Francesca Cutruzzolà

Serine, a nonessential amino acid classically defined as a precursor for protein synthesis and one-carbon metabolism, is increasingly recognized as a signaling metabolite that links the cellular metabolic status to regulatory decision-making. Intracellular serine availability is shaped by nutrient conditions, glycolytic flux, and activity of the serine synthesis pathway, and these fluctuations are sensed to elicit coordinated metabolic and signaling responses. This review discusses mechanisms by which serine modulates cell growth and stress responses, with particular emphasis on its interaction with central nutrient-sensing pathways, including mTORC1 and the integrated stress response. In parallel, serine-driven one-carbon metabolism is examined for its role in supporting nucleotide biosynthesis, methylation reactions, and redox homeostasis through folate-dependent pathways and NADPH generation, thereby coupling anabolic processes to the maintenance of redox balance and genome integrity. In addition to intracellular functions, serine contributes to intercellular signaling. Conversion of l-serine to d-serine mediates neuromodulatory activity via N-methyl-d-aspartate receptors, while serine availability also influences immune cell function, inflammatory signaling, and host–microbe interactions. Dysregulation of serine metabolism and signaling is further considered in the context of disease states, including cancer, neurodegeneration, and metabolic disorders. Together, these observations support a framework in which serine functions as an information-bearing metabolic signal that coordinates the biosynthetic capacity with cellular adaptation and intercellular communication.

Tumor metabolism, a crucial component in cancer progression, represents a potential prognostic and diagnostic platform in cancer detection. Here, we show that patient-specific stromal mesenchymal cells exhibit distinct behaviors in promoting either tumor growth or dissemination. Tumor-associated fibroblasts (TAFs) isolated from non-metastasized colorectal adenocarcinomas predominantly supported cancer cell proliferation, whereas TAFs isolated from metastatic adenocarcinomas facilitated cancer cell migration. An in vitro analysis of stromal paracrine factors revealed that variations in mitochondrial activity and the secretion of specific metabolites were closely associated with distinct tumor-stroma interactions. Among the oncometabolites identified, we validated amino acid expression in urine samples from 19 colon cancer patients to assess their potential as diagnostic biomarkers. Our results showed patient-specific alterations in oncometabolite expression, which were significantly different from those of healthy control individuals. The specificity, sensitivity, and accuracy analysis indicated 93–100% specificity, 74–82% sensitivity, and 84–89% accuracy of single metabolites in distinguishing cancer patients from healthy controls. While no false negatives were observed, urine samples from nine patients with various inflammatory conditions (including diverticulitis, appendicitis, and chronic gastritis) yielded false positives. Sensitivity analysis and the t-Distributed Stochastic Neighbor Embedding (t-SNE) nonlinear dimensionality reduction technique revealed distinct metabolite profiles for healthy controls, colon cancer patients, and patients diagnosed with inflammation. Overall, our findings suggest that the identified oncometabolites, when integrated into a biomarker panel, hold promise as a novel non-invasive tool for screening individuals at risk of cancer and inflammatory malignancies.

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% O₂) by integrating the Seahorse XFe96 Analyzer with an oxygen-controlled glove box. This approach permits precise control of oxygen availability and extends OCR analysis to low-oxygen regimes relevant to chronic infections and aquatic or engineered environments. Overall, our results establish a robust and versatile biophysical platform for probing bacterial respiratory dynamics and for phenotypic screening applications.

Here, we show, in vitro, predominant support of patient-specific tumor associated fibroblasts (TAFs) isolated from non-metastatic colorectal adenocarcinomas on cancer cell proliferation, whereas TAFs isolated from metastatic adenocarcinomas facilitated cancer cell migration. An in vitro analysis revealed that variations in mitochondrial activity and the secretion of specific metabolites correlated with patient diagnoses. Among the oncometabolites identified, we validated amino acid expression in urine samples from 19 colon cancer patients to assess their potential as diagnostic biomarkers. Our results suggested patient-specific alterations in oncometabolite expression, which were significantly different from those of healthy controls. The specificity, sensitivity, and accuracy analysis indicated 93–100% specificity, 74–82% sensitivity, and 84–89% accuracy of single metabolites in distinguishing cancer patients from controls. While no false negatives were observed, urine samples from nine patients with various inflammatory conditions (including diverticulitis, appendicitis, and chronic gastritis) yielded false positives. Sensitivity analysis and the t-Distributed Stochastic Neighbor Embedding (t-SNE) nonlinear dimensionality reduction technique revealed distinct metabolite profiles for healthy controls, colon cancer patients, and patients diagnosed with inflammation. Our findings suggest that the identified oncometabolites hold a promise as a novel non-invasive tool for screening individuals at risk of colorectal cancer.

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.

Heterozygous mutations in the Bone morphogenetic protein (BMP) type I receptor ACVR1, encoding activin-like kinase 2 (ALK2), underlie all cases of the rare genetic musculoskeletal disorder Fibrodysplasia Ossificans Progressiva (FOP). The most commonly found mutant ALK2 p.R206H receptor variant exhibits loss of auto inhibition of BMP signaling and can be activated by Activins, while wild-type receptors remain unresponsive. Consequently, the downstream chondrogenic signaling is enhanced, thus driving heterotopic ossification within soft connective tissues. Despite several investigational treatments being evaluated in clinical trials, no cure for FOP exists today. The cellular and molecular mechanisms underlying disease progression are still being deciphered. In this study, we show a close interplay between the mutant ALK2^R206H receptor signaling and dysregulation of the autophagic flux triggered by hypoxia. Mechanistically, reduced autophagic flux correlates with increased stability of ALK2^R206H, resulting in sustained signaling. Of note, we demonstrated that Rapamycin, under clinical investigation as a treatment for FOP, inhibits chondrogenic differentiation in an autophagy-dependent manner. Consistently, other pharmacological autophagy inducers, like Spermidine, can reduce ALK2^R206H driven chondrogenic differentiation in vitro. These results were verified in FOP patient-derived cells. In conclusion, this study shows that aberrant autophagic flux mediates sustained ALK2^R206H signaling, introducing a novel druggable target in FOP by reactivating autophagy.

Chronic lymphocytic leukemia (CLL) is a widespread type of leukemia that predominantly targets B lymphocytes, undermining the balance between cell proliferation and apoptosis. In healthy B cells, miR-15/16, a tandem of microRNAs, functions as a tumor suppressor, curbing the expression of the antiapoptotic B cell lymphoma 2 protein (Bcl-2). Conversely, in CLL patients, a recurring deletion on chromosome 13q14, home to the miR15-a and miR16-1 genes, results in Bcl-2 overexpression, thereby fostering the onset of the pathology. In the present research, a novel approach utilizing humanized ferritin-based nanoparticles was employed to successfully deliver miR15-a and miR-16-1 into MEG01 cells, a model characterized by the classic CLL deletion and overexpression of the human ferritin receptor (TfR1). The loaded miR15-a and miR16-1, housed within modified HumAfFt, were efficiently internalized via the MEG01 cells and properly directed into the cytoplasm. Impressively, the concurrent application of miR15-a and miR16-1 demonstrated a robust capacity to induce apoptosis through the reduction in Bcl-2 expression levels. This technology, employing RNA-loaded ferritin nanoparticles, hints at promising directions in the battle against CLL, bridging the substantial gap left by traditional transfection agents and indicating a pathway that may offer hope for more effective treatments.

SUMMARY: RNA 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.