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1.
Chem Sci ; 15(23): 8858-8872, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38873078

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.

2.
Molecules ; 27(20)2022 Oct 17.
Article in English | MEDLINE | ID: mdl-36296575

ABSTRACT

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated.


Subject(s)
Amino Acids , Vascular Endothelial Growth Factor A , Amino Acid Sequence , Peptides/chemistry , Circular Dichroism , Protein Conformation
3.
Biomolecules ; 12(6)2022 05 24.
Article in English | MEDLINE | ID: mdl-35740865

ABSTRACT

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.


Subject(s)
Porphyrins , Proteasome Endopeptidase Complex , Kinetics , Porphyrins/chemistry , Porphyrins/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proteolysis , Proteostasis
4.
FEBS J ; 289(6): 1591-1602, 2022 03.
Article in English | MEDLINE | ID: mdl-34689403

ABSTRACT

Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.


Subject(s)
Protein Folding , Proteins , Calorimetry, Differential Scanning , Circular Dichroism , Disulfides/chemistry , Humans , Protein Denaturation , Thermodynamics
5.
Bioorg Chem ; 116: 105379, 2021 11.
Article in English | MEDLINE | ID: mdl-34563997

ABSTRACT

The analysis of the forces governing helix formation and stability in peptides and proteins has attracted considerable interest in order to shed light on folding mechanism. We analyzed the role of hydrophobic interaction, steric hindrance and chain length on i, i + 3 position in QK peptide, a VEGF mimetic helical peptide. We focused on position 10 of QK, occupied by a leucine, as previous studies highlighted the key role of the Leu7-Leu10 interaction in modulating the helix formation and inducing an unusual thermodynamic stability. Leu10 has been replaced by hydrophobic amino acids with different side-chain length, hydrophobicity and steric hindrance. Ten peptides were, hence, synthesized and analyzed combining circular dichroism, calorimetry and NMR spectroscopy. We found that helical content and thermal stability of peptide QK changed when Leu10 was replaced. Interestingly, we observed that the changes in the helical content and thermal stability were not always correlated and they depend on the type of interaction (strength and geometry) that could be established between Leu7 and the residue in position 10.


Subject(s)
Peptides/chemistry , Vascular Endothelial Growth Factors/chemistry , Hydrophobic and Hydrophilic Interactions , Protein Conformation
6.
Eur J Med Chem ; 222: 113575, 2021 Oct 15.
Article in English | MEDLINE | ID: mdl-34130005

ABSTRACT

HPLW is a Vascular Endothelial Growth Factor (VEGF)-mimicking beta-hairpin peptide endowed of proangiogenic effect and showing valuable biomedical application in the proangiogenic therapy. However, the translational potential of HPLW is limited by its low metabolic stability, which would shorten the in vivo efficacy of the molecule. Here, we developed a peptide analog of HPLW, named HPLW2, that retains the structural and biological properties of the original peptide but features an impressive resistance to degradation by human serum proteases. HPLW2 was obtained by covalently modifying the chemical structure of the peptide with molecular tools known to impart protease resistance. Notably, the peptide was cyclized by installing an interstrand triazole bridge through Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) reaction. HPLW2 appears as a novel and promising drug candidate with potential biomedical application in the proangiogenic therapy as a low molecular weight drug, alternative to the use of VEGF. Our work points out the utility of the interstrand triazole bridge as effective chemical platform for the conformational and metabolic stabilization of beta-hairpin bioactive peptides.


Subject(s)
Peptides/chemistry , Vascular Endothelial Growth Factor A/chemistry , Cell Proliferation/drug effects , Cells, Cultured , Click Chemistry , Humans , Molecular Conformation , Peptides/pharmacology
7.
Int J Mol Sci ; 21(19)2020 Sep 29.
Article in English | MEDLINE | ID: mdl-33003385

ABSTRACT

The present study provides new evidence that cationic porphyrins may be considered as tunable platforms to interfere with the structural "key code" present on the 20S proteasome α-rings and, by consequence, with its catalytic activity. Here, we describe the functional and conformational effects on the 20S proteasome induced by the cooperative binding of the tri-cationic 5-(phenyl)-10,15,20-(tri N-methyl-4-pyridyl) porphyrin (Tris-T4). Our integrated kinetic, NMR, and in silico analysis allowed us to disclose a complex effect on the 20S catalytic activity depending on substrate/porphyrin concentration. The analysis of the kinetic data shows that Tris-T4 shifts the relative populations of the multiple interconverting 20S proteasome conformations leading to an increase in substrate hydrolysis by an allosteric pathway. Based on our Tris-T4/h20S interaction model, Tris-T4 is able to affect gating dynamics and substrate hydrolysis by binding to an array of negatively charged and hydrophobic residues present on the protein surface involved in the 20S molecular activation by the regulatory proteins (RPs). Accordingly, despite the fact that Tris-T4 also binds to the α3ΔN mutant, allosteric modulation is not observed since the molecular mechanism connecting gate dynamics with substrate hydrolysis is impaired. We envisage that the dynamic view of the 20S conformational equilibria, activated through cooperative Tris-T4 binding, may work as a simplified model for a better understanding of the intricate network of 20S conformational/functional states that may be mobilized by exogenous ligands, paving the way for the development of a new generation of proteasome allosteric modulators.


Subject(s)
Allosteric Regulation/genetics , Cations/metabolism , Porphyrins/metabolism , Proteasome Endopeptidase Complex/metabolism , Catalysis , Cations/pharmacology , Cytoplasm/genetics , Humans , Kinetics , Nuclear Magnetic Resonance, Biomolecular , Porphyrins/pharmacology , Proteasome Endopeptidase Complex/genetics , Protein Binding/drug effects
8.
J Pept Sci ; 26(2): e3231, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31749266

ABSTRACT

The binding process of A9 peptide toward HER2-DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2-DIVMP, multiple ligand-based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.


Subject(s)
Peptides/chemistry , Peptides/pharmacology , Receptor, ErbB-2/metabolism , Magnetic Resonance Spectroscopy , Protein Binding , Protein Domains , Receptor, ErbB-2/chemistry , Surface Plasmon Resonance
9.
Mol Biotechnol ; 61(11): 860-872, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31531759

ABSTRACT

Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains. VEGFR2 domain 7 has been shown to play a crucial role in receptor dimerization and regulation, therefore, representing a convenient target for the allosteric modulation of VEGFR2 activity. The ability to prepare a functional VEGFR2D7 domain represents the starting point to the development of novel VEGFR2 binders acting as allosteric inhibitors of receptor activity. Here, we describe a robust and efficient procedure for the preparation in E. coli of the VEGFR2 domain 7. The protein was obtained with a good yield and was properly folded. It was investigated in a biochemical and structural study, providing information on its conformational arrangement and in solution properties.


Subject(s)
Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/genetics , Allosteric Regulation , Circular Dichroism , Escherichia coli/metabolism , Magnetic Resonance Spectroscopy , Protein Domains/genetics , Protein Folding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Scattering, Radiation , Spectrometry, Fluorescence , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/isolation & purification
10.
Mol Biotechnol ; 61(7): 513-520, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31025286

ABSTRACT

VEGF-A/VEGFR2 complex is the major signaling pathway involved in angiogenesis and the inhibition of this axis retards tumor growth and inflammatory disorders progression, reducing vessel sprouting. Signaling by VEGFR2 requires receptor dimerization and a well-defined orientation of monomers in the active dimer. The extracellular portion of receptor is composed of seven Ig-like domains, of which D2-3 are the ligand binding domains, while D4 and D7, establishing homotypic contacts, allosterically regulate receptor activity. The allosteric targeting of VEGFR2 represents a promising alternative to study neovascular disorders overcoming drawbacks related to competition with VEGF. In this work, we expressed in bacterial host domain 4 of VEGFR2 (VEGFR2D4). After protein refolding, we characterized the purified domain and administered it in mice for monoclonal antibodies production. One of them, mAbD4, was tested in ELISA assays, showing a nanomolar affinity for VEGFR2D4. Finally, the methodology here described could contribute to the development of antibodies which can allosterically bind VEGFR2 and therefore to be used for imaging purposes or to modulate receptor signaling.


Subject(s)
Angiogenesis Inhibitors/immunology , Antibodies, Monoclonal , Recombinant Proteins/immunology , Vascular Endothelial Growth Factor Receptor-2/immunology , Allosteric Regulation , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/therapeutic use , Escherichia coli , Humans , Mice , Protein Domains/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/genetics
11.
Chemistry ; 24(44): 11461-11466, 2018 Aug 06.
Article in English | MEDLINE | ID: mdl-29799174

ABSTRACT

QK peptide is a vascular endothelial growth factor (VEGF)-mimetic molecule with significant proangiogenic activity. In particular, QK is able to bind and activate VEGF receptors (VEGFRs) to stimulate a functional response in endothelial cells. To characterize the peptide bioactivity and its molecular recognition properties, a detailed picture of the interaction between peptide QK and VEGF receptors is reported. By combining NMR spectroscopy studies in solution on the purified receptor and in the presence of intact endothelial cells, a molecular description of the binding interaction between peptide QK and VEGFR2 in the cellular context is obtained. These results reveal useful insights into the peptide biological mechanism, which opens the way to further optimization of this class of VEGF-mimicking peptides.


Subject(s)
Biomimetic Materials/chemistry , Peptides/chemistry , Receptors, Vascular Endothelial Growth Factor/chemistry , Vascular Endothelial Growth Factor A/chemistry , Endothelial Cells , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , Protein Conformation
12.
Brain ; 141(5): 1300-1319, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29490009

ABSTRACT

Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-ß signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-ß activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-ß/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.


Subject(s)
Carrier Proteins/metabolism , Cerebellar Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/physiology , Medulloblastoma/metabolism , Neoplasm Metastasis/physiopathology , PTEN Phosphohydrolase/metabolism , Adolescent , Animals , Carrier Proteins/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cerebellar Neoplasms/pathology , Child , Child, Preschool , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks , Humans , Infant , Male , Medulloblastoma/pathology , Mice , Mice, Inbred BALB C , Models, Molecular , Neoplasm Metastasis/genetics , PTEN Phosphohydrolase/genetics , Phosphoric Monoester Hydrolases , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Snail Family Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism
13.
J Med Chem ; 61(7): 2910-2923, 2018 04 12.
Article in English | MEDLINE | ID: mdl-29522685

ABSTRACT

Peptide-binding G protein-coupled receptors (GPCRs) are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based nuclear magnetic resonance (NMR) methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRF-CEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arises. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs.


Subject(s)
Neoplasms/metabolism , Receptors, CXCR4/antagonists & inhibitors , Animals , Cell Line , Cell Line, Tumor , Cell Survival , Chemokine CXCL12/antagonists & inhibitors , Chemokine CXCL12/metabolism , Cricetinae , Cricetulus , Epitope Mapping , Guanidine/metabolism , Humans , Leukemia, T-Cell/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Receptors, CXCR4/metabolism , Receptors, G-Protein-Coupled/metabolism
14.
Org Biomol Chem ; 16(5): 787-795, 2018 01 31.
Article in English | MEDLINE | ID: mdl-29319097

ABSTRACT

Molecular tools to stabilize the ß-hairpin conformation are needed as ß-hairpin peptides are useful molecules for pharmaceutical, biological and materials applications. We explored the use of a "triazole bridge", a covalent link between two ß-hairpin strands obtained through Cu-catalyzed alkyne-azide cycloaddition, combined with an aromatic-aromatic interaction. Highly conformationally stable peptides were identified by NMR screening of a small collection of cyclic peptides based on the Trpzip2 scaffold. The characteristic Trp-Trp interaction of Trpzip2 was replaced by a diagonal triazole bridge of variable length. NMR and CD analyses showed that triazole and indole rings could favorably interact to stabilize a ß-hairpin conformation. The conformational stabilization depends on the length of the triazole bridge and the reciprocal position between the aromatic rings. Combining aromatic interactions and the covalent inter-strand triazole bridge is a useful strategy to obtain peptides with a high ß-hairpin content.


Subject(s)
Peptides/chemistry , Triazoles/chemistry , Tryptophan/chemistry , Amino Acid Sequence , Catalysis , Copper/chemistry , Cycloaddition Reaction , Peptides/chemical synthesis , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Protein Conformation, beta-Strand , Protein Stability , Thermodynamics , Triazoles/chemical synthesis , Tryptophan/chemical synthesis
15.
Mol Biosyst ; 13(8): 1619-1629, 2017 Jul 25.
Article in English | MEDLINE | ID: mdl-28685787

ABSTRACT

The ability to modulate angiogenesis by chemical tools has several important applications in different scientific fields. With the perspective of finding novel proangiogenic molecules, we searched peptide sequences with a chemical profile similar to that of the QK peptide, a well described VEGF mimetic peptide. We found that residues 1617-1627 of the IQGAP1 protein show molecular features similar to those of the QK peptide sequence. The IQGAP1-derived synthetic peptide was analyzed by NMR spectroscopy and its biological activity was characterized in endothelial cells. These studies showed that this IQGAP1-derived peptide has a biological activity similar to that of VEGF and could be considered as a novel tool for reparative angiogenesis.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Peptides/pharmacology , Vascular Endothelial Growth Factor A/pharmacology , ras GTPase-Activating Proteins/chemistry , Amino Acid Sequence , Angiogenesis Inducing Agents/chemical synthesis , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Transformed , Cell Proliferation/drug effects , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Molecular Mimicry , Neovascularization, Physiologic/drug effects , Peptides/chemical synthesis , Protein Conformation, alpha-Helical , Swine , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
16.
Sci Rep ; 6: 31295, 2016 08 08.
Article in English | MEDLINE | ID: mdl-27498819

ABSTRACT

The angiogenic properties of VEGF are mediated through the binding of VEGF to its receptor VEGFR2. The VEGF/VEGFR interface is constituted by a discontinuous binding region distributed on both VEGF monomers. We attempted to reproduce this discontinuous binding site by covalently linking into a single molecular entity two VEGF segments involved in receptor recognition. We designed and synthesized by chemical ligation a set of peptides differing in length and flexibility of the molecular linker joining the two VEGF segments. The biological activity of the peptides was characterized in vitro and in vivo showing a VEGF-like activity. The most biologically active mini-VEGF was further analyzed by NMR to determine the atomic details of its interaction with the receptor.


Subject(s)
Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Binding Sites , Cell Movement , Cell Proliferation , Circular Dichroism , Crystallography, X-Ray , Human Umbilical Vein Endothelial Cells , Humans , Magnetic Resonance Spectroscopy , Mice , Mice, Inbred C57BL , Neovascularization, Pathologic , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, Secondary , Signal Transduction
17.
Chem Sci ; 7(2): 1286-1297, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-29910886

ABSTRACT

The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental (enzyme kinetics, UV stopped flow and NMR) and computational (bioinformatic analysis and docking studies) approaches to inspect proteasome inhibition by meso-tetrakis(4-N-methylpyridyl)-porphyrin (H2T4) and its two ortho- and meta-isomers. We show that in a first, fast binding event H2T4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 (Asp10, Phe9), α3 (Tyr5), α5 (Asp9, Tyr8), α6 (Asp7, Tyr6) and α7 (Asp9, Tyr8) subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the ß-rings. Of note, the proteasome inhibition by ortho- and meta-H2T4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome-ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.

18.
Sci Rep ; 5: 16651, 2015 Nov 25.
Article in English | MEDLINE | ID: mdl-26602442

ABSTRACT

HPLW, a designed VEGF (Vascular Endothelium Growth Factor) receptor-binding peptide, assumes a well folded ß-hairpin conformation in water and is able to induce angiogenesis in vivo. In this study, we investigated at atomic resolution the thermal folding/unfolding pathway of HPLW by means of an original multi-technique approach combining DSC, NMR, MD and mutagenesis analyses. In particular, careful NMR investigation of the single proton melting temperatures together with DSC analysis accurately delineate the peptide folding mechanism, which is corroborated by computational folding/unfolding simulations. The HPLW folding process consists of two main events, which are successive but do not superimpose. The first folding step initiates at 320 K upon the hydrophobic collapse of the Trp5 and Trp13 side-chains which stabilizes the concurrent ß-turn formation, whose COi-HNi + 3 hydrogen bond (Asp10 → Arg7) appears particularly stable. At 316 K, once the ß-turn is completely formed, the two ß-strands pair, very likely starting by Trp5 and Trp13, which thus play a key role also in the final step of the ß-hairpin folding. Overall, here we describe a multi-state hierarchical folding pathway of a highly structured ß-hairpin, which can be classified as a broken-zipper mechanism.


Subject(s)
Peptides/chemistry , Tryptophan/chemistry , Amino Acid Sequence , Calorimetry, Differential Scanning , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Dynamics Simulation , Mutagenesis , Peptides/genetics , Peptides/metabolism , Protein Folding , Protein Structure, Secondary , Protein Unfolding , Thermodynamics , Transition Temperature , Tryptophan/metabolism
19.
FEBS Lett ; 589(7): 798-804, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25701590

ABSTRACT

A change in the conformational plasticity of α-Synuclein (α-Syn) is hypothesised to be a key step in the pathogenic mechanism of Parkinson's disease (PD). Here, we report the study of extracellular α-Syn interaction with whole cells and membranes isolated from the neuronal SH-SY5Y cells, exploiting NMR and CD spectroscopies. In addition, the crosslinking agent DSG was used to freeze the conformational and oligomeric state of α-Syn in the presence of cells. These data, in a quasi-physiological environment, confirm the protein monomeric state with a propensity to adopt a transient alpha helical following interaction with biological membranes.


Subject(s)
Cell Membrane/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , Cell Line, Tumor , Circular Dichroism , Humans , Magnetic Resonance Imaging , Models, Molecular , Protein Binding , Protein Structure, Secondary
20.
Eur J Med Chem ; 91: 100-8, 2015 Feb 16.
Article in English | MEDLINE | ID: mdl-25240418

ABSTRACT

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.


Subject(s)
Agrobacterium tumefaciens/chemistry , Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DNA-Binding Proteins/chemistry , Molecular Docking Simulation , Zinc Fingers , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , Humans , Kinetics , Molecular Dynamics Simulation , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Species Specificity , Static Electricity , Structural Homology, Protein , Thermodynamics
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