacting proteins. In order to validate the score obtained, a PPI network with HsCaNB-1 was built. Interestingly, for TcCaNB and LmCaNB (and other orthologs in Leishmania, Figure S2), the substrate potential has not been determined, as well as for NFATC1 in H. sapiens.

CaN Regulatory and Catalytic Subunits of Obligate Intracellular Trypanosomatids Have a Differential Potential to Be Secreted by the Non-Classical Pathway
The predictions obtained for each of the subunits under study suggest that the subunits TcCaNA1, TcCaNB, and LsppCaNA2 with an NN-score of 0.682, 0.637, and 0.718, respectively, are secreted by the non-classical pathway. Only in the case of HsCaNB-1 does it have an NN-score of 0.548, close to the threshold proposed for mammalian sequences (Table 4).

Discussion
Trypanosomatid parasites have evolved to survive in different environments as their biology has developed, either in vector insects or in mammalian hosts. These different environments involve different parasite forms that could have conditioned the appearance of specific trypanosomatid phosphatases [58]. Regarding CaN, through the analysis of the TriTryp phosphatome, two groups of CaN were identified; one of them grouped with H. sapiens and S. cerevisae, and the second with less similarity to eukaryotic CaN and with characteristic features of kinetoplastids, and presenting some mutations in its catalytic residues, suggesting that they could be pseudophosphatases [38]. CaN-like activities have been observed in vitro in some of these recombinant A2 subunits [33].
The domain structures of the catalytic subunits of T. cruzi (TcCaNA1 and TcCaNA2), in which each one of them presents the CaNB-BD and catalytic domain, have already been identified and characterized [32,33,37]. On the other hand, in Leishmania, the same analysis has determined that it possesses the four characteristic domains of CaN [37,59]. In the present work, we seek to highlight the differences at the level of the primary structure of the subunits under study and the proposed regulatory mechanisms. In this regard, mammalian catalytic isoforms have a molecular mass (MW) ranging from 57 to 59 kDa, whereas lower eukaryotes vary from 57 to 71 kDa [17]. In addition, the pI of the TcCaNA2 and LsppCaNA2 subunits (8.13 and 6.37, respectively) deviate considerably from the range of pI observed for mammalian isoforms. In the case of the isoform α (pI = 5.6-5.8), β1 (pI = 5.3), β2 (pI = 5.6-5.8), however, the isoform γ (pI = 7.1) specific to the testis possesses a higher pI [60]. The latter suggests a differential subcellular distribution between the CaN subunits of these protozoa by virtue of pI [61,62].
A transcendent regulatory mechanism for the action of CaNA is cleavage by calpain. Curiously, calpains and calcineurins share the same subcellular distributions and exhibit equally high levels of activity after many of the same types of insults, thus Ca 2+ and CaN-sensitive protease are considered common effectors of Ca 2+ -induced dysfunctions and degenerations [63]. Calpain is capable of cleaving CaNA directly and increasing its phosphatase activity [64], generating more active 57, 48, and 45 kDa products in excitotoxic neurodegeneration models [65]. From the potential calpain cleaved sites analyzed by GPS-CDD in the CaNA subunits of T. cruzi and Leishmania spp., only TcCaNA1 and LsppCaNA1 possess a cleavage site located between the catalytic domain and the regulatory domain of CaN, which could upregulate CaN phosphatase activity by removing the regulatory domain [66] or parts of it [67], while the other sites could have other regulatory effects or impair CaN activity if they are located in the catalytic domain of CaNA. On the other hand, the insertion 243-VSGGSGSDYYTPSAGPSYGS-262 present in TcCaNA2, but not in TcCaNA1, has 4 of the 12 potential sites to be cleaved by calpain [33], while the insertion 235-YNNVEEPSGETYVPRLGLF-253 in LsppCaNA2 does not present any possibility of being cleaved. The regulation exerted by calpains in the biological cycle of T. cruzi has been studied through the use of inhibitors such as MDL28170, which stops the growth of epimastigote forms [68], in the same way as the role attributed to the TcCaNA2 in epimastigote proliferation [33]. Moreover, using the inhibitor MDL28170, it was shown that it decreases the viability of blood trypomastigotes and affects metacyclogenesis as well as the adherence of the parasite to the luminal surface of the triatomine gut [69,70]. It also inhibits the growth and viability of promastigotes of L. amazonensis [71]. The involvement of these enzymes is so great that there is evidence to suggest trypanosomal calpains as good drug targets [72]. Despite this, it is important to consider that inhibitors such as MDL28170 also act on host calpains, which is why studies are required to determine how pseudoproteolytic trypanosomal calpains function and how calpain inhibitors act against them [73]. This reinforces the important role of molecules associated with Ca 2+ , such as calpains and CaN, in basic cellular functions of the life cycle of T. cruzi and Leishmania spp.
It has been described that CaNB-BD in the catalytic subunit αδ of rat brain has four hydrophobic amino acid residues (Val 349 -Phe 350 and Phe 356 -Val 357 ), which are essential for the interaction between the catalytic subunit and the calcineurin regulatory subunit [20]. This configuration allows two hydrophobic peaks to be generated in the CaNB-BD. In the case of CaNB-BD of the CaNA subunits of T. cruzi and Leishmania, the Val 349 residue of the HsCaNAα subunit is not present in their corresponding protozoan orthologs. This makes the hydrophobic profile of the surrounding residues different, which suggests that the degree of differential hydrophobicity of CaNB-BD present in the CaNA of T. cruzi and Leishmania conditions the interaction of the invariant CaNB subunit with the CaNA1 or CaNA2 subunits in the conformation of the heterodimers.
The diversity of CaNA in Leishmania that present or not the CaM-BD may establish a differential role in the cellular processes of the parasite. Interestingly, a calciumindependent, but CaM-CaN-dependent signaling pathway has been proposed in regulating the inversion of the flagellar wave, acting antagonistically to the cAMP-dependent pathway, so both of these pathways could establish an equilibrium between flagelar or ciliary churning waveforms, allowing appropriate motility and responses of the parasite in its environment, crucial for its viability, survival, and infectivity [35].
Unlike the CaM-BD present in HsCaNAα, which has the following sequence: ARKEVIRNKIRAIGKMARVFSVLREES, with conserved residues with positive charge (in bold) and potential kinase phosphorylation site (underlined) [21], which is recognized in the ARVFSVLRE context to be phosphorylated in S by an unknown kinase and in LREES by PKA and as well as in brain CaNA [74], the CaM-BD in LsppCaNA1 and LsppCaNA1_var have an identity of 50% and 53%, respectively, with only four residues with a positive charge; however, they have a greater potential to be targeted for phosphorylation by kinases such as PKA and PKC, either in serine or threonine. It may be a regulatory mechanism of the CaM interaction of the parasite towards the LsppCaNA1 and LsppCaNA1_var subunits, as has been described for other CaM-binding proteins [75,76].
In relation to the AID present in HsCaNAα, it has been proposed as a pseudosubstrate exerting an action mechanism that blocks the active CaN site [24], together with evidence of the existence of additional autoinhibitory elements between CaM-BD and AID [77]. In fact, an autoinhibitory element present in the α and β isoforms of human CaNA called ASI (autoinhibitory segment) has been described, having the sequence 416 ARVFSVLR 423 ; importantly, it interacts with a hydrophobic groove formed at the junction of the CaNA and CaNB subunits [78]. In LsppCaNA1 and LsppCaNA1_var, the amino acid sequence of this region was assigned as SRMFHTLC. This element could have a similar role in Leishmania spp., as the ASI potential presents three nonpolar residues (M, F, and L) of the total of five present in the ASI in the isoforms α and β of human CaNA, and the basic character of the R residue is present in both sequences.
In the case of CaNB, the domains' architecture possessed by CaNB of these trypanosomatids is different, with three EF hand motifs for T. cruzi and four for Leishmania spp. [32,37]. These structural characteristics suggest a differential activation mechanism for their corresponding catalytic subunits, which could be closely related to the associated functional roles, such as invasion, proliferation, response to stress, and motility, among others [32][33][34][35]. Interestingly, the analysis of the domain architecture in Trypanosoma rangeli, a non-virulent trypanosome for mammals, suggests the presence of four EF-hand motifs with a potential role in the growth of epimastigotes, and of these four EF-hand motifs, only three fulfill the expected pattern of amino acid residues involved in coordinating Ca 2+ [79]. In the case of HsCaNB, the analysis using the Prosite program attributes a lower score to the EH-hand motives towards N-term, while these are higher towards C-term, according to what has already been evidenced [80]. Thus, CaNB in these protozoa, unlike its counterparts in mammals and fungi that have four complete EF-hand motifs, has only two complete typical EF-hands, presenting the following "motif signature":
The conformation pattern of the EF hand motifs (the first damaged and odd followed by two functional ones) of TcCaNB is also observed in oncomodulin and parvalbumin, proteins that are phylogenetically related to CaNB [81]. Therefore, these low affinity sites in the CaNB subunit of these protozoa would have a conformational role towards the CaNA subunits, as has been described for rat CaNB; high affinity sites are always saturated with Ca 2+ , while the low affinity sites are regulated by the concentration of Ca 2+ . Thus, the binding of Ca 2+ to the low affinity sites affects the conformation of CaNB and, when associated with CaNA, induces a conformational change in the regulatory domain, which leads to the exposure of the binding domain with CaM. This conformational change, necessary for the partial activation of the enzyme in the absence of CaM, allows it to become fully active when associated with CaM [80,82].
Through the generation of mutants in the E (acid) residue at position 12 of each loop that binds Ca 2+ to L (basic) of HsCaNB, the individual role of each EF hand motif was determined (by eliminating the Ca 2+ binding capacity in each of them), showing that those mutations in the EF-hand motifs 1 and 2 (towards N-term) suffered variations in their electrophoretic mobility depending on the presence or absence of Ca 2+ . Meanwhile, in those with mutations in EF-hand 3 and 4 motifs (towards C-term), no differences were observed in the electrophoretic pattern, suggesting that Ca 2+ binding in EF-hand motifs 1 and 2 varies according to their concentration, dynamically modulating the enzymatic function, while EF-hand 3 and 4 would always be saturated with Ca 2+ , fulfilling a structural role [80]. Moreover, 45 Ca 2+ exchange is faster in EF-hand 3 than EF-hand 4 mutated, indicating that these sites are not equivalent and that the effects on heterodimer formation are greater in the EF-hand 3; this could explain the differences observed in the two functional EF-hands of TcCaNB, establishing a structural role for EF-hand 3 and a conformational role for EF-hand 4, and for LsppCaNB, a structural role for EF-hand 3 and 4 and a conformational role for EF-hand 1 and 2.
With respect to the subcellular distribution of CaN, in mammals, it associates predominantly in the cytoplasm and in the synaptosomal cytosol [83]. It has also been associated with synthetic vesicles, suggesting that CaN binds unilamellar vesicles, this being a Ca 2+dependent mechanism [84]. Regarding the NFAT activation pathway, it is shown that Ca 2+ induces an association between CaN and NFAT, which results in the colocalization of both molecules in the nucleus [85]. It is important to mention the location and differential expression of isoforms of the CaNA, as is the case of neuronal isoforms α and β. Isoform α was visualized at the nuclear level, while isoform β was found in the cytoplasm of a wide variety of cells of the central nervous system (CNS) [86]. These results suggest that each isoform would present different sites of action in the neurons of the CNS and that this phenomenon has been conserved during evolution. A clear example of this differential functionality is that observed in mice lacking the isoform β, in which alterations are generated in the function and development on immune system [87], while the mice lacking the α isoform suffer kidney dysfunction [88]. Therefore, it is possible to affirm that CaN isoforms can be differentially expressed and/or the activity of each one of them can be differentially regulated, allowing their specific function.
Studies in yeast have shown that CaN dephosphorylates the transcription factor Crz1p in vitro and that its location is displaced from the cytosol towards the nucleus subsequent to the in vivo activation of CaN, demonstrating that CaN regulates the phosphorylation and localization of Crz1p, and identifies Crz1p as the first CaN substrate protein [30]. Furthermore, their observations reveal that the mechanism by which CaN regulates gene expression in yeast and mammalian cells is strikingly similar [30]. Curiously, in parasitic protists, they do not appear to possess yeast Crz1 or mammalian NFAT orthologs in their genomes and lack transcription factors [31], suggesting that the CaN cascade may function in ways other than factor-mediated regulatory mechanisms of transcription that control the virulence of parasitic protists owing to the considerable evolutionary distance compared with fungi [12]. However, some CaN targets have been proposed for Plasmodium falciparum including HSP90, actin-1, and phosphoglycerate kinase [89]; in T. cruzi, CaN would act during the process of cell invasion on protein targets of high molecular mass [32], while in Leishmania spp., it has not been addressed.
Regarding the characterization of some CaN subunits in kinetoplastids, this has been described in Leishmania spp. and in T. cruzi [58], and in T. brucei, there is information on the cellular localization by microscopy of the CaN subunits: for TbCaNA1 (Tb927.9.1540) in cytoplasm and flagellar cytoplasm; for TbCaNA2 (Tb927.10.6460) in cytoplasm, flagellar cytoplasm, and nuclear lumen; and for TbCaNB (ID Tb927.10.370) in endocytic compartmen and cytoplasm (see TrypTag.org database). In the case of Leishmania donovani, CaN has a cytosolic localization in promastigote forms that depends exclusively on Ca 2+ and CaM for its activity, which was biochemically evidenced [90]. In T. cruzi, the TcCaNA1 subunit presents an evidently nuclear cellular localization in epimastigotes and amastigotes [37], and TcCaNA2 presents a cytosolic localization in epimastigotes [33], and in the case of TcCaNB, a predominant localization was observed to be cytosolic with some accumulation in the kinetoplast (data not shown). These analyses support a probable co-distribution of the TcCaNA2 and TcCaNB subunits.
Through the in silico analysis of the amino acid sequences of the CaN subunits in T. cruzi and Leishmania spp., in the CELLO2GO tools [52] and WoLF PSORT [51], we sought to predict subcellular localization. Oddly, all CaN catalytic subunits of T. cruzi and Leishmania spp. have a marked predisposition for plasma membrane over cytoplasmic localization and, in the case of CaNA2, a strong extracellular component. These characteristics suggest that these phosphatases may be secreted and behave as virulence factors acting synergistically or independently in infected cells [91][92][93]. In the case of the TcCaNB, the analyses suggest that a localization in the chloroplast may be due to its proximity to the calcineurin B-like proteins present in plants, which have substitutions in their first EF-hand motif that seem to be unable to bind Ca 2+ [94] with the second incomplete EF-hand motif and the third and fourth complete motifs analyzed in Prosite ( Figure S3).
Studies have shown that several extracellular proteins can be exported without possessing a classical N-terminal signal peptide, such as FGF-1, FGF-2, IL-1, and galectins [53]. In the case of T. cruzi and Leishmania, studies indicate that they produce ectosomes, exosomes, and other soluble proteins not associated with vesicles from which they are released to deliver cargo on the host cells [95][96][97][98][99]. Studies on Leishmania spp. suggest that the exosome pathway is an unconventional protein secretion mechanism as most of the exosome proteins do not contain a predicted signal peptide [95]. The analyses carried out give a secretion potential through the non-classical pathway to TcCaNA1, TcCaNB, and LsppCaNA2 (NN-score > 0.6); in the case of LsppCaNA1 and HsCaNB, they have a close score, which argues in favor of the potential of these molecules to modulate the host cell, as in the case of some phosphatases described in T. cruzi [100]; particularly, the T. cruzi secretome has revealed the presence of Ca 2+ binding proteins such as calreticulin (gi|322823951) involved in host-parasite interaction, a calcium-binding protein (gi|487896), calmodulin (gi|10386) involved in cell signaling, and a calpain-type cysteine peptidase (gi|322830271) whose role is associated with proteolysis [97], with a characteristic of these proteins being the presence of EF-hand motifs. One of these proteins that has acquired a preponderant role as a modulator of the functions of the extracellular microenvironment is calreticulin (TcCRT), mediating the infectivity of the parasite to inhibit complement; it is also antiangiogenic and inhibits tumor development in vivo [101].
Several data have shown similar host responses against Leishmania and tumor progression [102,103]. Both parasite infection and tumor cells have to deal with the immune response to proliferate and survive, showing the importance of controlling immunological response favoring both models [104,105]. However, target-signaling mechanisms could function in opposite directions when activated by its heterologous molecules [106]. In this scenario, the release of an active CaN could participate by blocking or activating signaling pathways at far distances to promote resistance or susceptibility. This is not uncommon, as the discovery of the role of extracellular vesicles by transporting active molecules [107] shows a sophisticated and broad way of communication for parasites [9] and tumor cells [105]. Recently, HsCaNB has acquired interest as it has been attributed anti-tumor roles, categorized as immunity-mediated killing (extracellular role) and direct pro-apoptotic killing (intracellular role) [108]. In their extracellular role, it was determined that HsCaNB is a ligand of the αM integrin, which corresponds to a subunit of the heterodimeric integrin αMβ2, a receptor expressed mainly on the surface of innate immunity cells (macrophages, monocytes, neutrophils, and NK cells), suggesting that the levels of HsCaNB in the serum are important for the maintenance of the innate immune response and the immune surveillance of cancer, through the activation of the monocytemacrophage axis [109]. It was then shown that exogenous HsCaNB uptake depends on the TLR4/MD2/CD14 receptor complex, indicating a second membrane receptor (TLR4) plays a critical role in innate immunity and its connection with adaptive immunity [110]. Recently, recombinant HsCaNB (rhCNB) has been implicated in the inhibition of the proliferation of gastric cancer cells and hepatomas, through the induction of apoptosis and arrest of the cell cycle, as rhCNB promotes the expression of p53 and decreases the expression of cyclin B1 and cyclin-dependent kinase 1 (Cdk1), contributing to arrest in G2/M [111]. Through the generation of rhCNB truncates, the domain that mediates internalization called Trun3 was identified, which is captured by tumor cells and directed to tumors with almost the same efficiency as untruncated rhCNB, thus being a perfect antitumor candidate [112]. These studies suggest that these trypanosomatid CaNB subunits (particularly TcCaNB owing to their potential secretion) could play a role as mediators of the host immune response (among other potential mechanisms of action yet to be elucidated) during parasite infection and tumor formation.
With regard to the protein-protein interactions determined for TcCaNB and LmCaNB based on the parameters used, the predictions establish contact between the CaNB subunits and the CaNA subunits, and between the CaNB subunits and peptidylpropyl isomerases. In particular, in the case of TcCaNB, the predicted functional partners XP_810491.1 (allele of TcCLB.508413.40) and XP_808861.1 (allele of TcCLB.510755.138) are putative sequences that have a 98-99% identity with TcCaNA1 [37], while STRING does not predict the interaction between TcCaNB and TcCaNA2, as evidenced in vitro by far-Wester blotting [33]. Nevertheless, the other predicted functional partners were proteins with a peptidylprolil isomerase function FKBP type, among them XP_810893 described as TcMIP, which is homologous to other FK506 binding proteins [113], behaving as a virulence factor that is secreted only by trypomastigotes [114], and those parasites exposed to the cyclophilin TcCyP19-trialisin complex show a greater capacity to invade the host cell through activation the parasite CaN pathway [115]. Curiously, the localization of TcCaNB is mainly in the vicinity of the flagellar pocket (unpublished results) in infective and replicative forms, strongly suggesting that these T. cruzi proteins are secreted by events restricted to the flagellar pocket [116] such as the case of cruzipain [117].
Finally, the search for a new potential target as a parasitic CaN should have the objective of developing drugs that act specifically on pathogens while being non-immunosuppressive [12]. Another important aspect is that the drugs act directly on CaN, to avoid the effects of CsA and FK-506 on the peptidyl-prolyl-cis-trans isomerase activity of their related immunophilins (as mediators of the interaction with CaN), which is why the structure-based design of a highly selective inhibitor directed to the catalytic site of CaNA or on CaNB [118,119] are strategies that could specifically target T. cruzi and Leishmania.

Conclusions
In this work, it was possible to establish that the domain structure is diverse among the catalytic subunits of CaN of intracellular trypanosomatids, thus establishing potential different post-translational regulation mechanisms observed in the analysis of cleavage by calpains, or by the phosphorylation patterns in the regulatory domains of the catalytic subunits, particularly in the A1 subunits in Leishmania (LsppCaNA1 and LsppCaNA1_var). In the case of regulatory subunits, the domain structure is different, with LsppCaNB being more similar to the human regulatory subunit (HsCaNB-1) than to TcCaNB, although the binding affinity for Ca 2+ is conserved between TcCaNB and LsppCaNB. On the other hand, although the coupling sites to the immunophilin-immunosuppressive drug complexes are present in TcCaNB and LsppCaNB, the myristoylation potential is only found in HsCaNB-1.
Regarding the analysis of the subcellular distribution, the catalytic subunits of T. cruzi and Leishmania spp. preferentially (more than 50%) present a localization in the plasma membrane (with the exception of TcCaNA2a with only 27.2%), different from HsCaNAα, which is predominantly cytoplasmic (51.7%). In the case of regulatory subunits, the distribution was more heterogeneous, being more associated with chloroplast and cytoplasmic for LsppCaNB (33.8% and 26.5%, respectively) and preferentially in cytoplasm (48.6%) for TcCaNB, when comparing the nuclear/cytoplasmic localization of HsCaNB-1 (53.8% and 38.6%, respectively).
Besides the catalytic subunits, the interaction with molecules with peptidylprolyl isomerase activity, which is typical of cyclophilins and FKBPs, is confirmed in the analysis of TcCaNB and LsppCaNB potential interactors. Concerning the potential secretion, TcCaNA1, TcCaNB, and LsppCaNA2 can be secreted by the non-classical pathway, suggesting new extracellular roles for these protein phosphatases.
On the basis of these in silico data, differential CaN regulation mechanisms are established between these protozoa and its human counterpart, complementing the knowledge of this phosphatase, promoting the development of new potential pharmacological targets to combat neglected diseases caused by these intracellular trypanosomatids.