Biology subjects

Alonso, V. L.

Publications and source records attributed to Alonso, V. L..

7 recordsLinked to original sources

bioRxiv2026

Proteomic mapping of novel tubulin post-translational modifications in Trypanosoma cruzi cytoskeleton

Microtubules (MTs) play central roles in the organization and morphology of trypanosomatid parasites, forming highly specialized cytoskeletal structures such as the subpellicular corset, the flagellar axoneme, and the mitotic spindle. Functional specialization of MTs is regulated by the "tubulin code", which is defined by the combination of different - and {beta}-tubulin isotypes, a set of post-translational modifications (PTMs) and specific MT-binding proteins. Although multiple tubulin PTMs have been described in trypanosomatids using specific antibodies or mass spectrometry, to date no comprehensive mapping has been reported in Trypanosoma cruzi, the causative agent of Chagas Disease. In the present work, we performed a high-resolution proteomic analysis of PTMs present in - and {beta}-tubulin subunits of the T. cruzi Dm28c strain, using tubulin-enriched extracts obtained by in vitro polymerization. Multiple PTMs were identified, including acetylation, methylation, phosphorylation, and polyglutamylation, for which many modified amino acids had not been previously reported in trypanosomatids. Structural mapping of these modifications onto a predicted /{beta}-tubulin heterodimer showed that most modified residues are located in solvent-exposed regions of the protein. Together, these findings provide the first systematic map of tubulin PTMs in T. cruzi and support the existence of a complex tubulin code contributing to microtubule regulation in this parasite.

cell biology↗

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Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness with outdated treatments.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

CLASP1/2 REGULATE IMMUNE SYNAPSE MATURATION IN NATURAL KILLER CELLS

Natural killer (NK) cells are the first line of defense against viral infections and tumors. Their cytotoxic activity relies on the formation of an immune synapse (IS) with target cells. The lymphocyte function-associated antigen (LFA)-1 plays a central role in NK cell cytotoxicity by modulating NK-IS assembly and maturation. LFA-1 organization at the IS involves a Golgi-dependent mechanism, which has not been completely elucidated. CLIP-associating proteins (CLASP) 1/2 are microtubule plus-tip interacting proteins that control the dynamics of Golgi derived microtubules (GDMTs). In the present study, we found that CLASP1/2 depletion impaired LFA-1 organization at the IS and inhibited the polarization of the centrosome and the lytic granules towards the target cell. Our results also revealed the role of the Golgi apparatus as a microtubule organizing center (MTOC) in these cells. Furthermore, we found that, similarly to what was described in other cell types, NK cells require CLASP1/2 and AKAP350 for efficient nucleation of microtubules at the Golgi. Overall, this study uncovers the role of CLASP1/2 in the maturation of the lytic IS in NK cells, and presents evidence supporting the contribution of GDMTs in this process. Summary sentenceThe Golgi apparatus (GA) functions as a microtubule-organizing center (MTOC) in NK cells. During the recognition of tumoral cells by NK cells, CLASP1/2-mediated stabilization of GA-derived microtubules (GDMTs) facilitates vesicular LFA-1 (LFA-1v) trafficking toward the interaction surface, thereby promoting the immune synapse (IS) maturation.

immunology↗

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

Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes

Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as -tubulin and {beta}-tubulin. Also, I immunolocalized acetylated and tyrosinated -tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.

cell 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↗

bioRxiv2020

Alpha-tubulin acetylation in Trypanosoma cruzi: a dynamic instability of microtubules is required for replication and cell cycle progression

Trypanosomatids have a cytoskeleton arrangement that is simpler than what is found in most eukaryotic cells. However, it is precisely organized and constituted by stable microtubules. Such microtubules compose the mitotic spindle during mitosis, the basal body, the flagellar axoneme and the subpellicular microtubules, which are connected to each other and also to the plasma membrane forming a helical arrangement along the central axis of the parasite cell body. Subpellicular, mitotic and axonemal microtubules are extensively acetylated in Trypanosoma cruzi. Acetylation on lysine (K) 40 of -tubulin is conserved from lower eukaryotes to mammals and is associated with microtubule stability. It is also known that K40 acetylation occurs significantly on flagella, centrioles, cilia, basal body and the mitotic spindle in eukaryotes. Several tubulin posttranslational modifications, including acetylation of K40, have been catalogued in trypanosomatids, but the functional importance of these modifications for microtubule dynamics and parasite biology remains largely undefined. The primary tubulin acetyltransferase that delivers this modification was recently identified in several eukaryotes as Mec-17/ATAT, a Gcn5-related N-acetyltransferase. Here, we report that T. cruzi ATAT acetylates -tubulin in vivo and is capable of auto-acetylation. TcATAT is located in the cytoskeleton and flagella of epimastigotes and colocalizes with acetylated -tubulin in these structures. We have expressed TcATAT with an HA tag using the inducible vector pTcINDEX-GW in T. cruzi. Over-expression of TcATAT causes increased levels of the acetylated isoform, induces morphological and ultrastructural defects, especially in the mitochondrion, and causes a halt in the cell cycle progression of epimastigotes, which is related to an impairment of the kinetoplast division. Finally, as a result of TcATAT over-expression we observed that parasites became more resistant to microtubule depolymerizing drugs. These results support the idea that -tubulin acetylation levels are finely regulated for the normal progression of T. cruzi cell cycle.

cell biology↗