Schistosoma mansoni is a vector-borne intestinal parasite, endemic in ~70 tropical countries. Most of the World Health Organization interventions are based on mass drug administration (MDA) and sanitation. Since the parasite does not induce permanent immunity, reinfection rate is high, inducing scheduling of several MDA campaigns. To follow up after treatment and avoid blind MDA, providing a new rapid and cheap diagnostic test is needed, since the most used method is fifty years-old, lacks sensitivity and efficiency. Venom Allergen-Like proteins (SmVALs) have been previously identified as secreted/excreted proteins and potential biomarkers. Here we present the biophysical characterization of SmVAL11 and SmVAL13, their recognition by both specific antibodies and molecularly imprinted polymers (nanoMIPs), an easy-to-standardize alternative. The specific antibodies showed no cross-reactivity, while initial characterization of the nanoMIPs indicates a promising level of selectivity, establishing them as a reliable, cost-effective alternative. Therefore, our results are promising for the future development of a new cheap diagnostic test of intestinal schistosomiasis.

Quantitative metabolic profiling of bacterial communities provides biophysical insight into cellular respiration and energy transduction under controlled environmental conditions. The oxygen consumption rate (OCR) represents a direct, integrative readout of respiratory activity and has emerged as a sensitive phenotypic parameter for characterizing microbial physiology across different metabolic states. Here, we present an experimental protocol for measuring bacterial respiration using the Seahorse XFe96 Analyzer (Agilent), a microplate-based platform that enables high temporal resolution measurements of oxygen flux. Although originally developed for eukaryotic cell cultures, we adapted this technology to bacterial systems, allowing the simultaneous analysis of multiple strains in a medium-throughput format. The 96-well configuration, combined with sequential injection of up to four compounds, including metabolic substrates, stressors, and alternative electron acceptors, enables real-time perturbation of respiration and monitoring of dynamic OCR responses. To investigate bacterial respiration under physiologically and environmentally relevant oxygen tensions, OCR measurements were additionally performed under hypoxic conditions (5

In 2020 seven genetic variants of the mitochondrial serine hydroxymethyl transferase (SHMT2) were linked to a novel brain and heart developmental syndrome. SHMT2 is a pyridoxal 5′-phosphate (PLP) binding enzyme involved in one-carbon metabolism and mitochondrial redox homeostasis, which also shows several moonlighting functions and protein-protein interactions that are related to its ability to change its oligomeric state. Molecular dynamics suggested that these genetic variants may cause a range of different structural defects ultimately affecting the conformation of the active site, cofactor affinity and the oligomerization state. Here we present a systematic biochemical and structural analysis of these variants. We expressed and purified the SHMT2 mutants and evaluated their oligomeric state, PLP binding affinity, thermal stability, and catalytic activities compared to the wild-type enzyme. The crystal structures of the less active mutants were then solved to pinpoint the structural determinants of the defects highlighted in solution. The results are discussed considering the multi-functional role of SHMT2 and expand our current understanding of the enzyme’s sophisticated conformational and allosteric dynamics.

Targeting metabolic reprogramming is crucial for cancer treatment. Recent advances highlight RNA’s ability to directly regulate enzyme activity through riboregulation. In this study, we used an RNA-based approach to inhibit the mitochondrial enzyme Serine hydroxymethyltransferase 2 (SHMT2), which lacks a selective in vivo inhibitor. SHMT2, often overexpressed in various cancers, is pivotal in one-carbon metabolism, a pathway vital for cell proliferation. Our results show that RNA effectively inhibits SHMT2’s serine-to-glycine conversion in vitro (IC50 = 4.4 ± 0.2 nM). By using a mitochondrial import signal, we successfully delivered the inhibitory RNA into the mitochondria of lung cancer cells, reducing cell viability in vitro and tumor growth in vivo in a xenograft mouse model. These findings suggest that RNA-based strategies could be extended to selectively target other RNA-binding metabolic enzymes, offering potential solutions where small molecule inhibitors fall short or to counteract drug resistance.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme’s reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

SUMMARYRNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of these being characterized on structural grounds. Here we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme’s reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in the evolution of eukaryotic SHMT1 and the compartmentalization of one-carbon metabolism. The findings also provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT1), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex. We report the intracellular dynamics of the complex in cancer cells by an in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the thymidylate synthesis complex assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human thymidylate synthase and dihydrofolate reductase. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein-protein interactions. Lastly, our results regarding the activity of the complete thymidylate cycle in vitro may provide a useful tool with respect to developing drugs targeting the entire complex instead of the individual components.

Brain metastases are the most severe clinical manifestation of aggressive tumors. Melanoma, breast, and lung cancers are the types that prefer the brain as a site of metastasis formation, even if the reasons for this phenomenon still remain to be clarified. One of the main characteristics that makes a cancer cell able to form metastases in the brain is the ability to interact with the endothelial cells of the microvasculature, cross the blood–brain barrier, and metabolically adapt to the nutrients available in the new microenvironment. In this review, we analyzed what makes the brain a suitable site for the development of metastases and how this microenvironment, through the continuous release of neurotransmitters and amino acids in the extracellular milieu, is able to support the metabolic needs of metastasizing cells. We also suggested a possible role for amino acids released by the brain through the endothelial cells of the blood–brain barrier into the bloodstream in triggering the process of extravasation/invasion of the brain parenchyma.

Nutrient utilization and reshaping of metabolism in cancer cells is a well-known driver of malignant transformation. Less clear is the influence of the local microenvironment on metastasis formation and choice of the final organ to invade. Here we show that the level of the amino acid serine in the cytosol affects the migratory properties of lung adenocarcinoma (LUAD) cells. Inhibition of serine or glycine uptake from the extracellular milieu, as well as knockdown of the cytosolic one-carbon metabolism enzyme serine hydroxymethyltransferase (SHMT1), abolishes migration. Using rescue experiments with a brain extracellular extract, and direct measurements, we demonstrate that cytosolic serine starvation controls cell movement by increasing reactive oxygen species formation and decreasing ATP levels, thereby promoting activation of the AMP sensor kinase (AMPK) by phosphorylation. Activation of AMPK induces remodeling of the cytoskeleton and finally controls cell motility. These results highlight that cytosolic serine metabolism plays a key role in controlling motility, suggesting that cells are able to dynamically exploit the compartmentalization of this metabolism to adapt their metabolic needs to different cell functions (movement vs. proliferation). We propose a model to explain the relevance of serine/glycine metabolism in the preferential colonization of the brain by LUAD cells and suggest that the inhibition of serine/glycine uptake and/or cytosolic SHMT1 might represent a successful strategy to limit the formation of brain metastasis from primary tumors, a major cause of death in these patients.

The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.

The determination of the function of cells in zero-gravity conditions is a subject of interest in many different research fields. Due to their metabolic unicity, the characterization of the behaviour of erythrocytes maintained in prolonged microgravity conditions is of particular importance. Here, we used a 3D-clinostat to assess the microgravity-induced modifications of the structure and function of these cells, by investigating how they translate these peculiar mechanical stimuli into modifications, with potential clinical interest, of the biochemical pathways and the aging processes. We compared the erythrocyte's structural parameters and selected metabolic indicators that are characteristic of the aging in microgravity and standard static incubation conditions. The results suggest that, at first, human erythrocytes react to external stimuli by adapting their metabolic patterns and the rate of consumption of the cell resources. On longer timeframes, the cells translate even small differences in the environment mechanical solicitations into structural and morphologic features, leading to distinctive morphological patterns of aging.