Trypanosoma cruzi is a unicellular parasite that causes Chagas disease, which is endemic in the American continent but also worldwide, distributed by migratory movements. A striking feature of trypanosomatids is the polycistronic transcription associated with post-transcriptional mechanisms that regulate the levels of translatable mRNA. In this context, epigenetic regulatory mechanisms have been revealed to be of great importance, since they are the only ones that would control the access of RNA polymerases to chromatin. Bromodomains are epigenetic protein readers that recognize and specifically bind to acetylated lysine residues, mostly at histone proteins. There are seven coding sequences for BD-containing proteins in trypanosomatids, named TcBDF1 to TcBDF7, and a putative new protein containing a bromodomain was recently described. Using the Tet-regulated overexpression plasmid pTcINDEX-GW and CRISPR/Cas9 genome editing, we were able to demonstrate the essentiality of TcBDF2 in T. cruzi. This bromodomain is located in the nucleus, through a bipartite nuclear localization signal. TcBDF2 was shown to be important for host cell invasion, amastigote replication, and differentiation from amastigotes to trypomastigotes. Overexpression of TcBDF2 diminished epimastigote replication. Also, some processes involved in pathogenesis were altered in these parasites, such as infection of mammalian cells, replication of amastigotes, and the number of trypomastigotes released from host cells. In in vitro studies, TcBDF2 was also able to bind inhibitors showing a specificity profile different from that of the previously characterized TcBDF3. These results point to TcBDF2 as a druggable target against T. cruzi.

Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex, TcTINTIN, which is orthologous to human and yeast trimer independent of NuA4 for transcription interaction (TINTIN) complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component of TcTINTIN, TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity for TcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AF2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AF2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.

Little is known about acetylation signaling pathways in early branching organisms such as trypanosomatids in comparison to other unicellular eucaryotic species like yeast. In addition to important biological differences described in this deep branched organisms, serious bioinformatic limitations arise when facing the highly divergent sequences of the proteins involved, making it difficult to perform homology-based prediction of protein functions. The availability of several interactomic datasets, like those recently generated by Staneva et al for Trypanosoma brucei acetyl-lysine readers, writers and erasers and the public release of AlphaFold2 (AF2) and AF2-multimer could be envisaged as a shortcut to address this problem. These tertiary and quaternary structure predictions could be used as a tool with high predictive value when inferring protein function. In this work, we made use of public interactomic datasets to predict direct interactions using AF2-multimer and validated them by yeast two-hybrid (Y2H) assays. We focused on MRG domain-containing proteins of Trypanosoma cruzi and their interactions, typically found as structural part of histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes. Our results led us to identify TcTINTIN, a structurally conserved complex that is orthologous to the human and yeast TINTIN complexes, that cannot be identified by homology searches based only in the primary sequences. Our in silico and wet lab approach led us to determine a specific sequential assembly of this complex, involving the proteins TcMRGx (MORF4 Related Gene X), TcMRGBP (MRG Binding Protein) and TcBDF6 (Bromodomain Factor 6). We also found another MRG domain assembled in a trimeric complex formed between TcBDF5, TcBDF5BP (BDF5 Binding Protein) and TcBDF8. In this complex we describe a novel way in which an MRG domain participates in the interaction with two proteins binding two different surfaces, instead of just one as previously reported. Together, these results show that AF2-processed interactomic datasets can be used to identify chromatin-remodeling protein complexes previously unknown in deeply branched eukaryotes.