Structure-based mechanism of riboregulation of the metabolic enzyme SHMT1.

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.

A Solanum lycopersicum polyamine oxidase contributes to the control of plant growth, xylem differentiation, and drought stress tolerance.

Polyamines are involved in several plant physiological processes. In Arabidopsis thaliana, five FAD-dependent polyamine oxidases (AtPAO1 to AtPAO5) contribute to polyamine homeostasis. AtPAO5 catalyzes the back-conversion of thermospermine (T-Spm) to spermidine and plays a role in plant development, xylem differentiation, and abiotic stress tolerance. In the present study, to verify whether T-Spm metabolism can be exploited as a new route to improve stress tolerance in crops and to investigate the underlying mechanisms, tomato (Solanum lycopersicum) AtPAO5 homologs were identified (SlPAO2, SlPAO3, and SlPAO4) and CRISPR/Cas9-mediated loss-of-function slpao3 mutants were obtained. Morphological, molecular, and physiological analyses showed that slpao3 mutants display increased T-Spm levels and exhibit changes in growth parameters, number and size of xylem elements, and expression levels of auxin- and gibberellin-related genes compared to wild-type plants. The slpao3 mutants are also characterized by improved tolerance to drought stress, which can be attributed to a diminished xylem hydraulic conductivity that limits water loss, as well as to a reduced vulnerability to embolism. Altogether, this study evidences conservation, though with some significant variations, of the T-Spm-mediated regulatory mechanisms controlling plant growth and differentiation across different plant species and highlights the T-Spm role in improving stress tolerance while not constraining growth.

Beyond the barrier: the immune-inspired pathways of tumor extravasation.

Extravasation is a fundamental step in the metastatic journey, where cancer cells exit the bloodstream and breach the endothelial cell barrier to infiltrate target tissues. The tactics cancer cells employ are sophisticated, closely reflecting those used by the immune system for tissue surveillance. Remarkably, tumor cells have been observed to form distinct associations or clusters with immune cells where neutrophils stand out as particularly crucial partners. These interactions are not accidental; they are critical for cancer cells to exploit the immune functions of neutrophils and successfully extravasate. In another strategy, tumor cells mimic the behavior and characteristics of immune cells. They release a suite of inflammatory mediators, which under normal circumstances, guide the processes of endothelium reshaping and facilitate the entry and movement of immune cells within tissues. In this review, we offer a new perspective on the tactics employed by cancer cells to extravasate and infiltrate target tissues. We delve into the myriad mechanisms that tumor cells borrow, adapt, and refine from the immune playbook. Video Abstract.

Thymic-Epithelial-Cell-Dependent Microenvironment Influences Proliferation and Apoptosis of Leukemic Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological cancer characterized by the infiltration of immature T-cells in the bone marrow. Aberrant NOTCH signaling in T-ALL is mainly triggered by activating mutations of NOTCH1 and overexpression of NOTCH3, and rarely is it linked to NOTCH3-activating mutations. Besides the known critical role of NOTCH, the nature of intrathymic microenvironment-dependent mechanisms able to render immature thymocytes, presumably pre-leukemic cells, capable of escaping thymus retention and infiltrating the bone marrow is still unclear. An important challenge is understanding how leukemic cells shape their tumor microenvironment to increase their ability to infiltrate and survive within. Our previous data indicated that hyperactive NOTCH3 affects the CXCL12/CXCR4 system and may interfere with T-cell/stroma interactions within the thymus. This study aims to identify the biological effects of the reciprocal interactions between human leukemic cell lines and thymic epithelial cell (TEC)-derived soluble factors in modulating NOTCH signaling and survival programs of T-ALL cells and TECs. The overarching hypothesis is that this crosstalk can influence the progressive stages of T-cell development driving T-cell leukemia. Thus, we investigated the effect of extracellular space conditioned by T-ALL cell lines (Jurkat, TALL1, and Loucy) and TECs and studied their reciprocal regulation of cell cycle and survival. In support, we also detected metabolic changes as potential drivers of leukemic cell survival. Our studies could shed light on T-cell/stroma crosstalk to human leukemic cells and propose our culture system to test pharmacological treatment for T-ALL.

Ferritin-based disruptor nanoparticles: A novel strategy to enhance LDL cholesterol clearance via multivalent inhibition of PCSK9-LDL receptor interaction.

Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

Design of protein-binding peptides with controlled binding affinity: the case of SARS-CoV-2 receptor binding domain and angiotensin-converting enzyme 2 derived peptides.

The development of methods able to modulate the binding affinity between proteins and peptides is of paramount biotechnological interest in view of a vast range of applications that imply designed polypeptides capable to impair or favour Protein-Protein Interactions. Here, we applied a peptide design algorithm based on shape complementarity optimization and electrostatic compatibility and provided the first experimental in vitro proof of the efficacy of the design algorithm. Focusing on the interaction between the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor, we extracted a 23-residues long peptide that structurally mimics the major interacting portion of the ACE2 receptor and designed in silico five mutants of such a peptide with a modulated affinity. Remarkably, experimental KD measurements, conducted using biolayer interferometry, matched the in silico predictions. Moreover, we investigated the molecular determinants that govern the variation in binding affinity through molecular dynamics simulation, by identifying the mechanisms driving the different values of binding affinity at a single residue level. Finally, the peptide sequence with the highest affinity, in comparison with the wild type peptide, was expressed as a fusion protein with human H ferritin (HFt) 24-mer. Solution measurements performed on the latter constructs confirmed that peptides still exhibited the expected trend, thereby enhancing their efficacy in RBD binding. Altogether, these results indicate the high potentiality of this general method in developing potent high-affinity vectors for hindering/enhancing protein-protein associations.