Galectins, β-galactoside-binding soluble proteins, are involved in a multitude of biological functions and several diseases, so numerous galectin inhibitors, from small molecules to multivalent glycoconjugates, have been developed and investigated as tools for therapeutic applications. Notably, multivalent ligands on a biocompatible backbone offer a promising perspective for creating high-performance selective inhibitors with nanomolar affinity. Leveraging the oxidative polymerization of 5,6-dihydroxyindole (DHI), a key intermediate of eumelanin pigments, here, we present the synthesis and complete nuclear magnetic resonance characterization of a submicromolar multivalent ligand of galectin-3 based on a naturally biocompatible eumelanin backbone. The integration of several complementary techniques, namely, UV-vis spectrometry, dynamic light scattering, isothermal titration calorimetry, and biolayer interferometry, in the investigation of galectin-3-eumelanin-related ligand interactions, allowed us to calculate the KD and to propose a model for the protein-polymer interaction.

UBE2C (also known as UbcH10) is a ubiquitin-conjugating enzyme essential for mitotic progression and a potential therapeutic target in cancer. Here, we report a structure-based design and characterisation of peptides derived from a natural interacting partner (U1) aimed at modulating UBE2C activity. Biophysical and biochemical assays identified peptide 5 as a lead compound, capable of binding UBE2C with micromolar affinity and inhibiting the formation of the UBE2C-Ub thioester complex. Enhanced sampling molecular dynamics simulations revealed that peptide folding landscapes are correlated with activity, with active peptides sampling transient β-sheet conformations compatible with binding. To the best of our knowledge, this is the first report of a peptide inhibitor of UBE2C enzymatic activity.

Heavy metals (HMs) are naturally occurring elements which can be essential, such as zinc, copper, and iron, or non-essential, including cadmium, mercury, and lead. While essential metals serve as cofactors in critical enzymatic processes, elevated concentrations of both essential and non-essential HMs pose severe toxicity risks, primarily through oxidative stress, disruption of metal homeostasis, and biomolecular damage. Microorganisms have evolved diverse mechanisms to cope with metal-induced stress, including metal sequestration, enzymatic transformation, efflux systems, and surface immobilization. Among these, metallothioneins (Mts) are small, cysteine-rich proteins capable of high-affinity metal binding, contributing to cellular detoxification. Although Mts have been extensively studied in eukaryotes, knowledge of bacterial Mts remains limited, with characterized examples largely confined to cyanobacteria and a few other bacterial species. In this study, we identified a novel hybrid protein, TrxA, from Runella aurantiaca, containing a thioredoxin (Trx) domain fused to a Mt domain. The presence of the Trx domain may confer improved stability and solubility, supporting potential recombinant applications. In fact, the recombinant protein, named TrxMt, was heterologously expressed in Escherichia coli, displaying both disulfide-reducing activity and heavy metal–binding capability. Notably, TrxMt expression enhanced bacterial tolerance to multiple HMs, demonstrating its functional relevance in vivo. These findings expand the understanding of bacterial Mt diversity and suggest that TrxMt is a promising candidate for the bioremediation of heavy metal–contaminated environments, combining metal detoxification with favorable biochemical properties for industrial and environmental applications. • Identification of TrxA, a novel hybrid thioredoxin–metallothionein in R. aurantiaca • Recombinant protein TrxMt shows reductase activity and binds HMs • Overexpression of TrxMt enhances tolerance to different HMs in E. coli

Human galectin-3 (Gal-3), a β-galactoside binding lectin through its Carbohydrate Recognition Domain (CRD), is implicated in a wide range of cellular functions and is involved in critical biological processes including pathogen recognition, immune response, inflammation and fibrosis. Recently, Gal-3 has gained increasing attention for its role in pathological conditions such as cancer, where it influences cancer growth and progression, inflammatory processes and oxidative stress, opening the search for potential inhibitors. In this context, several naturally derived molecules have attracted particular interest, some of them being used in clinical trials. Here, we used the seeds of the legume Phaseolus coccineus as a green resource for bioactive compounds. The peptide-rich crude extracts were chemically characterized for their peptide and polyphenol contents, as well as their in vitro antioxidant activity, and the powerful obtained extract was tested for biological activities such as cytotoxicity and antioxidant and anti-inflammatory effects on cellular models. Furthermore, the interaction between the crude extract and the CRD of recombinant Gal-3 was verified with the aim of associating its biological effects with the inhibition of Gal-3 activity.

Chimera-type galectin-3 (Gal-3) is a β-galactoside-binding protein containing a single conserved carbohydrate-recognition domain, crucial in fibrosis and carcinogenesis. Selenium-based Gal-3 inhibitors have emerged as promising therapeutic agents, particularly for treating neoplastic diseases. Among them, a seleno-digalactoside (SeDG) substituted with a benzyl group at position 3 of both saccharide residues (benzyl 3,3'-seleno-digalactoside, SeDG-Bn), attracted considerable attention for its selectivity and potent inhibitory efficacy against Gal-3. NMR spectroscopy and molecular dynamics simulations were combined to investigate the binding of SeDG-Bn to Gal-3 at the molecular level. This approach revealed the recognized epitope, the binding mode within Gal-3 binding pocket and enabled the generation of a 3D model of the complex. Our findings show that the presence of a single benzyl group establishes hydrophobic contacts with amino acids in Gal-3 β-sheets S2 and S3, crucially enhancing the binding affinity compared to unmodified SeDG. The digalactose backbone orientation in Gal-3 binding site is partially modified by the benzyl group with respect to complexes with lactosamine and SeDG. These results provide valuable insights into the design of more potent and selective inhibitors for Gal-3, potentially contributing to new therapeutic strategies for conditions such as cancer and fibrosis.

In a recent study, we have identified BPH03 as a promising scaffold for the development of compounds aimed at modulating the interaction between PED/PEA15 (Phosphoprotein Enriched in Diabetes/Phosphoprotein Enriched in Astrocytes 15) and PLD1 (phospholipase D1), with potential applications in type II diabetes therapy. PED/PEA15 is known to be overexpressed in certain forms of diabetes, where it binds to PLD1, thereby reducing insulin-stimulated glucose transport. The inhibition of this interaction reestablishes basal glucose transport, indicating PED as a potential target of ligands capable to recover glucose tolerance and insulin sensitivity. In this study, we employ computational methods to provide a detailed description of BPH03 interaction with PED, evidencing the presence of a hidden druggable pocket within its PLD1 binding surface. We also elucidate the conformational changes that occur during PED interaction with BPH03. Moreover, we report new NMR data supporting the in-silico findings and indicating that BPH03 disrupts the PED/PLD1 interface displacing PLD1 from its interaction with PED. Our study represents a significant advancement toward the development of potential therapeutics for the treatment of type II diabetes.

The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein–protein interaction.

In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.

Given the significant involvement of galectins in the development of numerous diseases, the aim of the following work is to further study the interaction between galectin-3 (Gal3) and the LPS from Pseudomonas aeruginosa. This manuscript focused on the study of the interaction of the carbohydrate recognition domain of Gal3 with the LPS from Pseudomonas aeruginosa by means of different complementary methodologies, such as circular dichroism; spectrofluorimetry; dynamic and static light scattering and evaluation of the impact of Gal3 on the redox potential membranes of Escherichia coli and P. aeruginosa cells, as well as ITC and NMR studies. This thorough investigation reinforces the hypothesis of an interaction between Gal3 and LPS, unraveling the structural details and providing valuable insights into the formation of these intricate molecular complexes. Taken together, these achievements could potentially prompt the design of therapeutic drugs useful for the development of agonists and/or antagonists for LPS receptors such as galectins as adjunctive therapy for P. aeruginosa.

Members of the KCTD protein family play key roles in fundamental physio-pathological processes including cancer, neurodevelopmental/neuropsychiatric, and genetic diseases. Here, we report the crystal structure of the KCTD1 P20S mutant, which causes the scalp-ear-nipple syndrome, and molecular dynamics (MD) data on the wild-type protein. Surprisingly, the structure unravels that the N-terminal region, which precedes the BTB domain (preBTB) and bears the disease-associated mutation, adopts a folded polyproline II (PPII) state. The KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. Indeed, the BTB of each chain makes peculiar contacts with the preBTB and the C-terminal domain (CTD) of an adjacent chain. The BTB-preBTB interaction consists of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/-) helix discontinuous association. The inspection of the protein structure, along with the data emerged from the MD simulations, provides an explanation of the pathogenicity of the P20S mutation and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of KCTD1 in metal homeostasis.

In a recent study, we have identified BPH03 as a promising scaffold for the development of compounds aimed at modulating the interaction between PED/PEA15 (Phosphoprotein Enriched in Diabetes/Phosphoprotein Enriched in Astrocytes 15) and PLD1 (phospholipase D1), with potential applications in type II diabetes therapy. PED/PEA15 is known to be overexpressed in certain forms of diabetes, where it binds to PLD1, thereby reducing insulin-stimulated glucose transport. The inhibition of this interaction reestablishes basal glucose transport, indicating PED as a potential target of ligands capable to recover glucose tolerance and insulin sensitivity. In this study, we employ computational methods to provide a detailed description of BPH03 interaction with PED, evidencing the presence of a hidden druggable pocket within its PLD1 binding surface. We also elucidate the conformational changes that occur during PED interaction with BPH03. Moreover, we report new NMR data supporting the in-silico findings and indicating that BPH03 disrupts the PED/PLD1 interface displacing PLD1 from its interaction with PED. Our study represents a significant advancement toward the development of potential therapeutics for the treatment of type II diabetes.

The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.

Galectins, β-galactoside-binding proteins, play relevant roles in different biological processes; therefore, they are becoming emerging targets for diagnostic and therapeutic approaches [...]

The transcriptional regulator MucR from Brucella species controls the expression of many genes, including those involved in virulence, by binding AT-rich DNA regions. MucR and its homologs belong to the Ros/MucR family, whose members occur in α-proteobacteria. MucR is a recent addition to the family of histone-like nucleoid structuring (H-NS) proteins. Indeed, despite the lack of sequence homology, MucR bears many functional similarities with H-NS and H-NS-like proteins, structuring the bacterial genome and acting as global regulators of transcription. Here we present an integrated cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance, modeling and biochemical study shedding light on the functional architecture of MucR from Brucella abortus and its homolog Ml5 from Mesorhizobium loti. We show that MucR and Ml5 fold in a circular quaternary assembly, which allows it to bridge and condense DNA by binding AT-rich sequences. Our results show that Ros/MucR family members are a novel type of H-NS-like proteins and, based on previous studies, provide a model connecting nucleoid structure and transcription regulation in α-proteobacteria.