MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.

MucR belongs to a large protein family whose members regulate the expression of virulence and symbiosis genes in α-proteobacteria species. This protein and its homologs were initially studied as classical transcriptional regulators mostly involved in repression of target genes by binding their promoters. Very recent studies have led to the classification of MucR as a new type of Histone-like Nucleoid Structuring (H-NS) protein. Thus this review is an effort to put together a complete and unifying story demonstrating how genetic and biochemical findings on MucR suggested that this protein is not a classical transcriptional regulator, but functions as a novel type of H-NS-like protein, which binds AT-rich regions of genomic DNA and regulates gene expression.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

Copper (I) or (II) Replacement of the Structural Zinc Ion in the Prokaryotic Zinc Finger Ros Does Not Result in a Functional Domain.

A strict interplay is known to involve copper and zinc in many cellular processes. For this reason, the results of copper's interaction with zinc binding proteins are of great interest. For instance, copper interferences with the DNA-binding activity of zinc finger proteins are associated with the development of a variety of diseases. The biological impact of copper depends on the chemical properties of its two common oxidation states (Cu(I) and Cu(II)). In this framework, following the attention addressed to unveil the effect of metal ion replacement in zinc fingers and in zinc-containing proteins, we explore the effects of the Zn(II) to Cu(I) or Cu(II) replacement in the prokaryotic zinc finger domain. The prokaryotic zinc finger protein Ros, involved in the horizontal transfer of genes from A. tumefaciens to a host plant infected by it, belongs to a family of proteins, namely Ros/MucR, whose members have been recognized in different bacteria symbionts and pathogens of mammals and plants. Interestingly, the amino acids of the coordination sphere are poorly conserved in most of these proteins, although their sequence identity can be very high. In fact, some members of this family of proteins do not bind zinc or any other metal, but assume a 3D structure similar to that of Ros with the residues replacing the zinc ligands, forming a network of hydrogen bonds and hydrophobic interactions that surrogates the Zn-coordinating role. These peculiar features of the Ros ZF domain prompted us to study the metal ion replacement with ions that have different electronic configuration and ionic radius. The protein was intensely studied as a perfectly suited model of a metal-binding protein to study the effects of the metal ion replacement; it appeared to tolerate the Zn to Cd substitution, but not the replacement of the wildtype metal by Ni(II), Pb(II) and Hg(II). The structural characterization reported here gives a high-resolution description of the interaction of copper with Ros, demonstrating that copper, in both oxidation states, binds the protein, but the replacement does not give rise to a functional domain.

Characterization and cytotoxic activity of ribotoxin-like proteins from the edible mushroom Pleurotus eryngii.

Ribotoxin-like proteins (RL-Ps) represent a novel specific ribonuclease family found in edible mushrooms and are able to inhibit protein synthesis. Here, we report the characterization and cytotoxic effects of four novel RL-Ps, named eryngitins, isolated from fruiting bodies of the king oyster mushroom (Pleurotus eryngii). These proteins induced formation of α-fragment from rabbit ribosomes, characteristic of their enzymatic action. The two 15 kDa eryngitins (3 and 4) are considerably more thermostable than the 21 kDa ones (1 and 2), however their overall structural features, as determined by far-UV CD spectrometry, are similar. Complete in vitro digestibility by pepsin-trypsin, and lack of cytotoxicity towards human HUVEC cells suggest low toxicity of eryngitins, if ingested. However, eryngitins exhibit cytotoxic action against insect Sf9 cells, suggesting their possible use in biotechnological applications as bioinsecticides. This cytotoxicity was not enhanced in the presence of cytolytic protein complexes based on aegerolysin proteins from Pleurotus mushrooms.

Ribotoxic Proteins, Known as Inhibitors of Protein Synthesis, from Mushrooms and Other Fungi According to Endo's Fragment Detection.

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant 'ribosome inactivating proteins' (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo's assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the ß-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the ß-fragment experimentally detected by Endo's assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

An Updated Review of Bioactive Peptides from Mushrooms in a Well-Defined Molecular Weight Range.

Here, we report the current status of the bioactive peptides isolated and characterized from mushrooms during the last 20 years, considering 'peptide' a succession from to 2 to 100 amino acid residues. According to this accepted biochemical definition, we adopt ~10 kDa as the upper limit of molecular weight for a peptide. In light of this, a careful revision of data reported in the literature was carried out. The search revealed that in the works describing the characterization of bioactive peptides from mushrooms, not all the peptides have been correctly classified according to their molecular weight, considering that some fungal proteins (>10 kDa MW) have been improperly classified as 'peptides'. Moreover, the biological action of each of these peptides, the principles of their isolation as well as the source/mushroom species were summarized. Finally, this review highlighted that these peptides possess antihypertensive, antifungal, antibiotic and antimicrobial, anticancer, antiviral, antioxidant and ACE inhibitory properties.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Ribotoxin-like proteins from Boletus edulis: structural properties, cytotoxicity and in vitro digestibility.

Porcini are edible mushrooms widely used in cooking due to their extraordinary taste. Despite this, cases of food poisoning have been reported in the recent literature also for ingestion of porcini. Here, we report the isolation from Boletus edulis fruiting bodies of two novel ribotoxin-like proteins (RL-Ps), enzymes already studied in other organisms for their toxicity. These RL-Ps, named Edulitin 1 (16-kDa) and Edulitin 2 (14-kDa), show peculiar structural and enzymatic differences, which probably reflect their different bio-activities and a dose/time dependent toxicity (Edulitin 2) on normal and tumoral human cells. Particularly interesting is the resistance to proteolysis of Edulitin 2, for which it was observed that its toxicity was abolished only after heat treatment (90 °C) followed by proteolysis. As mushroom poisoning is a serious food safety issue, data here presented confirm the existence of toxins also in porcini and the importance of a proper cooking before their consumption.

Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.

This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

Zinc Fingers.

Zinc finger (ZF) domains, that represent the majority of the DNA-binding motifs in eukaryotes, are involved in several processes ranging from RNA packaging to transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding. While their amino acid composition varies from one domain to the other, a shared feature is the coordination of a zinc ion, with a structural role, by a different combination of cysteines and histidines. The classical zinc finger domain (also called Cys2His2) that represents the most common class, uses two cysteines and two histidines to coordinate the metal ion, and forms a compact ßßα architecture consisting in a ß-sheet and an α-helix. GAG-knuckle resembles the classical ZF, treble clef and zinc ribbon are also well represented in the human genome. Zinc fingers are also present in prokaryotes. The first prokaryotic ZF domain found in the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. It shows a Cys2His2 metal ion coordination sphere and folds in a domain significantly larger than its eukaryotic counterpart arranged in a ßßßαα topology. Interestingly, this domain does not strictly require the metal ion coordination to achieve the functional fold. Here, we report what is known on the main classes of eukaryotic and prokarotic ZFs, focusing our attention to the role of the metal ion, the folding mechanism, and the DNA binding. The hypothesis of a horizontal gene transfer from prokaryotes to eukaryotes is also discussed.

Effect of an additional N-terminal methionyl residue on enzymatic and antifungal activities of Ageritin purified from Agrocybe aegerita fruiting bodies.

Ageritin, a specific ribonuclease, damaging the largest rRNA in the highly conserved α-sarcin/ricin stem-loop (SRL) has been well characterized from edible mushroom Agrocybe aegerita. Given its peculiar characteristic, Ageritin is the prototype of a new ribotoxins family expressed in basidiomycetes. In this framework, we report the characterization of Met-Ageritin, an isoform of Ageritin with an additional N-terminal methionyl residue. This difference affects the enzymatic features of this toxin despite is able to release α-fragment when acting on yeast, rabbit or Trichoderma asperellum ribosomes. Met-Ageritin inhibits protein synthesis in vitro with an IC50 = 2.8 nM that is 21-fold lower than that of Ageritin, while not show endonuclease activity on DNA. Subsequently, we explored the antifungal activity of both isoforms against T. asperellum, pathogen for A. aegerita and Saccharomyces cerevisiae, used as eukaryotic model microorganism. The presence of an additional N-terminal methionyl residue in Met-Ageritin abolishes antifungal activity towards T. asperellum, while neither of two isoforms is able to inhibit the growth of S. cerevisiae. Overall, these data highlight the importance of the N-terminal region of this toxin that likely alters the conformational state of this enzyme considering the presence in this region of metal binding sites necessary for explicate enzymatic activity.

Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.

Ni(II), Hg(II), and Pb(II) Coordination in the Prokaryotic Zinc-Finger Ros87.

Zinc ion binding is a principal event in the achievement of the correct fold in classical zinc finger domains since the motif is largely unfolded in the absence of metal. In the case of a prokaryotic zinc finger, the larger ßßßαα domain contributes to the folding mechanism with a larger hydrophobic core. For these reasons, following the great amount of attention devoted to unveiling the effect of xenobiotic metal ion replacement in zinc fingers and in zinc-containing proteins in general, the prokaryotic zinc finger domain appears to be an interesting model for studying metal ion interaction with metalloproteins. Here, we explore the binding of Ni(II), Hg(II), and Pb(II) to Ros87, the DNA binding domain of the prokaryotic zinc finger protein Ros. We measured Ros87-metal ion dissociation constants and monitored the effects on the structure and function of the domain. Interestingly, we found that the protein folds in the presence of Ni(II) with important structural perturbations, while in the presence of Pb(II) and Hg(II) it does not appear to be significantly folded. Accordingly, an overall strong reduction in the DNA binding capability is observed for all of the examined proteins. Our data integrate and complement the information collected in the past few years concerning the functional and structural effects of metal ion substitution in classical zinc fingers in order to contribute to a better comprehension of the toxicity of these metals in biological systems.

Structural insights into nucleotide and protein sequence of Ageritin: a novel prototype of fungal ribotoxin.

Here, we report the amino acid sequence of Ageritin, the first ribotoxin-like protein from basidiomycetes (Agrocybe aegerita). This protein consists of 135 amino acid residues with a theoretical molecular mass of 14,801.80 Da (experimental mass 14,802.84 Da, [M + H+]+). Unlike both the classic ribotoxins and homologous RNases T1 family from ascomycetes, Ageritin has a single free cysteinyl residue and does not show homology with known RNases endowed with the specific enzymatic activity on the universally conserved Sarcin Ricin Loop. On the other hand, our 3D homology study shows that Ageritin has a structural core consisting of an antiparallel ß-sheet and an adjacent long α-helix, typical of ribotoxins and RNase T1 family, although the sheet has an orthogonal arrangement with respect to them. Thus, Ageritin is the first prototype of novel ribotoxin-like protein family from fungi.

Insight into the phylogenetic relationship and structural features of vertebrate myoglobin family.

Myoglobin (Mb) is studied to clarify the structure-function relationships in protein science. In this work, we report the results of a comparative analysis of amino acid sequences from 298 vertebrate Mbs. Forty-one high conserved residues were identified and seven of them were invariants [E18, G25, F43, V68, L72, H93 (proximal histidine) and H97]. E18 is the only invariant amino acid residue located out of the heme-pocket and Xe-cavities playing a role in interaction between the A and E-helices. A comparative analysis of several parameters related to amino acid composition shows an increase of average mass, accessible surface area and volume per residue from Actinopterygii to Mammalia and Aves. This may be due to an increased number of bulky residues reducing the non-specific cavities volume and thus improving the oxygen flow between the heme site and the outside of the protein. Finally, the phylogenetic analyses of Mb in vertebrates are consistent with an evolution that runs with the diversification of the species, but in which several episodes of gene duplication and lost have occurred, less frequently in the ancestors of great taxons, cartilaginous fishes and non-avian reptiles, most frequently in ray-finned fishes and mammals, and very frequently in birds.

A Combined NMR and Computational Approach to Determine the RGDechi-hCit-αv ß3 Integrin Recognition Mode in Isolated Cell Membranes.

The critical role of integrins in tumor progression and metastasis has stimulated intense efforts to identify pharmacological agents that can modulate integrin function. In recent years, αv ß3 and αv ß5 integrin antagonists were demonstrated to be effective in blocking tumor progression. RGDechi-hCit, a chimeric peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to recognize selectively αv ß3 integrin both in vitro and in vivo. High-resolution molecular details of the selective αv ß3 recognition of the peptide are certainly required, nonetheless RGDechi-hCit internalization limited the use of classical in cell NMR experiments. To overcome such limitations, we used WM266 isolated cellular membranes to accomplish a detailed NMR interaction study that, combined with a computational analysis, provides significant structural insights into αv ß3 molecular recognition by RGDechi-hCit. Remarkably, on the basis of the identified molecular determinants, we design a RGDechi-hCit mutant that is selective for αv ß5 integrin.

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells.

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.