D-Amino acids, once considered negligible in mammalian physiology, are now recognized as key neuromodulators. Among them, D-aspartate (D-Asp) regulates NMDAR and mGlu5 receptor function, influencing neurodevelopment, synaptic plasticity and cognition. In humans, postnatal brain D-Asp levels decline due to the upregulation of d-aspartate oxidase (hDASPO), the peroxisomal flavoenzyme responsible for D-Asp catabolism. Altered D-Asp homeostasis, characterized by reduced D-Asp and elevated hDASPO expression, has been associated with schizophrenia, supporting therapeutic strategies aimed at restoring physiological D-Asp levels. Although several small-molecule inhibitors of hDASPO have been reported, no potent or clinically viable candidates have emerged. Here, we investigated an alternative strategy based on targeted protein degradation to modulate hDASPO abundance. Guided by STD-NMR, we designed a series of heterobifunctional degraders integrating a hDASPO-binding ligand (olanzapine) with established E3 ligase recruiters (lenalidomide or VH032-Me) and aliphatic linkers of varying lengths. The resulting bifunctional compounds were synthesized and evaluated in biochemical assays for their ability to bind hDASPO. Most compounds retained micromolar inhibitory activity, indicating that derivatization at olanzapine position 10 preserves target engagement, with CRBN-recruiting analogues generally outperforming their VHL-based counterparts. Selected compounds were further investigated in cellular models, where PROTAC 17 demonstrated robust target engagement, positive cooperativity in ternary complex formation and dose-dependent degradation of hDASPO. Together, these findings establish the first proof-of-concept for hDASPO degradation via the PROTAC approach and provide a foundation for therapeutic strategies aimed at re-establishing D-Asp homeostasis in neuropsychiatric disorders characterized by NMDAR dysregulation.

Dimerization between the histone-fold domains (HFD) of two Plasmodium Oocyst Rupture Proteins (ORP1 and ORP2) is essential for oocyst rupture in the Anopheles mosquito vector host, representing a key event in parasite transmission to humans. Notably, ORPs are a rare example of HFD-containing proteins that operate outside the nucleus and that lack DNA-binding functions, typically associated with core histones and transcription factors hosting deviant histones. ORP HFD heterodimerization occurs at the outer capsule of the oocyst, immediately prior to rupture, thus providing a temporal window to administer dimerization blocking molecules. In this context, we present the first detailed structural analysis of the HFD ORP heterodimer, solved by X-ray crystallography at 3.1 Å resolution, and analyze the oligomerization interface as a possible druggable target. Targeting the mosquito phase of the parasite lifecycle remains an under-exploited avenue as present antimalarial therapies mainly target the human blood stages of infection. We employed a GAL4-based yeast two-hybrid (Y2H) combinatorial library of cyclic peptides (CPs) to identify six candidates that inhibit dimerization in vitro. Molecular docking simulations confirmed that all six CPs bind at the dimer interface, allowing us to rank them for further in vivo testing of their efficacy in blocking oocyst rupture.

The Ros/MucR family is constituted by proteins controlling the expression of genes crucial for the interaction with eukaryotic hosts. Ros/MucR family members were classified as H-NS-like proteins in α-proteobacteria, as they share fundamental features with H-NS proteins playing a pivotal role in controlling gene expression by structuring the bacterial genome. Here, we identified two new Ros/MucR family members in Sinorhizobium meliloti. They differ from classical MucR homologs since MucR2 lacks the circular oligomeric structure typical of other family members and MucR3 shows a concentration-dependent oligomerization ability with a low propensity to form circular particles, as shown by cryogenic electron microscopy. Moreover, MucR2 and MucR3 present a new zinc coordination sphere. The newly identified MucRs bind DNA, but lack the DNA bridging activity, which is crucial for structuring the bacterial genome. Using mass spectrometry, light scattering, NMR, EMSA and bridging assay, our study reports the first identification and characterization of two new MucRs and indicates that Ros/MucR family members control gene expression through distinct mechanisms. These results provide an important framework for future studies aimed at dissecting the interplay among MucR proteins and understanding how they can jointly orchestrate condition-dependent gene expression in bacterial species expressing multiple mucR homologous genes.

AIMS:Loss-of-function (LOF) mutations of the cardiac Na+ channel (SCN5A) are causatively associated with the Brugada Syndrome (BrS). However, the onset of Ventricular Fibrillation (VF) is a rare event, and critical factors favouring the pathological phenotype remain often elusive. This study explores how concomitant triggering conditions may impact on VF onset in a symptomatic proband carrying the S805L/SCN5A BrS mutation. METHODS AND RESULTS:Clinical, in-vitro, numerical, and structural analyses were performed. A 67-year-old male was resuscitated after cardiac arrest, and clinical analysis upon hospitalisation revealed severe hypokalaemia (2.5 mEq/L). The ECG showed a coved type-I BrS pattern and the SCN5A mutation (S805L) was identified. Patch-clamp studies carried out in a heterologous expression system (HEK293 cells) revealed that WT/S805L channels exhibit two different phenotypes (normal and LOF); the main parameter controlling this distribution is the cell membrane potential. A protected/normal behaviour was observed at -80 mV; conversely, LOF occurred at more negative potentials (-100/-120 mV). Further analyses in isolated outflow tract ventricular cardiomyocytes showed that hypokalaemia (and bradycardia) induced diastolic potential hyperpolarisation, thus favouring the Na+ current LOF. Computational and molecular modelling confirmed our findings and revealed the structural determinant of this alteration. CONCLUSION:WT/S805L Na+ channels exhibit either a LOF or a wild-type-like behaviour depending on the membrane potential. Since hypokalaemia and slow pacing rate induce cell hyperpolarisation and the associated LOF, they represent concurrent elements creating the scenario responsible for the VF and cardiac arrest. These results may represent an interpretative paradigm applicable to other BrS mutations.

Histone proteins play a central role in chromatin organization. In eukaryotes, the fundamental units of DNA packagingthe nucleosomal coresare assembled from histone dimers. The double histone fold (DHF) refers to a protein architecture in which two adjacent regions, each containing a histone fold, associate to form a histone pseudodimer. In the present study, by targeted sequence searches in protein databases and subsequent structural and phylogenetic investigations, we identified a large number of DHF proteins featuring a high or very high degree of identity with the amino acid sequences of both histones H3 and H4, which constitute a new class of eukaryotic DHF proteins. Strikinglysomehow in analogy with recently identified proteins encoded in some giant viruseswe found, as well, triplets of various kinds (i.e., proteins showing regions of homology with histones H3, histone H4, and an additional histone fold). We were also able to evidence the existence of unprecedented quadruplets encompassing two distinct DHF domains, as well as multiplets that include not only region of homology to nucleosome core histones, but also to the linker histone H1. Focus was put on the evolutionary scenarios for the origin of the newly identified proteins, as well as on the conservation of residues relevant for dimerization and DNA binding. Implications of our findings in fundamental areas of biochemistry are illustrated, and perspectives for future research directions are discussed.

Abstract The oocyst, the sporogonic stage of the malaria parasite located on the mosquito gut, is protected by a capsule, or cyst wall, which surrounds the plasma membrane. The capsule opens to release the infectious sporozoites. Previously, we identified two Oocyst Rupture Proteins (ORP1-2) that are essential for capsule excystation and sporozoite egress. Both ORPs contain a Histone-Fold Domain similar to the NF-YB and -YC subunits of the trimeric human transcription factor NF-Y. Here we identify a Plasmodium protein, named ORP3, as a third subunit of the ORP complex. Although ORP3 is largely unrelated to the NF-YA subunit responsible for trimerization with DNA-binding specificity of NF-Y, it retains the well conserved NF-YA helix A1, responsible for trimerization with the NF-YB/C dimer. A detailed phenotypic analysis of an orp3(-) mutant showed a defect in oocyst opening, as observed for the for orp1(-) and orp2(-) mutants. ORP3 localizes to the periphery of the oocyst, consistent with its position at the capsule. The functional importance of the conserved helix A1 of ORP3 was confirmed, as deletion of this helix abolishes oocyst excystation. The formation of a trimeric complex between a construct containing the helix A1 with the ORP1/2 dimer was further confirmed in vitro using a Yeast-3-hybrid approach. Finally, we confirmed the motility of orp(-) sporozoites, suggesting that the block in parasite transmission following injection into naïve mouse is likely due to a failure in developing into exoerythrocytic forms. Our data strengthen the hypothesis that Plasmodium has re-purposed the NF-Y complex fold and assembly for the unique biological function of promoting oocyst excystation and sporozoite release.

Brugada Syndrome (BrS) is a genetic disease associated with ventricular arrhythmias and is one of the causes of sudden cardiac death. In particular, dysfunctional cardiac Na + channels (SCN5A) represent the only mechanism supporting the autosomal dominant inheritance. A proband without previous medical history was referred to the intensive care unit after resuscitation showing ECG Type 1 BrS pattern and a significant hypokalemia (2.5 mEq/L). Genetic analysis identified a SCN5A de-novo mutation (S805L) in heterozygosis. This study investigates the causative association between the S805L mutation and the BrS event. First, we expressed wild type (WT) and/or mutated (Hetero and Homo) channels in HEK293T. The application of I/V protocols (hp -120 mV) revealed reduced I Na density in Homo and Hetero (-65% and -26% vs WT at -20 mV), indicating S805L as a loss-of-function (LOF) mutation. However, Hetero I Na density was comparable to WT at more depolarized hp (-80 mV), suggesting alterations in the voltage dependent availability of the channel. Accordingly, steady-state inactivation curves of both Hetero and Homo I Na were right shifted, indicating a gain-of-function (GOF) behaviour (Hetero availability: +13% vs WT at -80 mV). To investigate the possible role of hypokalemia in uncovering the LOF behaviour of the mutation, we reproduced in vitro the hypokalemic condition of the patient testing the effects of external K + (K + out ) on diastolic membrane potential (E diast ) of paced guinea-pig cardiomyocytes. Cells were isolated from the Right Ventricular Outflow Tract, known as site of BrS onset. In comparison to a normokalemic condition, E diast significantly hyperpolarized at 2.5 mM K + out and the effect was greater at low pacing rates (1 Hz) (-16.5 mV vs E diast at 5 mM K + and 4 Hz). Thus, the combination of bradycardia and hypokalemia might represent a mechanism able to unmask the LOF effect of S805L mutation in Hetero conditions. Computational approach on the human ventricular action potential confirmed these findings. In conclusion, S805L-SCN5A is, at the same time, a LOF and GOF mutation causing reduced channel expression with increased channel availability. While the balance between GOF-LOF effects guaranties a “protected” phenotype, the hypokalemic-induced cell hyperpolarization might reduce the GOF, leading to the appearance of the BrS event.

Nuclear Factor I (NFI) proteins were first identified in adenovirus DNA replication and later as regulators of gene transcription, stem cell proliferation, and differentiation. They play key roles in development, cancer and congenital disorders. Within the NFI family, NFI-X is critical for neural stem cell biology, hematopoiesis, muscle development, muscular dystrophies and oncogenesis. Here, we present the first structural characterization of the NFI transcription factor, NFI-X, both alone and bound to its consensus palindromic DNA site. Our analyses reveal a novel, MH1-like fold within NFI-X DNA-binding domain (DBD) and identify crucial structural determinants for activity, such as a Zn2+ binding site, dimeric assembly, activation mechanism and DNA-binding specificity. Given the >95% sequence identity within the NFI DBDs, our structural data are prototypic for the entire family; a NFI Rosetta Stone that allows decoding a wealth of biochemical and functional data and provides a precise target for drug design in a wider disease context. ### Competing Interest Statement The authors have declared no competing interest.

Nuclear Factor I (NFI) proteins are involved in adenovirus DNA replication and regulate gene transcription, stem cell proliferation, and differentiation. They play key roles in development, cancer, and congenital disorders. Within the NFI family, NFI-X is critical for neural stem cell biology, hematopoiesis, muscle development, muscular dystrophies, and oncogenesis. Here, we present the structural characterization of the NFI transcription factor NFI-X, both alone and bound to its consensus palindromic DNA site. Our analyses reveal a MH1-like fold within NFI-X DNA-binding domain (DBD) and identify crucial structural determinants for activity, such as a Zn²⁺ binding site, dimeric assembly, and DNA-binding specificity. Given the ~85% sequence identity within the NFI DBDs, our structural data are prototypic for the entire family, a NFI Rosetta Stone that allows decoding a wealth of biochemical and functional data and provides a precise target for drug design in a wider disease context.

Cold-active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here-reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298-331), whereas the dynamic behavior of loops flanking the active site (residues 47-61 and 407-413) may favor enzyme-substrate interactions at the optimal temperature of catalysis (Topt) by tethering together protein regions lining the active site. Stapling of the N-terminus onto the surface of the β-barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a "gatekeeping" hydrophobic residue (Leu178), located at the entrance of the active site.

A library of novel nicotinic acid derivatives, focusing on the modification of position 6 of the pyridine ring with (thio)ether functionalities, was mostly produced through an innovative green synthetic approach (Cyrene-based) and evaluated for their α-amylase and α-glucosidase inhibitory activity. Compounds 8 and 44 demonstrated micromolar inhibition against α-amylase (IC50 of 20.5 and 58.1 μM, respectively), with 44 exhibiting a remarkable ∼72% enzyme inactivation level, surpassing the efficacy of the control compound, acarbose. Conversely, 35 and 39 exhibited comparable inhibition values to acarbose against α-glucosidase (IC50 of 32.9 and 26.4 μM, respectively) and a significant enhancement in enzyme inhibition at saturation (∼80-90%). Mechanistic studies revealed that the most promising compounds operated through a noncompetitive inhibition mechanism for both α-amylase and α-glucosidase, offering advantages for function regulation over competitive inhibitors. These inhibitors may open a new perspective for the development of improved hypoglycemic agents for type 2 diabetes treatment.

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.

The Front Cover illustrates an efficient synthetic strategy for nucleophilic aromatic substitutions of nicotinic esters in Cyrene. This solvent, obtained from renewable cellulose waste and being non-toxic, non-mutagenic and biodegradable, integrates into Green Chemistry practices by aligning with sustainable principles and minimizing environmental impact. More information can be found in the Research Article by A. Citarella, V. Fasano et al.

The green solvent CyreneTM has emerged as a valuable substitute for conventional polar aprotic organic solvents such as DMF, DMSO and NMP (renowned for their toxicity and environmental concerns). However, in the presence of bases, Cyrene is prone to polymerization, thus potentially incompatible with reactions where a base is needed to generate reactive nucleophiles. In this study, we developed an efficient synthetic strategy for nucleophilic aromatic substitutions of nicotinic esters in Cyrene. The success of this protocol relies on a very short reaction time (only 15 minutes) which prevents polymerization from occurring. Indeed, Cyrene not only outperformed typical solvents such as DMF and DMSO, but also, being highly soluble in water, allowed an easy purification of the desired products by simple precipitation upon the addition of ice‐water.

High energy consumption in the nervous system requires a continuous supply of O2. This role is assisted by proteins from the globin super-family in the nerve cells of invertebrates, where 'nerve hemoglobins' (nHbs) are mainly present at mM concentrations and exhibit oxygen affinities comparable to those of vertebrate myoglobins. To gain insight into the structural bases of this function, we report the crystal structure of nHb from the Atlantic surf clam Spisula solidissima (SsHb), previously suggested to display a bis-histidyl hexa-coordinated heme in the deoxy state, high O2 affinity, and ligand binding cooperativity when assayed in situ. The crystallized protein forms a dimer through packing of a 4-helix bundle involving helices E and F of each subunit. The SsHb 'classic' globin fold displays bis-histidyl (His71(E7) and His103(F8)) hexa-coordination of the heme-Fe atom, with structural and dynamics variations found in the inter-helix hinge regions. Molecular Dynamics simulations of both monomeric and dimeric species in the bis-histidyl hexa-coordinated, deoxy penta-coordinated, and O2-bound hexa-coordinated states reveal distinct structural rearrangements at the interface between subunits in the dimer; these would affect the magnitude of the conformational fluctuations observed between monomer and dimer, and the topology of cavities within the protein matrix and at the interface. These results point to a distal site opening mechanism allowing access of the exogenous ligand to the heme and cast hypotheses on the dimer interface structural and dynamic properties that may support ligand binding cooperativity in dimeric SsHb.

EDITORIAL article Front. Mol. Biosci., 01 February 2023Sec. Structural Biology Volume 10 - 2023 | https://doi.org/10.3389/fmolb.2023.1145834

Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous β-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a β-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.