Introduction

Schistosomiasis is a neglected tropical parasitic disease of major public health importance [1–3] caused by blood flukes of the Schistosoma spp., with 258 million people requiring treatment worldwide, and 780 million at risk of infection (http://www.who.int/mediacentre/factsheets/fs115/en/). There is currently no effective vaccine against schistosomiasis [4] and the treatment of the disease relies on a single drug, praziquantel (PZQ). Because of the intensive use of PZQ, field observations show the appearance of schistosome strains resistant to PZQ [5, 6]. Thus, the development of new drugs is imperative.

We have used a “piggy-back” strategy that consists in identifying orthologues of proteins already targeted in other pathologies, like cancer. Among these, we have chosen to target enzymes involved in epigenetic processes [7] and in particular, histone deacetylases (HDAC), which are among the most studied epigenetic targets. Schistosoma mansoni possesses three class I HDACs (SmHDAC1, 3 and 8) and four class II HDACs, two class IIa and two class IIb enzymes [8, 9]. We have shown [10] that Trichostatin A (TSA), a pan-inhibitor of HDACs, induces hyperacetylation of histones, deregulates gene expression and induces the death of schistosome larvae and adult worms in culture. Schistosome HDACs are therefore promising targets for the development of new drugs against schistosomiasis, especially SmHDAC8 [11, 12]. Indeed, it is the only schistosome class I HDAC for which the structure of its catalytic pocket differs significantly from that of the human orthologue [13], allowing the development of selective inhibitors that are toxic for schistosome larvae (apoptosis and death) and adult worms (changes in the reproductive organs, separation of worm pairs and arrest of egg laying) in culture [13–15]. Moreover, transcript knockdown of SmHDAC8 leads to markedly reduced parasite viability and fecundity [13] suggesting that this enzyme is essential to parasite growth and development.

Human HDAC8 (hHDAC8) catalyzes the deacetylation of lysine residues within histone and non-histone proteins but its biological role has long remained elusive [11]. The two best-characterized non-histone substrates are the estrogen-related receptor (ERRα) and the structural maintenance chromosome 3 protein (SMC3). hHDAC8 interacts directly with ERRα in vivo and deacetylates ERRα in vitro, increasing its DNA binding affinity [16]. In the case of SMC3, a member of the cohesin complex, hHDAC8 is involved in its deacetylation that allows the recycling of the cohesin complex, and hHDAC8 mutations are linked with the Cornelia de Lange syndrome [17, 18]. Two recent studies using more systematic approaches identified novel hHDAC8 substrates. The first [19] detected seven proteins all of which are nuclear proteins, including SMC3, and this approach failed to identify histones or ERRα as substrates. The second study [20] detected 19 novel hHDAC8 substrates, not all of which were nuclear. Among them, the cohesin complex component SMC1A, but also all the protein substrates identified by Olson [19] were predicted by this approach. Clearly, each approach provides different information about the hHDAC8 substrates.

In order to characterize the protein partners of the 11 human HDACs, Joshi et al. [21] performed a global study of their interactions by establishing T-lymphoblast cell lines stably expressing Enhanced Green Fluorescent Protein (EGFP)-tagged hHDACs combined with proteomics and functional studies to identify hHDAC-containing protein complexes. They showed that in these cells hHDAC8 interacts with 15 proteins: four related to the cell cycle including SMC1A and 3, three related to protein and ion transport and 8 with other/unknown functions. However, although these proteins are members of hHDAC8-containing complexes, the methodology used cannot distinguish whether or not they interact directly with the enzyme. Moreover, human acetylome analysis [22] interestingly reveals that among these partner proteins, SMC1A, SMC3, SA2, SEC16A and NUP98 are acetylated but only SMC1A and SMC3 have been identified as hHDAC8 substrates [19, 20].

SmHDAC8 shows major structural differences compared to hHDAC8, most obviously in the presence of insertions within the catalytic domain that form loops at the surface of the protein [13] and therefore represent potential surfaces for protein-protein interactions. We therefore sought to determine whether SmHDAC8 interacts with the same or different proteins than hHDAC8. Since it is not possible to overexpress a tagged “bait” protein in schistosomes we decided to use two different methods to identify protein partners, yeast two-hybrid screening (Y2H) and co-immunoprecipitation experiments coupled to mass spectrometry (Co-IP/MS). The former technique characterizes direct protein-protein interactions and the latter identifies proteins that may interact directly or are members of protein complexes that interact with the target protein. They therefore yield different results, but in combination give an overall picture of the cellular processes in which SmHDAC8 participates. Our results show that SmHDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes, and contribute to the understanding of its status as a therapeutic target.

Methods

Results and discussion

The aim of our study was to identify SmHDAC8 partners in order to better apprehend its biological role and function. We used here two strategies to characterize the SmHDAC8 interactome.

The yeast two-hybrid (Y2H) screening using SmHDAC8 revealed a large number of positive clones, of which we sequenced 137 after the prescreening step. After manual correction and assembly using Sequencher, we characterized 49 different sequences, which were then identified and functionally annotated using Blast 2GO software (S1 Table).

Three independent co-immunoprecipitation (Co-IP) experiments were performed, using an anti-SmHDAC8 antibody (named IP1, IP2, and IP3). As a control, we performed Co-IP with a rat IgG antibody alone in each experiment. Mass spectrometry (MS) of the Co-IP proteins identified 1,500 different proteins (S2 Table). A significant degree of variation between the proteins identified in each experiment was noted, for which several reasons can be invoked. The three parasite protein extracts were each obtained from a pool of S. mansoni adult worms of both sexes and not from homogeneous cell cultures. Variations in protein expression between worm batches could induce differences between the three Co-IP/MS experiments. Moreover, S. mansoni is a complex multicellular parasite and protein quantities can vary between the different cellular types within a given individual as well as between different worms. Therefore, to take account of this relative variability between each extract, we chose to pool the results obtain for the three Co-IP/MS experiments IP1, IP2 and IP3. The possibility that the observed variability may have been due to non-specific interactions of our anti-SmHDAC8 antibody with other proteins can be discounted. A single band corresponding to the molecular weight of SmHDAC8 was detected on western blots of the immunoprecipitated material (S1 Fig) and the only HDAC detected by MS in the immunoprecipitates was SmHDAC8 (S2 Table). Of the 1,500 proteins for which peptides were detected we selected only those that fulfilled three criteria: (i) at least three peptides in the Co-IP experiment, (ii) with no more than two peptides in the control and (iii) with a spectral count ratio between Co-IP SmHDAC8 and control of greater than 3. After, this selection step we obtained 160 different proteins that were considered good candidates as SmHDAC8 partners (S2 Table).

Among the proteins identified by these two approaches, four are common between Y2H and Co-IP/MS: the Proliferation-associated protein 2G4, 38kDa (PA2G4, n°G4LXR6), Cathepsin-B1 (SmCB1, n°Q8MNY2), putative NADH-ubiquinone oxidoreductase (n°G4VK53) and microsomal glutathione S-transferase 3 (GST-3, n°G4VH65) (Fig 1).

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Fig 1. Venn diagram presenting the proteins identified by Y2H and Co-IP/MS as SmHDAC8 interactors.

Forty-nine and 160 different proteins were identified by Y2H and Co-IP/MS respectively. Four proteins are common between Y2H and Co-IP/MS: the Proliferation-associated protein 2G4, 38kDa (PA2G4, n°G4LXR6), Cathepsin-B1 (SmCB1, n°Q8MNY2), putative NADH-ubiquinone oxidoreductase (n°G4VK53) and microsomal glutathione S-transferase 3 (GST-3, n°G4VH65).

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Among these proteins, two illustrate the previously characterized roles in HDAC-dependent processes and/or the potential involvement of acetylation. (i) PA2G4 is highly conserved in eukaryotes. The human member of this family, ErbB3 binding protein 1 (Ebp1) was identified as a putative downstream member of an ErbB3-regulated signal transduction pathway [26]. More particularly, the C-terminal region (300–372) of Ebp1, which is important for transcriptional repression, was shown to bind HDAC2 and inhibitors of HDACs significantly reduced Ebp1-mediated transcriptional repression [27, 28]. Like its human counterpart, the interaction between PA2G4 and SmHDAC8 is mediated by its C-terminal moiety, corresponding to the fragment cloned in the Y2H screen. (ii) SmCB1 is an essential gut-associated peptidase that digests host blood proteins as a source of nutriments (note that we also detected, but only with the Co-IP/MS experiments, cathepsin D and L2 which are also part of the gut peptidase network) and is also a drug target because enzyme inhibition induced severity phenotypes in the parasite [29]. Moreover, SmCB1 is a promising vaccine candidate because its administration elicits protection against S. mansoni challenge infection in mice and hamsters [30, 31]. A direct link with HDACs has never been reported. Nevertheless, acetylome analysis from Schistosoma japonicum reveals that Cathepsin-B (n°Q7Z1I6) is acetylated on K241 and sequence alignments between SjCB1 and SmCB1 show that it is conserved.

The lack of convergence between Y2H screening and Co-IP/MS is perhaps not entirely surprising since they represent very different methods for interactome studies. Both strategies have weaknesses. In Y2H screening, direct interactions between two proteins are detected, but in some cases, these may be non-specific due to the juxtaposition of proteins or fragments that are never in contact within the cell. In the case of Co-IP/MS, some proteins cannot be identified because they are present in low quantities in the parasite extract and some proteins identified may be members of immunoprecipitated complexes and are not direct partners. The results obtained can therefore be considered as complementary and, taken together, provide an overall picture of the cellular processes in which SmHDAC8 is involved.

For some of the partner proteins identified only by Y2H and not by Co-IP/MS screening we carried out independent experiments to verify the interaction with SmHDAC8. For instance, the binding of SmCtBP, SmMBD2, tensin and actin-1 proteins to SmHDAC8 was verified by candidate-specific Y2H experiments. As expected, SmHDAC8 indeed interacted with SmCtBP and SmMBD2, as well as the other two proteins (Fig 2). These results are in agreement with available data for the human orthologues. (i) The human C terminal binding protein (CtBP) family members appear to mediate transcriptional repression in a histone deacetylase (HDAC)-dependent manner [32]. Some human class I HDACs, HDAC 1, 2 and 3 [33–35] and class II HDACs (HDAC4 and 5) [36] are present in the CtBP1 nuclear protein complex, but the possible involvement of hHDAC8 was not investigated. (ii) The methyl-CpG binding domain protein 2 (MBD2) binds to methylated DNA and represses transcription through the recruitment of NuRD co-repressor complex [37]. The MBD2-NuRD complex contains a histone deacetylase core composed of HDAC1/2, RbAp46/48 and MTA2 [38–41]. The MBD2 protein possesses, among others, an intrinsically disordered region (IDR) able to recruit RbAp48, HDAC2 and MTA2 [42]. More particularly, it is possible that HDACs bind directly to the MBD2 IDR, because the human HDAC interactome study reveals a specific interaction between MBD2 and HDAC1/2 [21]. The Database of Protein Disorders (DisProt, www.disprot.org) [43] predicts an IDR domain located between the MBD and the C-terminal coiled-coil domains of SmMBD2, which may be involved in SmHDAC8 binding.

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Fig 2. Confirmation of interactions by candidate-specific Y2H experiments.

In order to confirm the screening results, the isolated clones CtBP, MBD2, tensin and actin-1 (cloned into pGADT7 vector) transformed in the AH109 strain were mated with the bait construct SmHDAC8 pGBKT7 transformed in the Y187 strain. After incubation, diploid yeasts were plated on selective medium lacking leucine and tryptophan (left panel) and then on another high stringency selective medium lacking adenine, histidine, leucine and tryptophan (right panel) showing, as expected, that SmHDAC8 interacts with the clones tested.

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Focusing on the biological processes predicted with the blast2GO software for each protein identified, among the 49 partners identified with the Y2H screening (S1 Table), seven were of unknown function, although one encoded a peptide including an EGF-like domain and an IgG-like domain. One further sequence corresponded to a hitherto unannotated gene. The remaining proteins are involved in 21 different biological processes (S1 Table), and those represented by the largest numbers of different sequences are cytoskeleton organization, transcription regulation, metabolism, transport, cell cycle regulation, DNA repair and chromatin remodeling (Fig 3).

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Fig 3. Biological processes involving the proteins identified by Y2H and Co-IP/MS as SmHDAC8 interactors.

Histogram (left for Y2H and right for Co-IP/MS) showing the biological processes in which the identified protein partners of SmHDAC8 are involved. The processes were defined using the Blast2GO software. Bars represent the protein percentage belonging to one biological process. Red and black bars highlight common and specific processes between Y2H and Co-IP/MS, respectively.

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The 160 proteins identified by Co-IP/MS (S2 Table) are involved in 33 different biological processes, and those represented by the most different sequences are metabolic process, cytoskeleton organization, proteasome, proteolysis, translation regulation, transport, protein dephosphorylation and stress response (Fig 3).

These results show that some of these processes (22%) are common between our different experiments, and in particular metabolism and cytoskeleton organization (Fig 3). Moreover, they suggest that SmHDAC8 is a versatile deacetylase involved in both cytosolic (cytoskeleton organization, ribosome, proteolysis or proteasome) and nuclear (transcription regulation, DNA binding and repair, chromatin remodeling or cohesin complex) processes. This contrasts with the current knowledge of partners and substrates proteins of human HDAC8 (hHDAC8) that emphasizes nuclear functions. The global interactome study [21] that identified 15 partner proteins and the recent systematic studies of hHDAC8 substrates [19, 20] mainly identified nuclear partner proteins involved in the cohesin complex, transcriptional regulation and chromatin remodeling. Some extranuclear proteins were identified as partners, such as the Ca²⁺-dependent phospholipid binding protein Copine III [21] or substrates, such as the elongation factor 1 EF1α1 [20] but these were in the minority.

Here, the involvement of SmHDAC8 in extranuclear functions is illustrated by the direct interaction between SmHDAC8 and proteins involved in cytoskeleton organization like tensin, actin-1, actin 5c and Rho1 GTPase (S1 and S2 Tables). As previously mentioned, the binding of tensin and actin-1 proteins to SmHDAC8 was verified by candidate-specific Y2H experiments. As expected, SmHDAC8 indeed interacted with SmActin-1 and SmTensin (Fig 2). Interestingly, Waltregny et al. have shown that hHDAC8 interacts with smooth muscle alpha actin (but not beta-actin) to regulate cell contractility [44, 45]. Proteomic analyses have shown that all three human actin isoforms (alpha, beta and gamma) are acetylated [22, 46]. Moreover, the actin-associated proteins cortactin [47] and anillin [20] are hHDAC8 substrates. Several regulatory proteins of actin polymerization in human are also acetylated (gelsolin, CapZ, profilin and the Arp2/3 complex) [22] and we found some orthologues in our SmHDAC8 Co-IP/MS analyses (gelsolin, septin and a subunit of the Arp2/3 complex, S2 Table). Actin dynamics are also controlled by small GTPases of the Rho family, like RhoA, notably in the formation of focal adhesions and stress fibers [48–50]. RhoA was not identified by Joshi et al. [21] as interacting with hHDAC8 and does not seem to be acetylated in human [22], although it was found to be acetylated in Schistosoma japonicum [51]. Here, our Y2H screen shows the direct interaction between SmHDAC8 and SmRho1, the orthologue of human RhoA. Hence, it is possible that binding of SmHDAC8 to SmRho1 could participate in the control of the SmRho1 pathway.

Among all the hHDAC8 substrates and partners identified [19–21], only Sec proteins, involved in endoplasmic reticulum protein secretory pathways were also found to interact with SmHDAC8, although these are different and phylogenetically distinct proteins: Sec16A in human and Sec1 and Sec61β in S. mansoni (S2 Table).

Strikingly, we found no orthologues of the major hHDAC8 substrates/partners already characterized among the protein partners of SmHDAC8. This is exemplified by members of the cohesin complex, which is notably responsible for the correct separation of sister chromatids into two daughter cells during mitosis [52]. The tripartite cohesin ring is formed by two SMC (structural maintenance of chromosome) proteins SMC1A and SMC3, and the α-kleisin subfamily protein RAD21 [53–55]. Both SMC3 and SMC1A were found in the hHDAC8 interactome [21], as is SA2 (an accessory protein that binds to hRAD21). In their study, hRAD21 was also detected, but at a non-significant level. Unexpectedly, we did not identify any of the cohesin complex members present in S. mansoni using our Co-IP/MS strategy contrary to Joshi et al. One possible explanation is that they used cells overexpressing EGFP-tagged hHDACs that generated a higher quantity of hHDAC8 protein than is present under physiological conditions. Correct MS identification depends strongly on the quantity of immunoprecipitated protein, and unfortunately, we are unable to increase SmHDAC8 expression in S. mansoni. However, and interestingly, our Y2H screen shows a direct interaction of SmHDAC8 with SmRAD21. In order to confirm the Y2H screening result we carried out a candidate-specific Y2H experiment with the SmRAD21 clone and, as expected, SmHDAC8 interacts with SmRAD21, suggesting that SmHDAC8 forms an integral part of the cohesin complex and may have a central role in cell division, transcriptional regulation and DNA repair.

In conclusion, the protein partners of SmHDAC8 identified by our Y2H library screen and Co-IP/MS experiments are all orthologues of proteins not previously identified as substrates or partners of human HDAC8. While we did not necessarily expect to detect substrates using these methodologies, it is striking that none of the partner proteins detected by Joshi et al. were among the SmHDAC8 partners. We propose that the principal reason for this is that the methodology used in the previous study, with tagged HDACs (as bait to pull down protein complexes) expressed in a T-lymphoblast cell line, may address a limited subset of potential partners. In contrast, our Co-IP/MS study was performed on adult worm protein extracts and S. mansoni is a complex parasite encompassing a variety of cell types.

These Co-IP/MS approaches do not necessarily identify the proteins to which the HDACs physically bind because some proteins identified can be part of immunoprecipitated complexes. This is illustrated by the cohesin complex members identified by Joshi et al. (SMC1A, SMC3, and SA2) and that do not include RAD21, which only appeared as a non-significant binder. This is the reason why we also performed an Y2H screen using SmHDAC8 in order to identify proteins that bound directly to SmHDAC8. Our Y2H shows that SmHDAC8 physically binds to SmRAD21 and we suggest that the other cohesin complex components are pulled down by this interaction. Against this, it can be argued that the Y2H screen can identify non-specific interactions due to the juxtaposition of proteins or fragments that are never in contact within the cell. Again, this is why we have followed up the results of the initial screen with more detailed investigations of the interactions of two of the most interesting candidates, SmRAD21 and SmRho1, allowing us to confirm that both are bona fide partners of SmHDAC8.

Despite the fact that we did not identify the same protein partners as Joshi et al., it would be unwise to assume that all those identified in the Y2H screen and in the Co-IP/MS experiment are specific for the schistosome enzyme. It is probable that a number of their human orthologues will interact with hHDAC8 when investigated individually. Nevertheless, given the structural differences between the schistosome and human enzymes, and particularly the unstructured loops at the surface of SmHDAC8, encoded by insertions in the catalytic domain sequence, it can be assumed that specific partners of the latter are present. Some of the proteins we identified have no human orthologues and have unknown functions. Further work will determine which of the partner proteins are schistosome-specific, how they interact with SmHDAC8 and the role of these interactions within the parasite.

Supporting information