Publications

Molecular interactions between a diphenyl scaffold and PED/PEA15: Implications for type II diabetes therapeutics targeting PED/PEA15 – Phospholipase D1 interaction
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.
Mercurio I., D’Abrosca G., Della Valle M., Malgieri G., Fattorusso R., Isernia C., Russo L., Di Gaetano S., Pedone E.M., Pirone L., Del Gatto A., Zaccaro L., Alberga D., Saviano M., Mangiatordi G.F.
Computational and Structural Biotechnology Journal 2024 Vol. 23Link

Characterization of Surfactant Spheroidal Micelle Structure for Pharmaceutical Applications: A Novel Analytical Framework

We introduce an innovative theoretical framework tailored for the analysis of Pair Distribution Function (PDF) data derived from Small-Angle X-ray Scattering (SAXS) measurements of core-shell micelles. The new approach involves the exploitation of the first derivative of the PDF and the derivation of analytical equations to solve the core-shell micelle structure under the hypothesis of a spheroidal shape. These analytical equations enable us to determine the micelle’s aggregation number, degree of ellipticity, and contrast in electron density between the core-shell and shell-buffer regions after having determined the whole micelle size and its shell size from the analysis of the first derivative of the PDF. We have formulated an overdetermined system of analytical equations based on the unknowns that characterize the micelle structure. This allows us to establish a Figure of Merit, which is utilized to identify the most reliable solution within the system of equations.
De Caro L., Stoll T., Grandeury A., Gozzo F., Giannini C.

Pharmaceutics, 2024 ,16, 604 – Link

DeLA-DrugSelf: Empowering Multi-Objective De Novo Design through SELFIES Molecular Representation

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds ofIn this paper, we introduce DeLA-DrugSelf, an upgraded version of DeLA-Drug [J. Chem. Inf. Model. 62 (2022) 1411-1424], which incorporates essential advancements for automated multi-objective de novo design. Unlike its predecessor, which relies on SMILES notation for molecular representation, DeLA-DrugSelf employs a novel and robust molecular representation string named SELFIES (SELF-referencing embedded string). The generation process in DeLA-DrugSelf not only involves substitutions to the initial string representing the starting query molecule but also incorporates insertions and deletions. This enhancement makes DeLA-DrugSelf significantly more adept at executing data-driven scaffold decoration and lead optimization strategies. Remarkably, DeLA-DrugSelf explicitly addresses the SELFIES-related collapse issue, considering only collapse-free compounds during generation. These compounds undergo a rigorous quality metrics evaluation, highlighting substantial advancements in terms of drug-likeness, uniqueness, and novelty compared to the molecules generated by the previous version of the algorithm. To evaluate the potential of DeLA-DrugSelf as a mutational operator within a genetic algorithm framework for multi-objective optimization, we employed a fitness function based on Pareto dominance. Our objectives focused on target-oriented properties aimed at optimizing known cannabinoid receptor 2 (CB2R) ligands. The results obtained indicate that DeLA-DrugSelf, available as a user-friendly web platform (https://www.ba.ic.cnr.it/softwareic/delaself/), can effectively contribute to the data-driven optimization of starting bioactive molecules based on user-defined parameters.
Alberga D., Lamanna G., Graziano G., Delre P., Lomuscio M.C., Corriero N., Ligresti A., Siliqi., Saviano M., Contino M., Stefanachi A., Mangiatordi G.F.

Computers in Biology and Medicine 2024 – 10848 – Link

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as ‘agretti’, is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 μg/mL (1.4 μM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 μg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome- inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.
Babič M.N., Ragucci S., Leonardi A., Pavšič, Landi N., Križaj I., Gunde-Cimerman G., Sepčić K., Di Maro A.

Biomolecules, 2024, 13, 336;Link

Effect of active layer-by-layer edible coating on quality, biochemicals, and the antioxidant system in ready-to-eat ‘Williams’ pear fruit during cold storage This study tested an active edible coating on the quality and storage capacity of fresh-cut pear stored at 2 ±0.5 ◦C for 10 days by incorporating oxalic (OA) and citric acid (CA) into carboxymethyl cellulose (CMC) and sodium alginate (SA). The physicochemical parameters, secondary metabolite and amino acid contents, and antioxidant activities were determined. Furthermore, enzymes related to oxidative stress and browning, and relative markers were also analysed. The results showed that ready-to-eat pear coated with CMC+SA+OA+CA had a significant inhibitory effect on physiological decay, minimizing the TSS and pH increase and TA decrease. In addition, coating application improved the production of secondary metabolites. The activity of all enzymes involved in ROS detoxification was shown to be promoted by the application of the coating. The degree of browning and oxidative damage that increase progressively with increasing storage time in physiological condition, resulting inhibited by coating. Furthermore, this treatment significantly reduced the protein degradation. Therefore, using the CMC+SA+OA+CA edible coating has the potential to hinder decay and greatly extend the storage period of ’Williams’ pear fruit in cold storage.
Magri A., Landi N., Capriolo G., Di Maro A., Petriccione M.
Toxins, 2024-16(3) 29(2): 51 Link
Hortensins, Type 1 Ribosome-Inactivating Proteins from Seeds of Red Mountain Spinach: Isolation, Characterization, and Their Effect on Glioblastoma Cells Ribosome inactivating proteins (RIPs) are specific N-β-glycosylases that are well-characterized in plants. Their enzymatic action is to damage ribosomes, thereby blocking protein translation. Recently, several research groups have been working on the screening for these toxins in edible plants to facilitate the use of RIPs as biotechnological tools and biopesticides and to overcome public prejudice. Here, four novel monomeric (type 1) RIPs have been isolated from the seeds of Atriplex hortensis L. var. rubra, which is commonly known as edible red mountain spinach. These enzymes, named hortensins 1, 2, 4, and 5, are able to release the β-fragment and, like many other RIPs, adenines from salmon sperm DNA, thus, acting as polynucleotide:adenosine glycosidases. Structurally, hortensins have a different molecular weight and are purified with different yields (hortensin 1, ~29.5 kDa, 0.28 mg per 100 g; hortensin 2, ~29 kDa, 0.29 mg per 100 g; hortensin 4, ~28.5 kDa, 0.71 mg per 100 g; and hortensin 5, ~30 kDa, 0.65 mg per 100 g); only hortensins 2 and 4 are glycosylated. Furthermore, the major isoforms (hortensins 4 and 5) are cytotoxic toward human continuous glioblastoma U87MG cell line. In addition, the morphological change in U87MG cells in the presence of these toxins is indicative of cell death triggered by the apoptotic pathway, as revealed by nuclear DNA fragmentation (TUNEL assay).
Ragucci S., Russo V., Clemente A., Campanile M.G., Oliva M.A., Landi N., Pedone P.V., Arcella A., Di Maro A.
Postharvest Biology and Technology 212 (2024) 112873 Link
Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio
Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irre- versibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. Methods: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. Results: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. Conclusions: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.
Mottola F., Ragucci S., Carannante M., Landi N., Scudiero R., Di Maro A., Rocco L.
Front. Biasci. (Landmark Ed) 2024; 29(2): 51 Link

Intrinsically disordered proteins studied by NMR spectroscopy
Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of complex multi-domain proteins are now identified as a trend topic by the scientific community. NMR constitutes a unique investigation tool to access atom resolved information on their structural and dynamic properties, in isolation or upon interaction with potential partners (metal ions, small molecules, proteins, nucleic acids, membrane mimetics etc.). Their high flexibility and disorder, in contrast to more compact structures of globular protein domains, has a strong impact on NMR observables and NMR experiments should be tailored for their investigation. In this context, 13C direct detection NMR has become a very useful tool to contribute to IDPs/IDRs characterization at atomic resolution. 2D CON spectra can now be collected in parallel to 2D HN ones, and reveal information, which in some cases is not accessible through 2D HN spectra only, particularly when studying proteins in experimental conditions approaching physiological pH and temperature. The 2D HN/CON spectra are thus becoming a sort of identity card of an IDP/IDR in solution. Their simultaneous acquisition through multiple receiver NMR experiments is particularly useful to investigate the properties of highly flexible intrinsically disordered regions within complex multi-domain proteins, rather than in isolation as often performed to reduce the complexity of the system, an interesting perspective in the field.
Schiavina M., Bracaglia L., Bolognesi T., Rodella M.A., Tagliaferro G., Tino A.S., Pierattelli R., Felli I.C.,
Journal of Magnetic Resonance Open, 100143 (2023)  Link

Hunting down zinc(II)-binding sites in proteins with distance matrices
In recent years, high-throughput sequencing technologies have made available the genome sequences of a huge variety of organisms. However, the functional annotation of the encoded proteins often still relies on low-throughput and costly experimental studies. Bioinformatics approaches offer a promising alternative to accelerate this process. In this work, we focus on the binding of zinc(II) ions, which is needed for 5%-10% of any organism’s proteins to achieve their physiologically relevant form.
Laveglia V., Bazayeva M., Andreini C., Rosato A.
Bioinformatics, 39(11) (2023)  Link

Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths. Schiavina M.,Bracaglia L., Rodella M.A., Kümmerle R., Konrat R., Felli I.C., Pierattelli R. Nature Protocols 19, pages 406–440 (2024) Link

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3
Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.
Bargagna B., Banci L., Camponeschi F., 
International Journal of Molecular Science, 2023, 24(14), 11734 Link

Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs
The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.
Cerofolini L., Ravera E., Fischer C., Trovato A., Sacco F., Palinsky W., Angiuoni G., Fragai M., Baroni F., 
Anal. Chem. 2023, 95, 24, 9199-9206 Link

Studies of proline conformational dynamics in IDPs by 13C-detected cross-correlated NMR relaxation
Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole–dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.
Schiavina M.; Konrat Ruth; Ceccolini I.; Mateos B.; Konrat Robert; Felli I.C.; Pierattelli R.
Journal of Magnetic Resonance – Vol. 354, 107539 (2023) Link

Antifungal Activity of Ageritin, a Ribotoxin-like Protein from Cyclocybe aegerita Edible Mushroom, against Phytopathogenic Fungi
Ageritin from poplar mushrooms is a specific endonuclease that hydrolyzes a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA, thereby blocking protein synthesis. Considering the possible biotechnological use of this enzyme, here we report its antifungal activity against virulent fungi affecting crops of economic interest. Our results show that ageritin (200 μg/plug; ~13.5 nmole) inhibits the growth of Botrytis cinerea (57%), Colletotrichum truncatum (42%), and Alternaria alternata (57%), when tested on potato dextrose agar plates. At the same time, no effect was observed against Trichoderma harzianum (a fungus promoting beneficial effects in plants). To verify whether the antifungal action of ageritin against B. cinerea and T. harzianum was due to ribosome damage, we tested ageritin in vitro on partially isolated B. cinerea and T. harzianum ribosomes. Interestingly, ageritin was able to release the Endo’s fragment from both tested fungal ribosomes. We therefore decided to test the antifungal effect of ageritin on B. cinerea and T. harzianum using a different growth condition (liquid medium). Differently from the result in solid medium, ageritin can inhibit both B. cinerea and T. harzianum fungal growth in liquid medium in a concentration-dependent manner up to 35.7% and 38.7%, respectively, at the highest concentration tested (~200 μg/mL; 12 μM), and the analysis of RNA isolated from ageritin-treated cells revealed the presence of Endo’s fragment, highlighting its ability to cross the fungal cell wall and reach the ribosomes. Overall, these data highlight that the efficacy of antifungal treatment to prevent or treat a potential fungal disease may depend not only on the fungal species but also on the conditions of toxin application.
Ragucci S., Castaldi S., Landi N., Isticato R., Di Maro A.,
Toxins, 15(9) 578 (2023), Link

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies
Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin–ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.
Ragucci S., Landi N., Citores L., Iglesias R., Russo R., Clemente A., Saviano M., Pedone P.V., Chambery A., Ferreras J.M., Di Maro A.,
International Journal of Molecular Science, 2023, 24(19), 14435 Link

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes. Cerofolini L., Vasa K., Bianconi E., Salobehaj M., Cappelli G., Alice Bonciani., Licciardi G., Pérez-Ràfols A., Padilla-Cortés L., Antonacci S., Rizzo D., Ravera E., Viglianisi C., Calderone V., Parigi G., Luchinat C., Macchiarulo A., Menichetti S. Angewandte Chemie International Edition, Vol 62 Issue 31, Link

Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs
The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.
Cerofolini L., Ravera E., Fischer C., Trovato A., Sacco F., Palinsky W., Angiuoni G., Fragai M., Baroni F., 
Anal. Chem. 2023, 95, 24, 9199-9206 Link

Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3
Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.
Bargagna B., Banci L., Camponeschi F., 
International Journal of Molecular Science, 2023, 24(14), 11734 Link

Studies of proline conformational dynamics in IDPs by 13C-detected cross-correlated NMR relaxation
Intrinsically disordered proteins (IDPs) are significantly enriched in proline residues, which can populate specific local secondary structural elements called PPII helices, characterized by small packing densities. Proline is often thought to promote disorder, but it can participate in specific π·CH interactions with aromatic side chains resulting in reduced conformational flexibilities of the polypeptide. Differential local motional dynamics are relevant for the stabilization of preformed structural elements and can serve as nucleation sites for the establishment of long-range interactions. NMR experiments to probe the dynamics of proline ring systems would thus be highly desirable. Here we present a pulse scheme based on 13C detection to quantify dipole–dipole cross-correlated relaxation (CCR) rates at methylene CH2 groups in proline residues. Applying 13C-CON detection strategy provides exquisite spectral resolution allowing applications also to high molecular weight IDPs even in conditions approaching the physiological ones. The pulse scheme is illustrated with an application to the 220 amino acids long protein Osteopontin, an extracellular cytokine involved in inflammation and cancer progression, and a construct in which three proline-aromatic sequence patches have been mutated.
Schiavina M.; Konrat Ruth; Ceccolini I.; Mateos B.; Konrat Robert; Felli I.C.; Pierattelli R.
Journal of Magnetic Resonance – Vol. 354, 107539 (2023) Link

Sensitivity considerations on denoising series of spectra by singular value decomposition
In this manuscript researchers of CERM-Unifi demonstrated that – for challenging samples with limited NMR signal – a combined processing of the experiments allows for a time-efficient acquisition of nuclear relaxation experiments
Bruno F.; Fiorucci L.; Ravera E.
Magnetic Resonance in Chemistry – Vol. 61, P373-P379 (2023) Link

Spin Label Study of the Orientational Preferences of Lysozyme in a Bioinspired Silica Composite
Lysozyme can induce the formation of silica microparticles without the use of harsh chemical conditions. In previous work, researchers of CERM-Unifi proved that lysozyme remains in the composite and retains its structure. In this manuscript, using spin-labelling EPR spectroscopy, it is proven that lysozyme has some orientational preference in the resulting composite
Bruno F.,Gigli L.,Ravera E.
Journal of composites science – Vol.7(5), P188 (2023) Link

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays
Seed amplification assays (SAAs) are used to check for the presence of synuclein aggregates as a diagnostic tool for synucleinopathies, including Parkinson’s diesease. The outcome of the SAAs had been previously found to be sensitive to some components of the cerebrospinal fluids (CSF). Researchers of CERM-Unifi, in collaboration with University of Perugia, Istituto Neurologico Carlo Besta, and Amprion Inc. have here demonstrated that the determinant for the inhibition of SAAs giving false negative results is the concentration of lipoproteins
Bellomo G., Paciotti S.,Concha-Marambio L.,Rizzo D., Wojdala A.L., Chiasserini D., Gatticchi L., Cerofolini L., Giuntini S., De Luca C.M.G., Ma Y.,Farris C.M., Pieraccini G., Bologna S.,  Filidei M., Ravera E., Lelli M., Moda F., Fragai M., Parnetti L., Luchinat C.
Molecular Neurodegeneration – Vol 18, A20 (2023) Link