Background. In recent years, the resistance of pathogenic microorganisms to common antimicrobial ... more Background. In recent years, the resistance of pathogenic microorganisms to common antimicrobial agents is representing a severe public health problem. Moderate and wise use of antimicrobials and prevention of infections are the most effective methods for decreasing the spread and development of resistance. Therefore, the World Health Organization (WHO) intensified searching for new agents capable of fighting emerging bacteria. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), play a crucial role in innate immunity, representing one of the first barriers against external attack. In the present study, we evaluated the antibacterial activity of the Hylin-a1 peptide, derived from the frog skin of Heleioporus albopunctatus, against several strains of Gram-positive bacteria, e.g., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus mutans, and clinical isolates). Methods. Peptide toxicity was evaluated on human keratinocytes cells (HaCaT) by the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and time-killing assays were performed to deepen how the peptide exerted its action on bacterial cells. The antibacterial activity was evaluated following the broth microdilution method outlined by the National Committee on Clinical Laboratory Standards (NCCLS) using sterile 96-well microliter plates. Peptide concentrations were selected based on cytotoxicity calculated by MTT assay (50-0.39 µM). The antibacterial power was evaluated against Gram-positive bacteria, in detail S. aureus ATCC, S. epidermidis ATCC, E. faecalis ATCC, S. mutans ATCC. The antibacterial activity of Hylin-a1 was tested against clinical strains isolated from Microbiology Laboratory of the University of Campania "Luigi Vanvitelli".
Mitochondrial demise is a key feature of the progressive neuronal death contributing to acute and... more Mitochondrial demise is a key feature of the progressive neuronal death contributing to acute and chronic neurological disorders. Recent studies identified a pivotal role for the BH3-only protein Bid for such mitochondrial damage and delayed neuronal death after oxygen-glucose-deprivation, glutamate-induced excitotoxicity or oxidative stress in vitro, and after cerebral ischemia in vivo. Therefore, we developed new N-phenyl substituted thiazolidine-2,4-dione derivates as potent inhibitors of Bid-dependent neurotoxicity. The new compounds 6, 7 and 16 were identified as highly protective by extensive screening in a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). These compounds significantly prevent tBid-induced toxicity in the neuronal cell line, providing strong evidence that inhibition of Bid was the underlying mechanism of the observed protective effects. Furthermore, Bid-dependent hallmarks of mitochondrial dysfunction, such as loss of mitochondrial membrane potential, ATP depletion as well as impairments in mitochondrial respiration are significantly prevented by compound 6, 7 and 16. Therefore, the present study identifies a class of N-phenyl thiazolidinediones as novel Bid-inhibiting neuroprotective agents that provide promising therapeutic perspectives for neurodegenerative diseases, where Bid-mediated mitochondrial damage and associated intrinsic death pathways contribute to the underlying progressive loss of neurons.
International Journal of Molecular Sciences, May 6, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Biochimica Et Biophysica Acta - General Subjects, Dec 1, 2020
Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Induci... more Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370-394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing. Methods: We designed and synthesized a set of mono and bicyclic AIF(370-394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments. Results: We identified a stapled peptide analogue of AIF(370-394) with a tenfold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA. Conclusions: Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases. General significance: Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein−ligand interaction, and their utility for drug design.
The SARS-CoV-2 viral spike protein S receptorbinding domain (S-RBD) binds ACE2 on host cells to i... more The SARS-CoV-2 viral spike protein S receptorbinding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein−protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, β3, and β4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.
Background: Many stresses trigger cellular senescence, including telomere shortening, oncogene ac... more Background: Many stresses trigger cellular senescence, including telomere shortening, oncogene activation, DNAreplication stress, oxidative stress, and resistance to apoptosis. Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence, by binding to and inactivating p53, thereby leading to cellular senescence and inhibiting p53 mediated apoptosis. As a result, misregulation of FOXO4-p53 is linked to numerous diseases including cancers and aging-related diseases, pulmonary brosis and neurodegenerative diseases. Targeting of the FOXO4-p53 interface has been accomplished using a synthetic D retro inverse peptide (DRI), however recognition of the key molecular drivers of the interactions remains unknown. Understanding these interactions on a molecular level could lead to the development of small compounds, with enhanced pharmacological qualities, such as high stability, membrane permeability, and oral bioavailability, that mimics the activity of the DRI peptide. Methods and Results: Using an in silico model of the FOXO4-p53 interaction, we designed shorter peptides within the DRI to identify the key interactions. A biophysical assay demonstrates these shorter peptides retain strong binding a nity to p53, suggesting that the DRI's hot spot is located within these peptides. Conclusions: Taken together, this data provides information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence and provides the basis for the design of small molecules to inhibit this interaction.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of pr... more Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.
Anomalous protease activities are associated with many diseases. Great efforts are paid for selec... more Anomalous protease activities are associated with many diseases. Great efforts are paid for selecting specific protease modulators for therapeutic approaches. We have selected new modulators of enzyme activity by an homogeneous assay based on a doubly labeled small peptide used as substrate of trypsin. The substrate incorporates the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) at one end and an EDANS-quenching moiety (Dabcyl, (4-(4-dimethylaminophenylazo)-benzoic acid)) on the other end. Following cleavage by trypsin, the peptide-EDANS product is released interrupting the fluorescence resonance energy transfer effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335-345 nm and emission wavelengths at 485-510 nm. The method optimized, tested by detecting the strong inhibiting effect of α1-antitrypsin on trypsin activity, has been developed on 384 multi-well plates in a volume of 10 μL, using an automated platform. From the screening of a chemical library, four compounds that inhibit trypsin activity with IC50s in the micromolar range have been identified. Interestingly, the most active compound (M4) shows a chemical structure recapitulating that of other more potent inhibitors with thiourea and halogenated centers. Molecular docking studies show that M4 is a competitive inhibitor recognizing most residues within or nearby the catalytic pocket.
Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi ce... more Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi cell invasion,
Biochimica Et Biophysica Acta - Bioenergetics, Sep 1, 2018
Protein phosphorylation is a major regulatory mechanism of most cellular processes such as protei... more Protein phosphorylation is a major regulatory mechanism of most cellular processes such as protein synthesis, cell division, signal Abstracts e59
Emerging viruses pose an important global public health challenge, and early action is needed to ... more Emerging viruses pose an important global public health challenge, and early action is needed to control their spread. The Bunyaviridae family contains a great number of arboviruses which are potentially pathogenic for humans. For example, phleboviruses affect a large range of hosts, including humans and animals. Some infections usually have an asymptomatic course, but others lead to severe complications, such as Toscana virus, which is able to cause meningitis and encephalitis. Unfortunately, to date, no vaccines or antiviral treatments have been found. In the present study, we evaluated the effect of melittin-related peptides, namely the frog-derived RV-23 and AR-23, on sandfly fever Naples virus infection in vitro. Both peptides exhibited a strong antiviral activity by targeting the viral particles and blocking the virus–cell interaction. Their action was directed to an early phase of SFNV infection, in particular at viral adsorption on host cells, by interfering with the binding...
Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-bas... more Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z′ factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)-fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z′ factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min.
Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the t... more Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo. PREP1 (Pbx-regulating protein 1, aka PKNOX1), and MEIS1 (Myeloid ecotropic insertion site 1) are transcription factors belonging to the three amino acids loop extension (TALE) homeodomain family 1 , and play essential roles during embryonic development and cancer by associating to pre-B-cell leukemia (PBX) cofactors 2,3. PREP1 or MEIS1 dimerize with PBX1 and direct its DNA binding to specific and only partially overlapping sequences 4-8. Unveiling the PBX-interaction surface of PREP or MEIS would allow not only to better understand their molecular function but would also represent the basis for searching inactivating therapeutic molecules. PREP1 is essential in embryonic development 9,10 and in the adult functions as a tumour-suppressor 11. The tumour-suppressor role of PREP1 is associated with the maintenance of genomic stability 12 , control of DNA replication timing 13 , and protection of the nuclear envelope structure (Purushotaman D. et al., in preparation). Prep1 hypomorphic (Prep1 i/i) mouse fibroblasts accumulate DNA damage, chromosomal abnormalities and display increased basal and genotoxicity-induced apoptosis 12. Indeed, down-regulation of PREP1 induces DNA damage and consents oncogenes like Ras or Meis1 to evade senescence and transform 12. Prep1 i/i mice develop spontaneous tumours also at the heterozygous state and Prep1 haplo-insufficient mice accelerate the development of oncogenes-dependent tumours 9,11,14. Human tumours are low in PREP1 11,15 , while PREP1 overexpression slows-down the growth of tumour cells in vitro and in vivo 4. Because of these properties PREP1 is considered a tumour suppressor. In particular, overexpression of PREP1 inhibits the growth of MEIS1-dependent tumour cells, competing in vivo for the MEIS1 DNA-binding sites 4,5. All known functions of PREP1 depend on the association with PBX.
PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PR... more PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1 lM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization. Keywords PREP1 Á p160 Á Protein domain design Á Protein purification Á Circular dichroism spectroscopy Á Thermal stability Á Protein-protein interaction Electronic supplementary material The online version of this article (
Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpress... more Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpression affects glucose metabolism in several tissues. In particular, in skeletal muscle tissue the interaction of Prep1 with its cofactor p160 impairs GLUT4 expression and glucose uptake. In this study, we show that ceramides (C2cer), a class of lipids antagonizing insulin signalling, increase the levels of Prep1 and p160 in a dose and time-dependent fashion in L6 cells and induce their association by 80%. We find that C2cer exposure inhibits insulin receptor, IRS1 and Akt phosphorylation and reduces insulin-stimulated glycogen content and glucose uptake by 1.3-and 2.1-fold, respectively. The synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reduces in vitro Prep1-p160 binding in a dose-dependent way (IC 50 = 0.20μM). In C2cer-treated L6 cells, 10μM Prep1(54-72) restores insulin signalling impaired by ceramide treatment. Prep1 overexpressing L6 cells display similar metabolic alterations observed in ceramide-treated L6 cells and the presence of Prep1(54-72) mitigates these events. All these findings suggest that disruption of the Prep1/p160 molecular interaction enhances insulin sensitivity impaired by ceramides in skeletal muscle cells and indicate this complex as an important target for type 2 diabetes.
A novel Outer Membrane Vesicle-mediated mechanism of antimicrobial resistance of Klebsiella pneum... more A novel Outer Membrane Vesicle-mediated mechanism of antimicrobial resistance of Klebsiella pneumoniae BACKGROUND: Antibiotic resistance can occur through mutations or the acquisition of resistance gene determinants via horizontal gene transfer (HGT). The latter is the most important factor in the current AMR pandemic. Recent evidence has reported OMVs as a novel mechanism of HGT, although, the mechanisms and factors that regulate this process are unclear. This study shows, for the first time, the transfer of plasmids containing beta-lactamase resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). METHODS: Multisensitive standard K. pneumoniae (ATCC10031) was transformed through the calcium chloride method with pGR (high copy number) and PRM (low copy number) plasmids. OMVs were isolated from K. pneumoniae-pGR and-PRM colture and characterized via transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) and mass spectrometry. The plasmid concentration in the vesicular lumen was obtained by Real-time PCR. For gene transfer assays, multi-sensitive standard K. pneumoniae, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Burkholderia cepacia (B. cepacia) and Salmonella enterica (S. enterica) represent the recipient strains. These strains (10 7 CFU/mL) were incubated with 10 µg of OMV-K. pneumoniae for 8 hours at 37°C and, then, plated on LB agar supplemented with ampicillin. The bacterial colonies grown were counted for transformation efficiency and subjected to colony-PCR to detect the resistance genes.
Background. In recent years, the resistance of pathogenic microorganisms to common antimicrobial ... more Background. In recent years, the resistance of pathogenic microorganisms to common antimicrobial agents is representing a severe public health problem. Moderate and wise use of antimicrobials and prevention of infections are the most effective methods for decreasing the spread and development of resistance. Therefore, the World Health Organization (WHO) intensified searching for new agents capable of fighting emerging bacteria. Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), play a crucial role in innate immunity, representing one of the first barriers against external attack. In the present study, we evaluated the antibacterial activity of the Hylin-a1 peptide, derived from the frog skin of Heleioporus albopunctatus, against several strains of Gram-positive bacteria, e.g., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus mutans, and clinical isolates). Methods. Peptide toxicity was evaluated on human keratinocytes cells (HaCaT) by the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and time-killing assays were performed to deepen how the peptide exerted its action on bacterial cells. The antibacterial activity was evaluated following the broth microdilution method outlined by the National Committee on Clinical Laboratory Standards (NCCLS) using sterile 96-well microliter plates. Peptide concentrations were selected based on cytotoxicity calculated by MTT assay (50-0.39 µM). The antibacterial power was evaluated against Gram-positive bacteria, in detail S. aureus ATCC, S. epidermidis ATCC, E. faecalis ATCC, S. mutans ATCC. The antibacterial activity of Hylin-a1 was tested against clinical strains isolated from Microbiology Laboratory of the University of Campania "Luigi Vanvitelli".
Mitochondrial demise is a key feature of the progressive neuronal death contributing to acute and... more Mitochondrial demise is a key feature of the progressive neuronal death contributing to acute and chronic neurological disorders. Recent studies identified a pivotal role for the BH3-only protein Bid for such mitochondrial damage and delayed neuronal death after oxygen-glucose-deprivation, glutamate-induced excitotoxicity or oxidative stress in vitro, and after cerebral ischemia in vivo. Therefore, we developed new N-phenyl substituted thiazolidine-2,4-dione derivates as potent inhibitors of Bid-dependent neurotoxicity. The new compounds 6, 7 and 16 were identified as highly protective by extensive screening in a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). These compounds significantly prevent tBid-induced toxicity in the neuronal cell line, providing strong evidence that inhibition of Bid was the underlying mechanism of the observed protective effects. Furthermore, Bid-dependent hallmarks of mitochondrial dysfunction, such as loss of mitochondrial membrane potential, ATP depletion as well as impairments in mitochondrial respiration are significantly prevented by compound 6, 7 and 16. Therefore, the present study identifies a class of N-phenyl thiazolidinediones as novel Bid-inhibiting neuroprotective agents that provide promising therapeutic perspectives for neurodegenerative diseases, where Bid-mediated mitochondrial damage and associated intrinsic death pathways contribute to the underlying progressive loss of neurons.
International Journal of Molecular Sciences, May 6, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Biochimica Et Biophysica Acta - General Subjects, Dec 1, 2020
Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Induci... more Background: The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370-394)) provides a significant neuroprotection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical compounds targeting this complex are missing. Methods: We designed and synthesized a set of mono and bicyclic AIF(370-394) analogs containing both disulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments. Results: We identified a stapled peptide analogue of AIF(370-394) with a tenfold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA. Conclusions: Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic approaches in neurodegenerative diseases. General significance: Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conformational constraints on the protein−ligand interaction, and their utility for drug design.
The SARS-CoV-2 viral spike protein S receptorbinding domain (S-RBD) binds ACE2 on host cells to i... more The SARS-CoV-2 viral spike protein S receptorbinding domain (S-RBD) binds ACE2 on host cells to initiate molecular events, resulting in intracellular release of the viral genome. Therefore, antagonists of this interaction could allow a modality for therapeutic intervention. Peptides can inhibit the S-RBD:ACE2 interaction by interacting with the protein−protein interface. In this study, protein contact atlas data and molecular dynamics simulations were used to locate interaction hotspots on the secondary structure elements α1, α2, α3, β3, and β4 of ACE2. We designed a library of discontinuous peptides based upon a combination of the hotspot interactions, which were synthesized and screened in a bioluminescence-based assay. The peptides demonstrated high efficacy in antagonizing the SARS-CoV-2 S-RBD:ACE2 interaction and were validated by microscale thermophoresis which demonstrated strong binding affinity (∼10 nM) of these peptides to S-RBD. We anticipate that such discontinuous peptides may hold the potential for an efficient therapeutic treatment for COVID-19.
Background: Many stresses trigger cellular senescence, including telomere shortening, oncogene ac... more Background: Many stresses trigger cellular senescence, including telomere shortening, oncogene activation, DNAreplication stress, oxidative stress, and resistance to apoptosis. Forkhead box protein O4 (FOXO4) plays a pivotal role in cellular senescence, by binding to and inactivating p53, thereby leading to cellular senescence and inhibiting p53 mediated apoptosis. As a result, misregulation of FOXO4-p53 is linked to numerous diseases including cancers and aging-related diseases, pulmonary brosis and neurodegenerative diseases. Targeting of the FOXO4-p53 interface has been accomplished using a synthetic D retro inverse peptide (DRI), however recognition of the key molecular drivers of the interactions remains unknown. Understanding these interactions on a molecular level could lead to the development of small compounds, with enhanced pharmacological qualities, such as high stability, membrane permeability, and oral bioavailability, that mimics the activity of the DRI peptide. Methods and Results: Using an in silico model of the FOXO4-p53 interaction, we designed shorter peptides within the DRI to identify the key interactions. A biophysical assay demonstrates these shorter peptides retain strong binding a nity to p53, suggesting that the DRI's hot spot is located within these peptides. Conclusions: Taken together, this data provides information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence and provides the basis for the design of small molecules to inhibit this interaction.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of pr... more Parvulins, peptidyl-prolyl isomerase enzymes (PPIase), catalyze the cis-trans isomerization of prolyl bonds in polypeptides, contributing to folding and function regulation of many proteins. Among Parvulins, Par17, exclusively expressed in hominids, is the least examined in terms of structure, catalytic function and cellular activity. Setting the conditions for the preparation of recombinant active Par17 may therefore significantly foster future studies. Here, we comparatively evaluated the impact of several parameters, including host strains, culture media, isopropyl ß-D-1-thiogalactopyranoside concentration, post-induction incubation time and temperature, on the overexpression of Par17 in E. coli cells. A similar approach was also comparatively adopted for the preparation of the recombinant full-length Pin1 protein, the most representative Parvulin, and the catalytic domains of both enzymes. Proteins were efficiently expressed and purified to homogeneity and were subjected to a structural characterization by Size Exclusion Chromatography and Circular Dichroism. Moreover, a single-step homogeneous protease-based fluorimetric assay, potentially scalable in HTS format, has been developed for determining the peptidyl-prolyl cis-trans isomerase activity of recombinant Parvulins. Results obtained show that proteins are folded and active. These new data mark an important milestone for progressing the investigation of Parvulins.
Anomalous protease activities are associated with many diseases. Great efforts are paid for selec... more Anomalous protease activities are associated with many diseases. Great efforts are paid for selecting specific protease modulators for therapeutic approaches. We have selected new modulators of enzyme activity by an homogeneous assay based on a doubly labeled small peptide used as substrate of trypsin. The substrate incorporates the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) at one end and an EDANS-quenching moiety (Dabcyl, (4-(4-dimethylaminophenylazo)-benzoic acid)) on the other end. Following cleavage by trypsin, the peptide-EDANS product is released interrupting the fluorescence resonance energy transfer effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335-345 nm and emission wavelengths at 485-510 nm. The method optimized, tested by detecting the strong inhibiting effect of α1-antitrypsin on trypsin activity, has been developed on 384 multi-well plates in a volume of 10 μL, using an automated platform. From the screening of a chemical library, four compounds that inhibit trypsin activity with IC50s in the micromolar range have been identified. Interestingly, the most active compound (M4) shows a chemical structure recapitulating that of other more potent inhibitors with thiourea and halogenated centers. Molecular docking studies show that M4 is a competitive inhibitor recognizing most residues within or nearby the catalytic pocket.
Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi ce... more Binding of triazole-linked galactosyl arylsulfonamides to galectin-3 affects Trypanosoma cruzi cell invasion,
Biochimica Et Biophysica Acta - Bioenergetics, Sep 1, 2018
Protein phosphorylation is a major regulatory mechanism of most cellular processes such as protei... more Protein phosphorylation is a major regulatory mechanism of most cellular processes such as protein synthesis, cell division, signal Abstracts e59
Emerging viruses pose an important global public health challenge, and early action is needed to ... more Emerging viruses pose an important global public health challenge, and early action is needed to control their spread. The Bunyaviridae family contains a great number of arboviruses which are potentially pathogenic for humans. For example, phleboviruses affect a large range of hosts, including humans and animals. Some infections usually have an asymptomatic course, but others lead to severe complications, such as Toscana virus, which is able to cause meningitis and encephalitis. Unfortunately, to date, no vaccines or antiviral treatments have been found. In the present study, we evaluated the effect of melittin-related peptides, namely the frog-derived RV-23 and AR-23, on sandfly fever Naples virus infection in vitro. Both peptides exhibited a strong antiviral activity by targeting the viral particles and blocking the virus–cell interaction. Their action was directed to an early phase of SFNV infection, in particular at viral adsorption on host cells, by interfering with the binding...
Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-bas... more Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z′ factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)-fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z′ factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min.
Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the t... more Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo. PREP1 (Pbx-regulating protein 1, aka PKNOX1), and MEIS1 (Myeloid ecotropic insertion site 1) are transcription factors belonging to the three amino acids loop extension (TALE) homeodomain family 1 , and play essential roles during embryonic development and cancer by associating to pre-B-cell leukemia (PBX) cofactors 2,3. PREP1 or MEIS1 dimerize with PBX1 and direct its DNA binding to specific and only partially overlapping sequences 4-8. Unveiling the PBX-interaction surface of PREP or MEIS would allow not only to better understand their molecular function but would also represent the basis for searching inactivating therapeutic molecules. PREP1 is essential in embryonic development 9,10 and in the adult functions as a tumour-suppressor 11. The tumour-suppressor role of PREP1 is associated with the maintenance of genomic stability 12 , control of DNA replication timing 13 , and protection of the nuclear envelope structure (Purushotaman D. et al., in preparation). Prep1 hypomorphic (Prep1 i/i) mouse fibroblasts accumulate DNA damage, chromosomal abnormalities and display increased basal and genotoxicity-induced apoptosis 12. Indeed, down-regulation of PREP1 induces DNA damage and consents oncogenes like Ras or Meis1 to evade senescence and transform 12. Prep1 i/i mice develop spontaneous tumours also at the heterozygous state and Prep1 haplo-insufficient mice accelerate the development of oncogenes-dependent tumours 9,11,14. Human tumours are low in PREP1 11,15 , while PREP1 overexpression slows-down the growth of tumour cells in vitro and in vivo 4. Because of these properties PREP1 is considered a tumour suppressor. In particular, overexpression of PREP1 inhibits the growth of MEIS1-dependent tumour cells, competing in vivo for the MEIS1 DNA-binding sites 4,5. All known functions of PREP1 depend on the association with PBX.
PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PR... more PREP1/p160 is a protein complex with relevant physiopathological roles in vivo. p160 regulates PREP1 transcriptional activity by preventing the formation of other PREP1-containing complexes, whereas PREP1 regulates p160 activity by increasing its stability. This induces the repression of the insulin-regulated glucose transporter GLUT4 dampening insulin sensitivity. In spite of the considerable amount of functional studies performed on the PREP1/p160 complex in vivo, a biochemical and structural characterization of this complex has not been so far undertaken, given the poor stability of the recombinant full-length proteins. Here, we report the design and preparation of PREP1 and p160 domains together with preliminary structural and binding studies. PREP1, residues 45-155, and p160, residues 20-160, have been expressed and purified as folded, monomeric domains. The two domains show both all-alpha secondary structures, as demonstrated by CD studies and are endowed with unusually high thermal stabilities. We have also estimated for the first time the PREP1-p160 interaction strength finding that the two recombinant domains interact with a KD ranging between about 0.3 and 1 lM. Altogether, data suggest that the selected PREP1 and p160 domains are structurally independent and that their structure is underlined by high stability and a prevailing alpha-helical organization. Keywords PREP1 Á p160 Á Protein domain design Á Protein purification Á Circular dichroism spectroscopy Á Thermal stability Á Protein-protein interaction Electronic supplementary material The online version of this article (
Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpress... more Prep1 is a homeodomain transcription factor belonging to the TALE protein family. Its overexpression affects glucose metabolism in several tissues. In particular, in skeletal muscle tissue the interaction of Prep1 with its cofactor p160 impairs GLUT4 expression and glucose uptake. In this study, we show that ceramides (C2cer), a class of lipids antagonizing insulin signalling, increase the levels of Prep1 and p160 in a dose and time-dependent fashion in L6 cells and induce their association by 80%. We find that C2cer exposure inhibits insulin receptor, IRS1 and Akt phosphorylation and reduces insulin-stimulated glycogen content and glucose uptake by 1.3-and 2.1-fold, respectively. The synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reduces in vitro Prep1-p160 binding in a dose-dependent way (IC 50 = 0.20μM). In C2cer-treated L6 cells, 10μM Prep1(54-72) restores insulin signalling impaired by ceramide treatment. Prep1 overexpressing L6 cells display similar metabolic alterations observed in ceramide-treated L6 cells and the presence of Prep1(54-72) mitigates these events. All these findings suggest that disruption of the Prep1/p160 molecular interaction enhances insulin sensitivity impaired by ceramides in skeletal muscle cells and indicate this complex as an important target for type 2 diabetes.
A novel Outer Membrane Vesicle-mediated mechanism of antimicrobial resistance of Klebsiella pneum... more A novel Outer Membrane Vesicle-mediated mechanism of antimicrobial resistance of Klebsiella pneumoniae BACKGROUND: Antibiotic resistance can occur through mutations or the acquisition of resistance gene determinants via horizontal gene transfer (HGT). The latter is the most important factor in the current AMR pandemic. Recent evidence has reported OMVs as a novel mechanism of HGT, although, the mechanisms and factors that regulate this process are unclear. This study shows, for the first time, the transfer of plasmids containing beta-lactamase resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). METHODS: Multisensitive standard K. pneumoniae (ATCC10031) was transformed through the calcium chloride method with pGR (high copy number) and PRM (low copy number) plasmids. OMVs were isolated from K. pneumoniae-pGR and-PRM colture and characterized via transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) and mass spectrometry. The plasmid concentration in the vesicular lumen was obtained by Real-time PCR. For gene transfer assays, multi-sensitive standard K. pneumoniae, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Burkholderia cepacia (B. cepacia) and Salmonella enterica (S. enterica) represent the recipient strains. These strains (10 7 CFU/mL) were incubated with 10 µg of OMV-K. pneumoniae for 8 hours at 37°C and, then, plated on LB agar supplemented with ampicillin. The bacterial colonies grown were counted for transformation efficiency and subjected to colony-PCR to detect the resistance genes.
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