Biology subjects

Cricco, J. A.

Publications and source records attributed to Cricco, J. A..

6 recordsLinked to original sources

bioRxiv2026

Functional Mapping of the Trypanosoma cruzi Serinome by Fluorophosphonate Activity-Based Protein Profiling

Serine hydrolases (SHs) constitute one of the largest enzyme superfamilies in eukaryotes, yet their roles in Trypanosoma cruzi, the causative agent of Chagas disease, remain largely uncharacterized. Here, we report an activity-based chemoproteomic map of the T. cruzi epimastigote serinome by combining genome-informed in silico curation with whole-cell activity-based protein profiling (ABPP) using a panel of cell-permeable fluorophosphonate (FP)-alkyne probes. Whole-cell labelling followed by label-free quantitative proteomics (LFQ-MS), identified 37 enriched SH-like proteins, including 35 with conserved or partially conserved catalytic triad/dyad features, spanning lipases, peptidases, esterases, and previously uncharacterized hydrolases. The 35 SHs represent approximately 63 % of the 56 predicted SHs retained after catalytic-site curation. Domain architecture analysis revealed broad structural diversity, while orthologue-based localization data suggested association with multiple subcellular compartments, including glycosomal, mitochondrial, and endosomal localizations. Gene Ontology enrichment highlighted lipid metabolic and catabolic processes as dominant functional themes, and protein-protein interaction network analysis supported functional connectivity among the captured enzymes. Several identified SHs, including oligopeptidase B, prolyl oligopeptidase Tc80, serine carboxypeptidase CPB1, and phospholipase A1 (PLA1) have previously been characterized in trypanosomatids as virulence factors and as mediator of host-pathogen interactions. Together, these findings establish a fluorophosphonate-based chemoproteomic resource for the kinetoplastid community and prioritize probe-accessible active T. cruzi SHs for future functional validation and antiparasitic inhibitor discovery.

biochemistry↗

bioRxiv2026

A First-In-Class Broad Spectrum Inhibitor of Copper Exporting P1B-type ATPases

Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a first-in-class inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu+ entry site to the translocation pathway. In silico docking against the Xenopus ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu+ entry site, and selectively inhibited Escherichia coli CopA ATPase activity and Cu+ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu+ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu+-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu+-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms. Significance StatementCopper-transporting P1B-type ATPases are essential for copper homeostasis in all domains of life, yet have lacked pharmacological inhibitors. This work identifies MKV3 as the first small-molecule inhibitor of Cu+-ATPases in bacteria, fungi, plants and animals, and defines a conserved, druggable Cu+ entry pocket that governs metal delivery to the transmembrane pathway. MKV3s ability to potentiate copper-mediated killing in multidrug-resistant bacterial pathogens highlights its potential as an antimicrobial adjuvant, while its attenuation of mammalian ATP7A/B function offers promise in oncology and copper-related diseases. Collectively, these findings establish a new tool for targeting of Cu+-ATPases with wide-ranging applications across biological systems.

cell biology↗

bioRxiv2025

Transcriptomic response to different heme sources in Trypanosoma cruzi

Heme is an essential molecule for most organisms, yet some parasites, like Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize it. These parasites must acquire heme from their hosts, making this process critical for their survival. In the midgut of the insect vector, T. cruzi epimastigotes are exposed to both hemoglobin (Hb) and free heme resulting from its degradation. Despite the importance of this nutrient, how different heme sources influence parasite gene expression remains poorly understood. Here, we showed that heme restitution either as hemin or Hb to heme-starved parasites induces an early and distinct transcriptional response in T. cruzi epimastigotes. Using RNA sequencing at 4- and 24-hours post-supplementation, we identified gene subsets commonly or uniquely regulated by each heme source, including genes putatively linked to heme acquisition and metabolism. We also presented here the first studies focused on CRAL/TRIO domain-containing protein (TcCRAL/TRIO), a novel heme responsive hemoprotein identified from this study. Our results provide a more detailed picture of T. cruzi biology and highlights heme acquisition as a promising point of vulnerability. These findings may ultimately contribute to the identification of potential molecular targets for the development of new therapeutic strategies against Chagas disease.

biochemistry↗

bioRxiv2024

Copper trafficking in Trypanosoma cruzi: the transcriptional response of candidates to balance toxicity and recruitment

Trypanosoma cruzi (Chagas disease) depends on acquiring nutrients and cofactors, like copper (Cu), from its hosts. Cu is essential for aerobic organisms, but it can also be toxic, so its transport and storage must be regulated. In the present study, we characterized the effects of changes in Cu availability on growth, intracellular ion content, and oxygen consumption. Our results show that Cu is essential for epimastigote proliferation and for metacyclogenesis, while intracellular amastigotes suffered from Cu stress during infection. We identify several genes potentially involved in Cu metabolism among which orthologs of the conserved P-type Cu ATPases involved in Cu export and loading of secreted enzymes were found and named TcCuATPase. TcCuATPase transcription is regulated during infective stages and by Cu availability in epimastigotes. No homologs were identified for the high affinity importer CTR1 instead we propose that the iron transport TcIT a ZIP family transporter is involved in Cu uptake based on its transcriptional response to Cu. Further canonical Cu targets (based on homology to yeast and mammals) such as the iron reductase TcFR and the cupro-oxidase TcFet3 are up regulated during infective stages and under intracellular Cu stress. We also demonstrated that Cu, iron, and heme metabolisms are related. In sum, Cu metabolism is essential in T. cruzi life cycle. Even though cytosolic Cu-chaperons are still missing, we propose a model for Cu transport and intracellular distribution in T. cruzi including conserved factors such as TcCuATPase and others such as TcFR and TcIT playing novel functions.

microbiology↗

bioRxiv2023

TcHRG plays a central role in orchestrating heme uptake in Trypanosoma cruzi epimastigotes

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating TcHRG expression when a free heme source is added to axenic culture. Herein, we explore the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. It was found that the parasites endogenous TcHRG (protein and mRNA) responds similarly to bound (hemoglobin) and free (hemin) heme. Additionally, the overexpression of TcHRG leads to an increase in intracellular heme content. The localization of TcHRG is also not affected in parasites supplemented with hemoglobin as the sole heme source. Endocytic null epimastigotes do not show a significant difference in growth profile, intracellular heme content and TcHRG protein accumulation compared to WT when feeding with hemoglobin or hemin as a source of heme. These results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes controls heme homeostasis by modulating TcHRG expression independently of the source of available heme.

biochemistry↗

bioRxiv2020

Trypanosoma cruzi senses intracellular heme levels and regulates heme homeostasis by modulating TcHTE protein expression

Heme is an essential cofactor for many biological processes in aerobic organisms. Unlike most organisms, which can synthesize it de novo through a conserved pathway, the etiological agent of Chagas disease, Trypanosoma cruzi, as well as other trypanosomatids relevant for human health, are heme auxotrophs; thereby they must import it from the hosts. TcHTE protein is involved in T. cruzi heme transport, although its specific role remains elusive. In the present work we studied endogenous TcHTE in the different life cycle stages of the parasite in order to gain insight in its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote). We have also demonstrated that T. cruzi epimastigotes can sense intracellular heme content by an unknown mechanism and regulates heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of T. cruzi biology as well as other trypanosomatids, pointing out this pathway as a novel drug target for therapeutics.

biochemistry↗