40 results on '"Pedone EM"'
Search Results
2. NMR backbone dynamics studies of human PED/PEA-15 outline protein functional sites
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FARINA B, PIRONE L, RUSSO, Luigi, VIPARELLI F, DOTI N, PEDONE C, PEDONE EM, FATTORUSSO, Roberto, Farina, B, Pirone, L, Russo, Luigi, Viparelli, F, Doti, N, Pedone, C, Pedone, Em, and Fattorusso, Roberto
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protein-protein interaction ,PEDPEA-15 protein ,NMR spectroscopy ,death domain ,backbone dynamic - Abstract
PEDPEA-15 (phosphoprotein enriched in diabetesphosphoprotein enriched in astrocytes) is a ubiquitously expressed protein and a key regulator of cell growth and glucose metabolism. PEDPEA-15 mediates both homotypic and heterotypic interactions and is constituted by an N-terminal canonical death effector domain and a C-terminal tail. In the present study, the backbone dynamics of PEDPEA-15 via 15N R1 and R2 and steady-state [1H]-15N NOE measurements is reported. The dynamic parameters were analyzed using both Lipari-Szabo model-free formalism and a reduced spectral density mapping approach. The results obtained define a polar and charged surface of the death effector domain characterized by internal motions in the micro- to millisecond timescale, which is crucial for the multiple heterotypic functional protein-protein interactions in which PEDPEA-15 is involved. The present study contributes to a better understanding of the molecular basis of the PEDPEA-15 functional interactions and provides a more detailed surface for the design and development of PEDPEA-15 binders. © 2010 FEBS.
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- 2010
3. Structural Insight of the Full-Length Ros Protein: A Prototype of the Prokaryotic Zinc-Finger Family
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Luigi Russo, Gaetano Malgieri, Paolo V. Pedone, Ilaria Baglivo, Luciano Pirone, Gianluca D'Abrosca, Carla Isernia, Antonella Paladino, Roberto Fattorusso, Rosa Iacovino, Rinaldo Grazioso, Emilia Pedone, Marica Sassano, D'Abrosca, G, Paladino, A, Baglivo, I, Russo, L, Sassano, M, Grazioso, R, Iacovino, R, Pirone, L, Pedone, Em, Pedone, Pv, Isernia, C, Fattorusso, R, and Malgieri, G
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Models, Molecular ,0301 basic medicine ,lcsh:Medicine ,Virulence ,010402 general chemistry ,01 natural sciences ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,NMR spectroscopy ,Bacterial Proteins ,Protein Domains ,Transcription (biology) ,Amino Acid Sequence ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,Gene ,Zinc finger ,Binding Sites ,Multidisciplinary ,biology ,Chemistry ,lcsh:R ,Zinc Fingers ,Nuclear magnetic resonance spectroscopy ,Agrobacterium tumefaciens ,Cell cycle ,biology.organism_classification ,0104 chemical sciences ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,Protein structure predictions ,biology.protein ,lcsh:Q ,Molecular modelling ,Protein A - Abstract
Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56–142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-‘like’ gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1–55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.
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- 2020
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4. Heterotypic Sam-Sam Association between Odin-Sam1 and Arap3-Sam: Binding Affinity and Structural Insights
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Pasqualina Liana Scognamiglio, Daniela Marasco, Maurizio Pellecchia, Emilia Pedone, Marilisa Leone, Luciano Pirone, Flavia Anna Mercurio, Mercurio, Fa, Marasco, Daniela, Pirone, L, Scognamiglio, Pl, Pedone, Em, Pellecchia, M, and Leone, M.
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GTPase-activating protein ,Stereochemistry ,GTPase-Activating Proteins ,Organic Chemistry ,Isothermal titration calorimetry ,Plasma protein binding ,GTPase ,Biology ,Biochemistry ,Molecular Docking Simulation ,Article ,Protein Structure, Tertiary ,Protein–protein interaction ,Humans ,Molecular Medicine ,Ankyrin repeat ,Molecular Biology ,Sterile alpha motif ,Adaptor Proteins, Signal Transducing ,Protein Binding - Abstract
Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2-receptor (EphA2-Sam). Both Arap3-Sam and EphA2-Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2-Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin-Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2-Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3-Sam and Odin-Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin-Sam1 and Arap3-Sam might bind with a topology that is common to several Sam-Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3-Sam associations.
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- 2012
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5. Solution structure of the first Sam domain of Odin and binding studies with the EphA2 receptor
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Daniela Marasco, Maurizio Pellecchia, Flavia Anna Mercurio, Luciano Pirone, Marilisa Leone, Emilia Pedone, Mercurio, Fa, Marasco, Daniela, Pirone, L, Pedone, Em, Pellecchia, M, and Leone, M.
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Models, Molecular ,Molecular model ,SPR ,In Vitro Techniques ,Endocytosis ,Biochemistry ,Article ,Humans ,Protein Interaction Domains and Motifs ,Sam domain ,Surface plasmon resonance ,Receptor ,Nuclear Magnetic Resonance, Biomolecular ,Adaptor Proteins, Signal Transducing ,Chemistry ,Chemical shift ,Receptor, EphA2 ,Isothermal titration calorimetry ,Surface Plasmon Resonance ,Recombinant Proteins ,Solutions ,Crystallography ,Multiprotein Complexes ,Biophysics ,Thermodynamics ,Ankyrin repeat ,Sterile alpha motif - Abstract
The EphA2 receptor plays key roles in many physiological and pathological events, including cancer. The process of receptor endocytosis and the consequent degradation have attracted attention as possible means of overcoming the negative outcomes of EphA2 in cancer cells and decreasing tumor malignancy. A recent study indicates that Sam (sterile alpha motif) domains of Odin, a member of the ANKS (ankyrin repeat and sterile alpha motif domain-containing) family of proteins, are important for the regulation of EphA2 endocytosis. Odin contains two tandem Sam domains (Odin-Sam1 and -Sam2). Herein, we report on the nuclear magnetic resonance (NMR) solution structure of Odin-Sam1; through a variety of assays (employing NMR, surface plasmon resonance, and isothermal titration calorimetry techniques), we clearly demonstrate that Odin-Sam1 binds to the Sam domain of EphA2 in the low micromolar range. NMR chemical shift perturbation experiments and molecular modeling studies point out that the two Sam domains interact with a head-to-tail topology characteristic of several Sam-Sam complexes. This binding mode is similar to that we have previously proposed for the association between the Sam domains of the lipid phosphatase Ship2 and EphA2. This work further validates structural elements relevant for the heterotypic Sam-Sam interactions of EphA2 and provides novel insights for the design of potential therapeutic compounds that can modulate receptor endocytosis.
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- 2012
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6. Solution Structure and Backbone Dynamics of the K18G/R82E Alicyclobacillus acidocaldarius Thioredoxin Mutant: A Molecular Analysis of Its Reduced Thermal Stability
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Roberto Fattorusso, Emilia Pedone, and Carla Isernia, Benedetto Di Blasio, Michele Saviano, Mosè Rossi, Simonetta Bartolucci, Carlo Pedone, Paola Di Lello, Oliver Ohlenschläger, Marilisa Leone, Leone, M, DI LELLO, P, Ohlenschlager, O, Pedone, Em, Bartolucci, Simonetta, Rossi, Mose', DI BLASIO, B, Pedone, Carlo, Saviano, M, Isernia, C, Fattorusso, R., Leone, M., DI LELLO, P., Ohlenschlaeger, O., Pedone, E. M., Bartolucci, S., Rossi, M., DI BLASIO, B., Pedone, C., Saviano, M., Isernia, Carla, Fattorusso, Roberto, Ohlenschlaeger, O, and Rossi, M
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Models, Molecular ,Hot Temperature ,Molecular Sequence Data ,Mutant ,Biology ,Biochemistry ,Protein Structure, Secondary ,backbone dynamic ,Thioredoxins ,Bacterial Proteins ,Oxidoreductase ,nmr spectroscopy ,Enzyme Stability ,Thermal stability ,Thioredoxin ,Nuclear Magnetic Resonance, Biomolecular ,Thermostability ,chemistry.chemical_classification ,Thermophile ,thermostability ,Crystallography ,chemistry ,Mutation ,Biophysics ,Alicyclobacillus acidocaldarius ,Mesophile - Abstract
No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.
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- 2004
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7. Circular oligomeric particles formed by Ros/MucR family members mediate DNA organization in α-proteobacteria.
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Chaves-Sanjuan A, D'Abrosca G, Russo V, van Erp B, Del Cont-Bernard A, Capelli R, Pirone L, Slapakova M, Sgambati D, Fattorusso R, Isernia C, Russo L, Barton IS, Roop RM 2nd, Pedone EM, Bolognesi M, Dame RT, Pedone PV, Nardini M, Malgieri G, and Baglivo I
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- DNA, Bacterial metabolism, DNA, Bacterial genetics, Brucella abortus genetics, Brucella abortus metabolism, Models, Molecular, Cryoelectron Microscopy, Gene Expression Regulation, Bacterial, Protein Binding, AT Rich Sequence, Mesorhizobium genetics, Mesorhizobium metabolism, Alphaproteobacteria metabolism, Alphaproteobacteria genetics, Bacterial Proteins metabolism, Bacterial Proteins genetics, Bacterial Proteins chemistry, DNA-Binding Proteins metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics
- Abstract
The transcriptional regulator MucR from Brucella species controls the expression of many genes, including those involved in virulence, by binding AT-rich DNA regions. MucR and its homologs belong to the Ros/MucR family, whose members occur in α-proteobacteria. MucR is a recent addition to the family of histone-like nucleoid structuring (H-NS) proteins. Indeed, despite the lack of sequence homology, MucR bears many functional similarities with H-NS and H-NS-like proteins, structuring the bacterial genome and acting as global regulators of transcription. Here we present an integrated cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance, modeling and biochemical study shedding light on the functional architecture of MucR from Brucella abortus and its homolog Ml5 from Mesorhizobium loti. We show that MucR and Ml5 fold in a circular quaternary assembly, which allows it to bridge and condense DNA by binding AT-rich sequences. Our results show that Ros/MucR family members are a novel type of H-NS-like proteins and, based on previous studies, provide a model connecting nucleoid structure and transcription regulation in α-proteobacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2024
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8. Exploring a Potential Optimization Route for Peptide Ligands of the Sam Domain from the Lipid Phosphatase Ship2.
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Vincenzi M, Mercurio FA, La Manna S, Palumbo R, Pirone L, Marasco D, Pedone EM, and Leone M
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- Humans, Ligands, Protein Binding, Binding Sites, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases metabolism, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases chemistry, Receptor, EphA2 metabolism, Receptor, EphA2 chemistry, Receptor, EphA2 genetics, Peptides chemistry, Peptides metabolism, Peptides pharmacology, Sterile Alpha Motif
- Abstract
The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein-protein interaction.
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- 2024
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9. Structural studies of KCTD1 and its disease-causing mutant P20S provide insights into the protein function and misfunction.
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Balasco N, Ruggiero A, Smaldone G, Pecoraro G, Coppola L, Pirone L, Pedone EM, Esposito L, Berisio R, and Vitagliano L
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- Humans, Amino Acid Sequence, Models, Molecular, Molecular Dynamics Simulation, Protein Domains, Structure-Activity Relationship, Co-Repressor Proteins chemistry, Co-Repressor Proteins genetics, Co-Repressor Proteins metabolism, Mutation
- Abstract
Members of the KCTD protein family play key roles in fundamental physio-pathological processes including cancer, neurodevelopmental/neuropsychiatric, and genetic diseases. Here, we report the crystal structure of the KCTD1 P20S mutant, which causes the scalp-ear-nipple syndrome, and molecular dynamics (MD) data on the wild-type protein. Surprisingly, the structure unravels that the N-terminal region, which precedes the BTB domain (preBTB) and bears the disease-associated mutation, adopts a folded polyproline II (PPII) state. The KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. Indeed, the BTB of each chain makes peculiar contacts with the preBTB and the C-terminal domain (CTD) of an adjacent chain. The BTB-preBTB interaction consists of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/-) helix discontinuous association. The inspection of the protein structure, along with the data emerged from the MD simulations, provides an explanation of the pathogenicity of the P20S mutation and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of KCTD1 in metal homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)
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- 2024
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10. Inhibition of the EphA2-Sam/Ship2-Sam Association through Peptide Ligands: Studying the Combined Effect of Charge and Aromatic Character.
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Vincenzi M, Mercurio FA, Palumbo R, La Manna S, Pirone L, Marasco D, Pedone EM, and Leone M
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- Ligands, Humans, Models, Molecular, Protein Binding, Amino Acid Sequence, Receptor, EphA2 metabolism, Receptor, EphA2 antagonists & inhibitors, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases metabolism, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases antagonists & inhibitors, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases chemistry, Peptides chemistry, Peptides pharmacology, Peptides metabolism, Sterile Alpha Motif
- Abstract
The Sam (sterile alpha motif) domain from the lipid phosphatase Ship2 binds the Sam domain from the EphA2 receptor to negatively regulate receptor endocytosis and degradation. This interaction is primarily linked to pro-oncogenic effects. We report on the design and evaluation of EphA2-Sam/Ship2-Sam peptide inhibitors provided with positive charges and different aromatic characters. Starting from the sequence of previously identified Ship2-Sam targeting peptides, an in silico approach was set up to predict higher affinity peptide ligands. A few peptides were experimentally tested through an interdisciplinary approach. Interaction studies were performed by nuclear magnetic resonance spectroscopy and biolayer interferometry. 3D models of Ship2-Sam/peptide complexes were predicted by AlphaFold2. Cell-based assays were carried out to investigate whether such peptide sequences might have an influence on EphA2 signaling. The approach led to the identification of novel Ship2-Sam ligands and shed further light on original approaches to design inhibitors of the Ship2-Sam/EphA2-Sam interaction.
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- 2024
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11. Molecular interactions between a diphenyl scaffold and PED/PEA15: Implications for type II diabetes therapeutics targeting PED/PEA15 - Phospholipase D1 interaction.
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Mercurio I, D'Abrosca G, Della Valle M, Malgieri G, Fattorusso R, Isernia C, Russo L, Di Gaetano S, Pedone EM, Pirone L, Del Gatto A, Zaccaro L, Alberga D, Saviano M, and Mangiatordi GF
- Abstract
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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)
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- 2024
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12. Structural characterization of PHOX2B and its DNA interaction shed light on the molecular basis of the +7Ala variant pathogenicity in CCHS.
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Diana D, Pirone L, Russo L, D'Abrosca G, Madheswaran M, Benfante R, Di Lascio S, Caldinelli L, Fornasari D, Acconcia C, Corvino A, Ventserova N, Pollegioni L, Isernia C, Di Gaetano S, Malgieri G, Pedone EM, and Fattorusso R
- Abstract
An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)
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- 2024
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13. MucR protein: Three decades of studies have led to the identification of a new H-NS-like protein.
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Baglivo I, Malgieri G, Roop RM 2nd, Barton IS, Wang X, Russo V, Pirone L, Pedone EM, and Pedone PV
- Abstract
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., (© 2024 John Wiley & Sons Ltd.)
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- 2024
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14. Editorial: Special Issue "Galectins: Structure, Function and Therapeutic Inhibitors".
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Pedone EM, Di Gaetano S, and Capasso D
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- Galectins
- Abstract
Galectins, β-galactoside-binding proteins, play relevant roles in different biological processes; therefore, they are becoming emerging targets for diagnostic and therapeutic approaches [...].
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- 2024
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15. MucR from Sinorhizobium meliloti : New Insights into Its DNA Targets and Its Ability to Oligomerize.
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Slapakova M, Sgambati D, Pirone L, Russo V, D'Abrosca G, Valletta M, Russo R, Chambery A, Malgieri G, Pedone EM, Dame RT, Pedone PV, and Baglivo I
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- Bacterial Proteins genetics, Bacterial Proteins metabolism, Transcription Factors metabolism, DNA genetics, DNA metabolism, Symbiosis, Gene Expression Regulation, Bacterial, Repressor Proteins genetics, Sinorhizobium meliloti genetics
- Abstract
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.
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- 2023
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16. SARS-CoV-2 ORF8 dimerization and binding mode analysis with class I MHC: computational approaches to identify COVID-19 inhibitors.
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Selvaraj C, Dinesh DC, Pedone EM, Alothaim AS, Vijayakumar R, Rudhra O, and Singh SK
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- Humans, Dimerization, COVID-19, SARS-CoV-2
- Abstract
SARS-CoV-2 encodes eight accessory proteins, one of which, ORF8, has a poorly conserved sequence with SARS-CoV and its role in viral pathogenicity has recently been identified. ORF8 in SARS-CoV-2 has a unique functional feature that allows it to form a dimer structure linked by a disulfide bridge between Cys20 and Cys20 (S-S). This study provides structural characterization of natural mutant variants as well as the identification of potential drug candidates capable of binding directly to the interchain disulfide bridge. The lead compounds reported in this work have a tendency to settle in the dimeric interfaces by direct interaction with the disulfide bridge. These molecules may disturb the dimer formation and may have an inhibition impact on its potential functional role in host immune evasion and virulence pathogenicity. This work provides detailed insights on the sequence and structural variability through computational mutational studies, as well as potent drug candidates with the ability to interrupt the intermolecular disulfide bridge formed between Cys20 and Cys20. Furthermore, the interactions of ORF8 peptides complexed with MHC-1 is studied, and the binding mode reveals that certain ORF8 peptides bind to MHC-1 in a manner similar to other viral peptides. Overall, this study is a narrative of various computational approaches used to provide detailed structural insights into SARS-CoV-2 ORF8 interchain disulfide bond disruptors., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
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- 2023
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17. Screening a Molecular Fragment Library to Modulate the PED/PEA15-Phospholipase D1 Interaction in Cellular Lysate Environments.
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Farina B, Pirone L, D'Abrosca G, Della Valle M, Russo L, Isernia C, Sassano M, Del Gatto A, Di Gaetano S, Zaccaro L, Malgieri G, Pedone EM, and Fattorusso R
- Subjects
- Binding Sites, Biological Transport, Cellular Microenvironment, Glucose, Humans, Insulin Resistance, Ligands, Molecular Docking Simulation, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Conformation, Thermodynamics, Apoptosis Regulatory Proteins chemistry, Astrocytes chemistry, Diabetes Mellitus, Type 2 metabolism, Peptide Fragments chemistry, Phospholipase D chemistry, Small Molecule Libraries chemistry
- Abstract
The overexpression of PED/PEA15, the phosphoprotein enriched in diabetes/phosphoprotein enriched in the astrocytes 15 protein (here referred simply to as PED), observed in some forms of type II diabetes, reduces the transport of insulin-stimulated glucose by binding to the phospholipase D1 (PLD1). The inhibition of the PED/PLD1 interaction was shown to restore basal glucose transport, indicating PED as a pharmacological target for the development of drugs capable of improving insulin sensitivity and glucose tolerance. We here report the identification and selection of PED ligands by means of NMR screening of a library of small organic molecules, NMR characterization of the PED/PLD1 interaction in lysates of cells expressing PLD1, and modulation of such interactions using BPH03, the best selected ligand. Overall, we complement the available literature data by providing detailed information on the structural determinants of the PED/PLD1 interaction in a cellular lysate environment and indicate BPH03 as a precious scaffold for the development of novel compounds that are able to modulate such interactions with possible therapeutic applications in type II diabetes.
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- 2021
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18. Exploring the Ability of Cyclic Peptides to Target SAM Domains: A Computational and Experimental Study.
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Mercurio FA, Di Natale C, Pirone L, Vincenzi M, Marasco D, De Luca S, Pedone EM, and Leone M
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- Humans, Molecular Docking Simulation, Peptide Library, Protein Binding, Protein Conformation, Peptides, Cyclic chemistry, Peptides, Cyclic metabolism, Receptor, EphA2 metabolism, Sterile Alpha Motif
- Abstract
Sterile alpha motif (SAM) domains are protein interaction modules with a helical fold. SAM-SAM interactions often adopt the mid-loop (ML)/end-helix (EH) model, in which the C-terminal helix and adjacent loops of one SAM unit (EH site) bind the central regions of another SAM domain (ML site). Herein, an original strategy to attack SAM-SAM associations is reported. It relies on the design of cyclic peptides that target a region of the SAM domain positioned at the bottom side of the EH interface, which is thought to be important for the formation of a SAM-SAM complex. This strategy has been preliminarily tested by using a model system of heterotypic SAM-SAM interactions involving the erythropoietin-producing hepatoma kinase A2 (EphA2) receptor and implementing a multidisciplinary plan made up of computational docking studies, experimental interaction assays (by NMR spectroscopy and surface plasmon resonance techniques) and conformational analysis (by NMR spectroscopy and circular dichroism). This work further highlights how only a specific balance between flexibility and rigidity may be needed to generate modulators of SAM-SAM interactions., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
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- 2020
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19. Cystatin B Involvement in Synapse Physiology of Rodent Brains and Human Cerebral Organoids.
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Penna E, Cerciello A, Chambery A, Russo R, Cernilogar FM, Pedone EM, Perrone-Capano C, Cappello S, Di Giaimo R, and Crispino M
- Abstract
Cystatin B (CSTB) is a ubiquitous protein belonging to a superfamily of protease inhibitors. CSTB may play a critical role in brain physiology because its mutations cause progressive myoclonic epilepsy-1A (EPM1A), the most common form of progressive myoclonic epilepsy. However, the molecular mechanisms underlying the role of CSTB in the central nervous system (CNS) are largely unknown. To investigate the possible involvement of CSTB in the synaptic plasticity, we analyzed its expression in synaptosomes as a model system in studying the physiology of the synaptic regions of the CNS. We found that CSTB is not only present in the synaptosomes isolated from rat and mouse brain cortex, but also secreted into the medium in a depolarization-controlled manner. In addition, using biorthogonal noncanonical amino acid tagging (BONCAT) procedure, we demonstrated, for the first time, that CSTB is locally synthesized in the synaptosomes. The synaptic localization of CSTB was confirmed in a human 3D model of cortical development, namely cerebral organoids. Altogether, these results suggest that CSTB may play a role in the brain plasticity and open a new perspective in studying the involvement of CSTB deregulation in neurodegenerative and neuropsychiatric diseases.
- Published
- 2019
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20. Molecular basis of the scalp-ear-nipple syndrome unraveled by the characterization of disease-causing KCTD1 mutants.
- Author
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Smaldone G, Balasco N, Pirone L, Caruso D, Di Gaetano S, Pedone EM, and Vitagliano L
- Subjects
- Amino Acid Substitution, Benzothiazoles metabolism, Co-Repressor Proteins metabolism, Genes, Dominant, Humans, Models, Molecular, Protein Conformation, Protein Denaturation, Protein Domains, Protein Stability, Recombinant Proteins metabolism, Transcription Factor AP-2 metabolism, Abnormalities, Multiple genetics, Co-Repressor Proteins genetics, Ear, External abnormalities, Hypospadias genetics, Muscle Hypotonia genetics, Mutation, Missense, Nipples abnormalities, Point Mutation, Scalp abnormalities
- Abstract
The scalp-ear-nipple (SEN) syndrome is an autosomal-dominant disorder characterized by cutis aplasia of the scalp and malformations of breast, external ears, digits, and nails. Genetic analyses have shown that the disease is caused by missense mutations of the KCTD1 protein, although the functional/structural basis of SEN insurgence is hitherto unknown. With the aim of unravelling the molecular basis of the SEN syndrome associated with KCTD1 mutations we here expressed and characterized several disease causing mutants. A preliminary dissection of the protein provides insights into the role that individual domains play in KCTD1 stability. The characterization of SEN-causing mutants indicates that, although the mutation sites are located in distant regions of the BTB domain or of the pre-BTB region, all of them are unable to interact with the transcription factor AP-2α, a well-known KCTD1 biological partner. Notably, all mutations, including the one located in the pre-BTB region, produce a significant destabilization of the protein. The structural role of the pre-BTB region in KCTD1 and other proteins of the family is corroborated by its sequence conservation in orthologs and paralogs. Interestingly, SEN-causing mutations also favor the tendency of KCTD1 to adopt structural states that are characterized by the ability to bind the β-amyloid fluorescent dye thioflavin T. The formation of aggregation-prone species may have important implications for the disease etiology. Collectively, these findings provide an intriguing picture of the functional and structural alterations induced by KCTD1 mutations that ultimately lead to disease.
- Published
- 2019
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21. Design and analysis of EphA2-SAM peptide ligands: A multi-disciplinary screening approach.
- Author
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Mercurio FA, Di Natale C, Pirone L, Marasco D, Calce E, Vincenzi M, Pedone EM, De Luca S, and Leone M
- Subjects
- Amino Acid Sequence, Binding Sites, Humans, Ligands, Nuclear Magnetic Resonance, Biomolecular, Peptide Library, Peptides blood, Peptides metabolism, Protein Stability, Receptor, EphA2 metabolism, Sterile Alpha Motif, Drug Design, Molecular Docking Simulation, Peptides chemistry, Receptor, EphA2 chemistry
- Abstract
EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
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22. Identifying the region responsible for Brucella abortus MucR higher-order oligomer formation and examining its role in gene regulation.
- Author
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Pirone L, Pitzer JE, D'Abrosca G, Fattorusso R, Malgieri G, Pedone EM, Pedone PV, Roop RM 2nd, and Baglivo I
- Subjects
- DNA, Bacterial genetics, Gene Deletion, Point Mutation genetics, Prokaryotic Cells physiology, Zinc Fingers genetics, Bacterial Proteins genetics, Brucella abortus genetics, Gene Expression Regulation, Bacterial genetics
- Abstract
MucR is a member of the Ros/MucR family of prokaryotic zinc-finger proteins found in the α-proteobacteria which regulate the expression of genes required for the successful pathogenic and symbiotic interactions of these bacteria with the eukaryotic hosts. The structure and function of their distinctive zinc-finger domain has been well-studied, but only recently the quaternary structure of the full length proteins was investigated demonstrating their ability to form higher-order oligomers. The aim of this study was to identify the region of MucR involved in higher-order oligomer formation by analysing deletion and point mutants of this protein by Light Scattering, and to determine the role that MucR oligomerization plays in the regulatory function of this protein. Here we demonstrate that a conserved hydrophobic region at the N-terminus of MucR is responsible for higher-order oligomer formation and that MucR oligomerization is essential for its regulatory function in Brucella. All these features of MucR are shared by the histone-like nucleoid structuring protein, (H-NS), leading us to propose that the prokaryotic zinc-finger proteins in the MucR/Ros family control gene expression employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators.
- Published
- 2018
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23. Sam domain-based stapled peptides: Structural analysis and interaction studies with the Sam domains from the EphA2 receptor and the lipid phosphatase Ship2.
- Author
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Mercurio FA, Pirone L, Di Natale C, Marasco D, Pedone EM, and Leone M
- Subjects
- Amino Acid Sequence, Drug Discovery, Humans, Models, Molecular, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases chemistry, Protein Binding drug effects, Receptor, EphA2 chemistry, Sterile Alpha Motif drug effects, Peptides chemistry, Peptides pharmacology, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases metabolism, Protein Interaction Maps drug effects, Receptor, EphA2 metabolism
- Abstract
Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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24. MucR binds multiple target sites in the promoter of its own gene and is a heat-stable protein: Is MucR a H-NS-like protein?
- Author
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Baglivo I, Pirone L, Malgieri G, Fattorusso R, Roop Ii RM, Pedone EM, and Pedone PV
- Abstract
The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti . MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein.
- Published
- 2018
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25. Folding mechanisms steer the amyloid fibril formation propensity of highly homologous proteins.
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Malgieri G, D'Abrosca G, Pirone L, Toto A, Palmieri M, Russo L, Sciacca MFM, Tatè R, Sivo V, Baglivo I, Majewska R, Coletta M, Pedone PV, Isernia C, De Stefano M, Gianni S, Pedone EM, Milardi D, and Fattorusso R
- Abstract
Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml1
53-149 and zinc-lacking Ml452-151 . The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153-149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml452-151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452-151 and Ml153-149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml153-149 has formed only amorphous aggregates and Ml452-151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition diseases.- Published
- 2018
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26. The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors.
- Author
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Mercurio FA, Di Natale C, Pirone L, Iannitti R, Marasco D, Pedone EM, Palumbo R, and Leone M
- Subjects
- Antineoplastic Agents pharmacology, Binding Sites, Cell Line, Tumor, Drug Design, Escherichia coli, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Male, Membrane Proteins, Models, Molecular, Necrosis chemically induced, Necrosis metabolism, Nuclear Magnetic Resonance, Biomolecular, Peptides chemistry, Peptides pharmacology, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases chemistry, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics, Preliminary Data, Prostatic Neoplasms drug therapy, Prostatic Neoplasms metabolism, Protein Binding, Receptor, EphA2 chemistry, Receptor, EphA2 genetics, Saccharomyces cerevisiae Proteins, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases antagonists & inhibitors, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases metabolism, Receptor, EphA2 antagonists & inhibitors, Receptor, EphA2 metabolism, Sterile Alpha Motif drug effects
- Abstract
The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.
- Published
- 2017
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27. Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability.
- Author
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Baglivo I, Pirone L, Pedone EM, Pitzer JE, Muscariello L, Marino MM, Malgieri G, Freschi A, Chambery A, Roop Ii RM, and Pedone PV
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, Biofilms growth & development, Brucella abortus genetics, Colony Count, Microbial, Gene Expression Regulation, Bacterial, Genes, Bacterial, Mesorhizobium genetics, Mutation genetics, Netropsin metabolism, Phenotype, Plankton growth & development, Protein Binding, AT Rich Sequence genetics, Bacterial Proteins metabolism, Brucella abortus metabolism, DNA, Bacterial genetics, Mesorhizobium metabolism, Protein Multimerization
- Abstract
Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium lo ti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.
- Published
- 2017
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28. Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?
- Author
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Mercurio FA, Costantini S, Di Natale C, Pirone L, Guariniello S, Scognamiglio PL, Marasco D, Pedone EM, and Leone M
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Cataract genetics, Circular Dichroism, Humans, Mass Spectrometry, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Insertional, Neoplasm Proteins chemistry, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Nuclear Magnetic Resonance, Biomolecular, Peptide Fragments chemical synthesis, Peptide Fragments chemistry, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases chemistry, Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases metabolism, Protein Binding, Protein Interaction Mapping, Protein Structure, Secondary, Receptor, EphA2 genetics, Receptor, EphA2 metabolism, Recombinant Fusion Proteins chemistry, Structure-Activity Relationship, Receptor, EphA2 chemistry
- Abstract
Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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29. Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners.
- Author
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Pirone L, Smaldone G, Esposito C, Balasco N, Petoukhov MV, Spilotros A, Svergun DI, Di Gaetano S, Vitagliano L, and Pedone EM
- Subjects
- Amino Acid Sequence, Animals, Biophysical Phenomena, Circular Dichroism, Cullin Proteins chemistry, Cullin Proteins genetics, Drosophila Proteins chemistry, Drosophila Proteins genetics, Drosophila melanogaster genetics, Drosophila melanogaster metabolism, Drosophila melanogaster physiology, Golgi Matrix Proteins, Humans, Membrane Proteins chemistry, Membrane Proteins genetics, Protein Binding, Protein Denaturation, Scattering, Small Angle, Sequence Homology, Amino Acid, Temperature, X-Ray Diffraction, Cullin Proteins metabolism, Drosophila Proteins metabolism, Homeostasis, Membrane Proteins metabolism, Sleep physiology
- Abstract
The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation., (Copyright © 2016. Published by Elsevier B.V.)
- Published
- 2016
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30. Targeting EphA2-Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues.
- Author
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Mercurio FA, Marasco D, Di Natale C, Pirone L, Costantini S, Pedone EM, and Leone M
- Subjects
- Amino Acid Sequence, Circular Dichroism, Drug Design, Kinetics, Magnetic Resonance Spectroscopy, Molecular Dynamics Simulation, Peptides chemical synthesis, Peptides metabolism, Protein Binding, Protein Structure, Secondary, Receptor, EphA2 genetics, Receptor, EphA2 metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Solid-Phase Synthesis Techniques, Sterile Alpha Motif, Surface Plasmon Resonance, Peptides chemistry, Receptor, EphA2 chemistry
- Abstract
The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam-Sam interactions. Because EphA2-Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam-Sam interactions involving EphA2., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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31. Peptide Fragments of Odin-Sam1: Conformational Analysis and Interaction Studies with EphA2-Sam.
- Author
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Mercurio FA, Di Natale C, Pirone L, Scognamiglio PL, Marasco D, Pedone EM, Saviano M, and Leone M
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Amino Acid Sequence, Humans, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Trifluoroethanol chemistry, Water chemistry, Adaptor Proteins, Signal Transducing chemistry, Peptide Fragments chemistry, Peptide Fragments metabolism, Receptor, EphA2 chemistry, Receptor, EphA2 metabolism
- Abstract
Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
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32. Cullin 3 Recognition Is Not a Universal Property among KCTD Proteins.
- Author
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Smaldone G, Pirone L, Balasco N, Di Gaetano S, Pedone EM, and Vitagliano L
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Amino Acid Sequence, Calorimetry, Cell Cycle Proteins, Chromatography, Gel, Circular Dichroism, Cullin Proteins chemistry, Cullin Proteins genetics, Humans, Molecular Dynamics Simulation, Molecular Sequence Data, Potassium Channels chemistry, Potassium Channels genetics, Potassium Channels metabolism, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Repressor Proteins chemistry, Repressor Proteins genetics, Sequence Alignment, Transferases, Cullin Proteins metabolism, Repressor Proteins metabolism
- Abstract
Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6(BTB) and KCTD11(BTB) bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12(BTB) and KCTD15(BTB) do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2β3 loop between KCTD11(BTB) and KCTD12(BTB) is sufficient to abolish the ability of KCTD11(BTB) to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.
- Published
- 2015
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33. Molecular recognition of Cullin3 by KCTDs: insights from experimental and computational investigations.
- Author
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Balasco N, Pirone L, Smaldone G, Di Gaetano S, Esposito L, Pedone EM, and Vitagliano L
- Subjects
- Amino Acid Sequence, Cullin Proteins chemistry, Humans, Models, Molecular, Molecular Sequence Data, Potassium Channels chemistry, Protein Binding, Protein Conformation, Sequence Homology, Amino Acid, Substrate Specificity, Ubiquitination, Cullin Proteins metabolism, Molecular Dynamics Simulation, Potassium Channels metabolism, Ubiquitin metabolism
- Abstract
Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5-Cul3)(5) pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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34. Mapping functional interaction sites of human prune C-terminal domain by NMR spectroscopy in human cell lysates.
- Author
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Diana D, Smaldone G, De Antonellis P, Pirone L, Carotenuto M, Alonzi A, Di Gaetano S, Zollo M, Pedone EM, and Fattorusso R
- Subjects
- Carrier Proteins metabolism, Cell Biology, Drug Discovery, Gelsolin chemistry, Glycogen Synthase Kinase 3 metabolism, Glycogen Synthase Kinase 3 beta, Humans, Magnetic Resonance Spectroscopy, NM23 Nucleoside Diphosphate Kinases metabolism, Phosphoric Monoester Hydrolases, Carrier Proteins chemistry, Glycogen Synthase Kinase 3 chemistry, NM23 Nucleoside Diphosphate Kinases chemistry
- Abstract
Get well prune: The C-terminal third domain of h-prune is largely unfolded and involved in relevant protein-protein interactions, particularly with Nm23-H1 (see figure), GSK-3β and gelsolin. This study shows that protein functions mediated by protein-protein interactions can be accurately followed in cell lysates by using fast NMR spectroscopy, which could be easily used for a very efficient NMR drug-discovery strategy., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2013
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35. Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy.
- Author
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Farina B, Doti N, Pirone L, Malgieri G, Pedone EM, Ruvo M, and Fattorusso R
- Subjects
- Apoptosis Regulatory Proteins, Enzyme-Linked Immunosorbent Assay, Humans, Intracellular Signaling Peptides and Proteins metabolism, Peptide Fragments metabolism, Phospholipase D metabolism, Phosphoproteins metabolism, Protein Conformation, Intracellular Signaling Peptides and Proteins chemistry, Magnetic Resonance Spectroscopy, Peptide Fragments chemistry, Phospholipase D chemistry, Phosphoproteins chemistry
- Abstract
PED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
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36. Molecular organization of the cullin E3 ligase adaptor KCTD11.
- Author
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Correale S, Pirone L, Di Marcotullio L, De Smaele E, Greco A, Mazzà D, Moretti M, Alterio V, Vitagliano L, Di Gaetano S, Gulino A, and Pedone EM
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Cycle Proteins, Cullin Proteins chemistry, Cullin Proteins genetics, HEK293 Cells, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed methods, Protein Binding genetics, Protein Isoforms chemistry, Protein Isoforms genetics, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Transferases, Ubiquitin chemistry, Potassium Channels chemistry, Potassium Channels genetics, Ubiquitin-Protein Ligases chemistry
- Abstract
The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)
- Published
- 2011
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37. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.
- Author
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Canettieri G, Di Marcotullio L, Greco A, Coni S, Antonucci L, Infante P, Pietrosanti L, De Smaele E, Ferretti E, Miele E, Pelloni M, De Simone G, Pedone EM, Gallinari P, Giorgi A, Steinkühler C, Vitagliano L, Pedone C, Schinin ME, Screpanti I, and Gulino A
- Subjects
- Acetylation, Animals, Cell Cycle Proteins, Cell Line, Cell Line, Tumor, Cells, Cultured, Chromatin Immunoprecipitation, Cullin Proteins genetics, Electrophoresis, Polyacrylamide Gel, Hedgehog Proteins genetics, Histone Deacetylase 1 genetics, Histone Deacetylase 1 metabolism, Histone Deacetylase 2 genetics, Histone Deacetylase 2 metabolism, Histone Deacetylases genetics, Humans, Immunoblotting, Immunohistochemistry, Kruppel-Like Transcription Factors genetics, Kruppel-Like Transcription Factors metabolism, Medulloblastoma genetics, Medulloblastoma metabolism, Mice, NIH 3T3 Cells, Nerve Tissue Proteins genetics, Oncogene Proteins genetics, Protein Binding, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction genetics, Spectrometry, Mass, Electrospray Ionization, Trans-Activators genetics, Transferases, Zinc Finger Protein GLI1, Zinc Finger Protein Gli2, Cullin Proteins metabolism, Hedgehog Proteins metabolism, Histone Deacetylases metabolism, Nerve Tissue Proteins metabolism, Oncogene Proteins metabolism, Signal Transduction physiology, Trans-Activators metabolism
- Abstract
Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.
- Published
- 2010
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38. The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain.
- Author
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Malgieri G, Russo L, Esposito S, Baglivo I, Zaccaro L, Pedone EM, Di Blasio B, Isernia C, Pedone PV, and Fattorusso R
- Subjects
- Agrobacterium tumefaciens genetics, Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Binding Sites, Cysteine genetics, Cysteine metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Histidine genetics, Histidine metabolism, Hydrophobic and Hydrophilic Interactions, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Tertiary, Repressor Proteins genetics, Repressor Proteins metabolism, Sequence Alignment, Agrobacterium tumefaciens chemistry, Bacterial Proteins chemistry, DNA chemistry, DNA metabolism, DNA-Binding Proteins chemistry, Protein Folding, Repressor Proteins chemistry, Zinc Fingers
- Abstract
The first putative prokaryotic Cys(2)His(2) zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys(2)His(2) zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys(2)His(2) zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys(2)His(2) zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9-66), is arranged in a betabetabetaalphaalpha topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on (15)N R(1), (15)N R(2), and heteronuclear (15)N-{(1)H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain.
- Published
- 2007
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39. Computational analysis of the thermal stability in thioredoxins: a molecular dynamics approach.
- Author
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Pedone EM, Bartolucci S, Rossi M, and Saviano M
- Subjects
- Amino Acid Sequence, Bacillus, Computer Simulation, Escherichia coli, Hot Temperature, Models, Molecular, Molecular Sequence Data, Protein Conformation, Sequence Alignment, Sequence Analysis, Solutions, Thermodynamics, Bacterial Proteins chemistry, Mathematical Computing, Thioredoxins chemistry
- Abstract
The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability. In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.
- Published
- 1998
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- View/download PDF
40. Conformational studies of retro-inverso peptides: the crystal and molecular structure of the hydantoin from H-Ala-g-Ala-mGly-OBzl.
- Author
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Benedetti E, Pedone EM, Kawahata NH, and Goodman M
- Subjects
- Amides chemistry, Amino Acid Sequence, Crystallization, Crystallography, X-Ray, Hydrogen Bonding, Models, Molecular, Molecular Sequence Data, Molecular Structure, Hydantoins chemistry, Oligopeptides chemistry, Protein Conformation
- Abstract
The crystal structure of the hydantoin 1-[(S)-1'-aminoethylmalonyl benzyl ester]-(S)-4-methylimidazolidin-2,5-dione (1) derived from the peptide H-Ala-gAla-mGly-OBzl, having the retro-inverso modification of the Ala-Gly bond, has been determined by x-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2(1) with a = 6.539, b = 14.721, c = 17.101 A, z = 4. The structure was solved by direct methods and refined with anisotropic thermal factors to a final R value of 0.067 for the 947 observed reflections. Reversal of the Ala-Gly amide bond perturbs the folding tendency of the backbone shown by the parent peptide t-BuCO-Ala-Gly-NHiPr. The gem-diamino residue, gAla, and the malonyl moieties are found in the helical and the extended conformations, respectively. Intramolecular hydrogen bonding is not observed. The molecules in the crystal are held together by the formation of two intermolecular hydrogen bonds of the N-H ... O=C type with N ... O distances of 2.86 and 3.17 A, respectively.
- Published
- 1995
- Full Text
- View/download PDF
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