Paolo Vincenzo Pedone

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.

Models designed to study protein/metal ion interaction are helpful to provide insights into the rules of metal ion selectivity. In this context, the prokaryotic zinc finger family Ros/MucR offers an example of several naturally occurring homologues binding a structural zinc ion with coordination spheres characterized by different amino acid arrays. In particular, Ros87, the zinc binding domain of the protein Ros from A. tumefaciens, binds Zn(II) with a classical Cys ₂ His ₂ coordination sphere, but most of its homologues show a substitution of the second cysteine by an aspartate. In this study, the binding properties to Zn(II), Co(II) and Cd(II) of the protein Ros87‐C27D, Ros87 mutant with a CysAspHis ₂ coordination sphere, are investigated by means of UV–vis, CD and NMR spectroscopies. Dissociation constants, structural effects and the resulting mechanisms of folding are compared with the wild‐type protein bearing the classical Cys ₂ His ₂ coordination sphere. CysAspHis ₂ coordination sphere induces a two‐state mechanism of folding in the presence of all three different metals, while, differently in the case of Ros87 complexed to Zn(II) or Co(II), the presence of the second cysteine in the coordination sphere leads to the formation of a stable metal binding folding intermediate. Our study underlines how the interplay between the different metal ions and the coordinating amino acid sets is determinant in defining the different Kds and the folding pathway of a given protein.

‘Asprinio’ grape is used to produce a white wine from the Campania region, known as ‘Asprinio’ (DOC since 1993). A plethora of approaches was adopted to characterize the organic compounds (e.g., free amino acids and other metabolites) and microbial population (bacteria and fungi) in ‘Asprinio’ grape must by collecting samples from three different vineyards cultivated with the ‘alberata’ training system. The average free amino acid content of ‘Asprinio’ grape must showed quantitative variations, but no significant statistical differences were found. On average, proline was the most abundant free amino acid (~282 mg/L; 47.9%), followed by arginine (~66 mg/L; 11.5%) and glutamine (~25.2 mg/L; 4.2%). On the other hand, the total polyphenol content (TPC) of ‘Asprinio’ grape must was different, like their antioxidant activity, which increased when the TPC content was higher. Moreover, 1D and 2D NMR spectra highlighted the presence of high sugars amount (in particular glucose) as well as trans-caftaric acid, trans-coutaric acid, trans-fertaric acid, and the cis-isomers of these cinnamate esters. Finally, the evaluation of the microbial communities in the ‘Asprinio’ grape must revealed the presence of several representative bacterial phyla—mainly Bacteroidota, Proteobacteria, and Actinobacteriota—as well as various fungal genera, including Cladosporium, Hanseniaspora, Aspergillus, and Saccharomyces. Notably, these microorganisms, which contribute to the fermentation process and influence the final wine flavor, have been found in different proportions in the grape musts analyzed. Our results contribute to increasing knowledge of the ‘Asprinio’ grape, an indigenous vine of Southern Italy.

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.

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).

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.

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.

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 sequence of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, was 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 exhibit 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 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 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.

The shelterin component telomeric repeat-binding factor 2 (TRF2) is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2 _TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, resulting in a telomeric DNA damage response. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2 _TRFH domain. We identify APOD53 as our most promising compound. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2 _TRFH domain and induces phenotypes consistent with TRF2 _TRFH domain mutants. These include induction of a telomeric DNA damage response in the absence of fusions, increased telomeric replication stress, and impaired recruitment of regulator of telomere elongation helicase 1 (RTEL1) and structure-specific endonuclease subunit (SLX4) to telomeres. We demonstrate that APOD53 impairs cell growth in both a telomerase-positive and an ALT cell line, while sparing the viability of non-cancerous cells. Finally, we find that co-treatment with APOD53 and the G4 stabilizer RHPS4 further exacerbates telomere replication stress.

White button mushroom (Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes. In addition, bisporitin shows magnesium-dependent endonuclease activity and displays a similar far-UV CD spectrum as ageritin, the prototype of RL-Ps, isolated from Cyclocybe aegerita fruiting bodies. Interestingly, bisporitin is the first member of RL-Ps to have noticeably lower thermal stability (T_m = 48.59 ± 0.98 °C) compared to RL-Ps isolated in other mushrooms (T_m > 70 °C). Finally, this protein is only partially hydrolyzed in an in vitro digestive system and does not produce adverse growing effects on eukaryotic cell lines. This evidence paves the way for future investigations on possible bioactivities of this RL-P in the digestive system.