Biology subjects

Rodriguez Araya, E.

Publications and source records attributed to Rodriguez Araya, E..

5 recordsLinked to original sources

bioRxiv2026

Structural Insights into Bromodomain-Containing Complexes from Trypanosoma cruzi Revealed by Proximity Labeling and Stoichiometric Space Exploration

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness with outdated treatments. Bromodomain factors (BDFs) are essential proteins that recognize acetylated lysines and have strong therapeutic potential. They form part of epigenetic complexes that regulate chromatin accessibility and, therefore, gene expression. However, little is known about their structure in trypanosomatids. Here, we used a combination of experimental and bioinformatic approaches to infer the stoichiometry and structure of T. cruzi bromodomain-containing complexes. By reconstructing the proximity networks of five BDFs using TurboID-directed proximity labeling, we identified highly interconnected components that assemble into the CRKT and NuA4 complexes. Using novel structure prediction strategies that systematically explore the stoichiometric space, we inferred that CRKT assembles into three distinct modules and NuA4 in two, with different degrees of interaction dynamics. The core module of CRKT contains two copies of each component, including BDF3, BDF5, and BDF8, arranged in a subcomplex with central symmetry. The catalytic module of CRKT has three subunits, including the histone acetyltransferase 2 (HAT2), while the BET (bromodomain and extra-terminal) module has one unit of both BDF4 and BDF1. The catalytic module of NuA4 closely resembles the yeast piccolo-NuA4 module and contains HAT1, while the TINTIN module associates with the catalytic module via the C-terminal domain of BDF6. These insights shed light on the structure and composition of epigenetic complexes in trypanosomatids, opening new avenues for rational drug design aimed at disrupting their function.

molecular biology↗

bioRxiv2025

Context-Dependent Protein Structure Prediction Analysis and Stoichiometry Inference with MultimerMapper

Advances in artificial intelligence (AI) have transformed the field of protein structure, notably with the accuracy level reached by AlphaFold in the prediction of monomeric and multimeric protein structures. However, while cloud implementations have broadened access to these methods, there is still a lack of non-AI tools to systematically interpret and analyze the resulting data, especially under varying modeling contexts. Here, we present MultimerMapper, a pipeline and software suite designed to extract, integrate, analyze, and visualize the behavior of multimeric systems in ensembles of overlapping protein structure predictions. It utilizes statistics and graph theory, combined with novel approaches, to interpret how protein-protein interfaces and protein conformation behave across different modeling contexts. Starting from a list of input sequences, MultimerMapper guides users through the generation and interpretation of structure prediction ensembles. It produces interactive 2D and 3D visualizations, highlights higher-order subassemblies, infers probable stoichiometries, and identifies alternative interaction modes and conformational changes associated with the presence or absence of specific proteins. MultimerMapper is cross-platform, freely available, and easily integrates with existing workflows. It offers a new perspective on the dynamic nature of predicted protein complexes, supporting researchers in the exploration of functional mechanisms and assembly paths.

bioinformatics↗

bioRxiv2024

Identification of novel Bromodomain inhibitors of Trypanosoma cruzi Bromodomain Factor 2 (TcBDF2) using a fluorescence polarization-base high-throughput assay

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and participate in regulating DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350 ranked compounds were further analyzed in a dose-response assay. From this analysis, 7 compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significative trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 M validating these assays to search for bromodomain inhibitors.

microbiology↗

bioRxiv2022

Deciphering divergent trypanosomatid nuclear complexes by analyzing interactomic datasets with AlphaFold2 and genetic approaches

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-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on 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 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 AlphaFold2-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 AlphaFold2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/516520v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@16e7750org.highwire.dtl.DTLVardef@15b3b76org.highwire.dtl.DTLVardef@9cf1e9org.highwire.dtl.DTLVardef@1ce0ee0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Essential bromodomain TcBDF2 as a drug target against Chagas disease

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 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 that 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.

cell biology↗